Surface Characterisation of Bacterial Cells Relevant to Mineral Industry

Home , Acidophile (histology), Bacillus (shape), Lipoteichoic acid, Periplasm

Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals

Prashant K. Sharma

Division of Mineral Processing Luleå University of Technology SE-97187, Luleå Sweden

November, 2001

SUMMARY Biomineral beneficiation concerns the manner in which different microorganisms bring about the enrichment of an ore matrix. It involves the selective removal of undesirable mineral constituents from an ore through microbe-mineral interactions in the processes such as selective flotation and flocculation. The adhesion of microorganisms to minerals result in alteration of surface chemistry of minerals relevant to beneficiation process due to a consequence of the formation of a biofilm on the surface or biocatalysed surface oxidation or reduction products. Physico-chemical properties of microbial cell surface influence their adhesion behaviour, therefore the physico-chemical characterisation of microbial cell is essential in order to fully understand and control the biomineral beneficiation process. Different bacteria have different surface properties and adaptation of bacteria by growing them in presence of minerals introduces further changes in their surface properties. The pure strains of Thiobacillus thiooxidans, Thiobacillus ferrooxidans (T.f.) along with sulphur grown T.f. and Paenibacillus polymyxa (P.p.) along with chalcopyrite, pyrite, galena and sphalerite adapted strains are used in the study. The charge characteristics of mineral and bacterial cell surfaces are determined using electrokinetic measurements and the chemical composition is determined using FT-IR, FT-Raman and XPS spectroscopy. Hydrophobicity of the mineral and bacterial cell surface is evaluated using contact angle measurements, adhesion to organic solvents and surface energy evaluation. The surface energy of bacterial cells has been evaluated using different physico-chemical approaches - Fowkes, Equation of state, Geometric mean and Lifshitz-van der Waals/Acid-Base (LW-AB) approach. A detailed analysis of the physico-chemical approaches available to evaluate the bacterial cells surface energy from liquid contact angles is performed using literature data on 147 different microbial cells. It has been concluded that Geometric mean and Equation of state approaches evaluate similar surface energy values. But both of them give inconsistent results as the surface energy values change with the use of different liquid contact angles. Surface energy evaluated using LW-AB approach gives most detailed information of the bacterial cell surface. This approach is effected by mathematical instability but contact angle with the three liquids - Water, Formamide and Methyleneiodide/α-Bromonapthelene evaluates the most consistent results. The electron donor characteristics evaluated by LW-AB approach can differentiate between the gram-negative and gram-positive bacterial species. The other advantage of LW-AB approach is the fact that extended DLVO approach could be used to study the adhesion of bacterial cells on mineral surfaces. The polar components of the surface energy of mineral were lower than bacterial cells. Bacterial cells had a very high electron donating characteristics but for mineral surface it was on the lower side. Adhesion of microbial cells on mineral surface has been studied by constructing adsorption isotherms and indirectly by FT-IR spectroscopy and electrokinetic studies. Adsorption studies, electro-kinetic studies, IR-spectroscopic

I studies and change in the flotation behaviour of the minerals experimentally showed that the adhesion of bacteria is taking place on the mineral surface. The bacterial adhesion is theoretically assessed by thermodynamic and extended DLVO calculations. The extended DLVO approach is found to be more effective in predicting the adhesion behaviour than the expectations from the thermodynamic approach. The thermodynamic approach yields no bacterial adhesion on minerals and this discrepancy could be the result of inadequate description of the phenomena, which strongly depends on the distance of separation between bacterial cell and mineral surface. The adhesion predictions by the DLVO approach are able to partially explain the adhesion and hence the bioflotation results of pyrite and chalcopyrite. Extended DLVO also shows that on account of high bacterial surface energy their aggregation is not feasible. But due to the hydrophobicity of pyrite and chalcopyrite, their aggregation is possible. Mineral-adapted bacterial cells showed marked differences in their surface properties from the unadapted ones. Paenibacillus polymyxa cells became more hydrophilic and gained the electron-accepting characteristic after adaptation, where as for Thiobacillus ferrooxidans the IEP shifted to higher pH value. Single mineral Hallimond flotation is performed for chalcopyrite and pyrite after interaction with microbial cells and in presence and absence of collector. Microbial cells were able to successfully depress pyrite flotation and not chalcopyrite. Hence, a separation among pyrite and chalcopyrite is possible by bioflotation.

Keywords: Bacterial adhesion, Bioprocessing, Biobeneficiation, bioflotation, microorganisms, sulphide minerals, Zeta-potential, Contact angle, surface energy, DLVO, XPS, FT-IR, FT-Raman

ACKNOWLEDGEMENTS

First of all, I would like to express my sincere gratitude to my supervisor, Associate Professor K. Hanumantha Rao for all his help and patience.

I would like to thank Prof. Eric Forssberg for making it possible for me to come here and pursue my doctoral studies.

Sincere thanks to Prof. K.A. Natarajan, Indian Institute of Science, Bangalore, India for introducing me to the field of bio-processing and his continued help, fruitful discussions and suggestions.

I would also like to acknowledge The Swedish Foundation for International Co- operation in Research and Higher Education (STINT), Sweden for financial support for the project “Bio-mineral and bio-hydrometallurgical processing”. I would also like to acknowledge the financial support from the KKS (Stiftelsen för Kunskaps- och Kompetensutveckling) Företagsforskarskola inom berg- och mineralteknik in the later period of my studies.

I would like to thank Ulf Nordström, Maine Ranheimer, Birgitta Nyberg and Björg Tangen Lundmark for their help in the lab.

Thanks to all my friends and colleagues (alphabetically)- Andreas Krig, Aruna Thakur, Hamid-Reza Manouchehri, Maneesh Singh, Nourreddine Menad, Philippe Lingois, Tarun Kundu and Vidyadhar Ari for the time spent together.

Last but most important, I would like to thank my wife, Aradhana, for her patience with me especially during the preparation of this thesis. I would also like to thank my parents in India for their continued support.

Prashant K. Sharma November 2001

III

CONTENTS

Contents Page

Summary I Acknowledgements III

1. Introduction 1

2. Minerals and Microbes 5 2.1 The Cell 5 2.2 Bacterial Strains: Their Growth and Adaptation 8 2.3 Minerals and Reagents 12 Paper I 13

3. Surface Characterisation 23 3.1 Bacterial Cell Surface Structure 23 3.2 Bacterial Cell Surface Properties and its Characterisation 30 Paper II 53 Paper III 77 Paper IV 89

4. Microbial Adhesion and Adsorption on Mineral Surfaces 201 4.1 Microbial Adhesion Mechanisms 201 4.2 Factors Effecting Adhesion 202 4.3 Thermodynamic Aspects of Adhesion 204 4.4 Colloidal Aspects of Adhesion 205 4.5 Mineral Microbe Interaction: Experimental Studies 209 Paper V 213 Paper VI 233

5. Biobeneficiation: Bioflotation 245 Paper VII 251 Paper VIII 259 Paper IX 279

6. Conclusions 289

7. References 291

List of Publications 297

Chapter 1: Introduction

1. Introduction Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001

Minerals exist in nature abundantly in the earth's crust in the form of ore bodies, i.e., in association with other minerals. In order to extract metals from minerals by hydro- and pyro-metallurgical methods, it is very important to concentrate the mineral from the ore. Mineral processing is the branch of science, which concerns itself with the processes of mineral separation from ore. Conventionally, physico-chemical methods are used in mineral processing, but now a days, biotechnological processing routes are sought to solve the problems associated with lean grade ores and where the traditional methods fail to separate the minerals from complex ores. Two major areas, which are making advances in minerals bioprocessing, are bio-leaching and bio-beneficiation (Fig. 1.1). Bio-leaching can be defined as a hydrometallurgical dissolution process assisted by microorganisms for the recovery of metals from their ores/concentrates. Major activity has been in bio-leaching of sulphide minerals and chemolithoautotrophic bacteria have been used for the bioleaching process. Over the past three decades bio-leaching has come a long way and is now economically competitive, many processes have been commercialised and are in use. Whereas, bio-beneficiation is relatively a new area and a new term which, has recently has been defined as “bio-beneficiation involves the selective removal of undesired mineral constituents from an ore through interaction with microorganisms, enriching the solid residue with respect to the desired mineral phase” (Natarajan, 1998). The subject of this thesis lies in the broad area of minerals bio-processing and more specifically in bio-beneficiation. Bio-beneficiation uses the conventional wet mineral processing methods, which separate minerals based on their surface property differences, in presence of bio-reagents. The most common methods are flotation and settling (flocculation) which are termed as bio-flotation and bio-flocculation in the bio-processing terminology. The bio-reagents used are the microbial cultures, microbial cells and microbial metabolites (aqueous environment in which microbes grow). The two major factors, which contribute to selectivity in bio-flotation and bioflocculation processes, are selective adhesion of microbial cells on the mineral surface, which forms a biofilm and causes alteration on the mineral surface, and secondly, selective interaction of attached microbial cells with the added chemical reagents (Fig. 1.1). Adhesion of microbial cells depends upon its electrostatic, van der Waals and acid/base interactions with the mineral surface. All these interactions are function of the microbial surface properties like, surface charge, surface hydrophobicity, the van der Waals component of microbial cell surface energy. Chapter 3 deals with the surface characterisation aspects of microbial cell and mineral surface. Adhesion of microbial cells on the mineral surface causes the formation of biofilm and imparting its own surface properties to the mineral. Adhesion also leads to bio-catalysed oxidation or reduction of the mineral surface in the case of chemolithotrophic microorganisms, generation of surface-active bio-reagents or adsorption of metabolic products. All these processes lead to alteration in surface properties of the mineral.

1. Introduction Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001

Minerals Bioprocessing

Bioleaching Biobeneficiation

Bioflotation Bioflocculation Flotation

Interaction of adsorbed Chapter 5 microbial cells and

Factors Biobeneficiation imparting altered mineral surface selectivity with added chemical reagents for flotaiton or flocculation Selective adhesion of microbes on mineral surface Alteration of surface chemistry of mineral

Microbial adhesion ter 4 ter p Adhesion Microbial Cha van der Waals interactions Acid-base Interactions Electrostatic interactions

Depends upon

surface composition Surface Hydrophobicity Chapter 3 Characterisation surface charge Cells Mineral

Microbes and Minerals Chapter 2

Figure 1.1 Biobeneficiation and its dependence on physico-chemical characteristics of the microbial cell surface; the figure also shows the layout of this Thesis

Chemical reagents like collectors, flocculants, dispersants and frothers, which are used in the process of flotation and flocculation, interact with the surface of the mineral. After the formation of a biofilm on the mineral surface and other surface

1. Introduction Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001 change caused by the microbial adhesion, the interaction of these chemical reagents will be different and hence the selectivity again is a function of microbial surface charge, hydrophobicity and surface composition (Fig. 1.1). Therefore, in order to fully understand the mechanism behind any selectivity achieved in the bio-beneficiation process, it is very important to know the physico-chemical surface properties of the minerals and microbial cell surface. The total understanding of the process demands that the affects of microbial interaction could be predicted and hence theoretical models are required to quantify the affects. Theoretical models to understand and predict the microbial adhesion exists based on thermodynamics and colloidal chemistry, explained in detain in Chapter 4. But it is very difficult to quantify or predict the final surface property changes or final surface composition of the mineral after microbial adhesion. Present work covers only a part of the whole scope as described previously. The main emphasis of the thesis is the various techniques for the surface characterisation of microbial cell regarding their charge, hydrophobicity and chemical composition. The characterisation has been performed on chemolithoautotrophs – Thiobacillus ferrooxidans (T.f.) and Thiobacillus thiooxidans (T.t.) and heterotroph – Paenibacillus polymyxa (P.p.). Another major portion of the thesis is dedicated to the surface energy evaluation of the microbial cells using different approaches, a detailed analysis is performed using about 147 contact angle data from literature and measured in laboratory. Bio-beneficiation of sulfide mineral form the other objective of the study out of which bio-flotation of chalcopyrite and pyrite has been performed using T.f. and P.p. There has been a lot of activity in the field of bio-leaching and therefore exhaustive literature on the bio-leaching is available. But the subject of biobeneficiation is relatively new and hence literature is not so forthcoming. Since bioleaching was the prominent one among bio-leaching and bio-beneficiation, chemolithotrophs were the first type of microbes, which were used in the biobeneficiation studies also. Desulphurisation of coal by depression of pyrite in the flotation separation of coal from associated minerals is shown by Misra et al. (1996) and Attia et al. (1993) using Thiobacillus ferrooxidans. Yelloji Rao et al. (1992) reported separation of sulphuric acid treated galena from sphalerite using Thiobacillus ferrooxidans. Mycobacterium phlei, a gram positive, rod shaped prokaryotic cell has been observed to be a flocculent for phosphates, hematite and coal fines (Raichur et al. 1996; Smith et al., 1991). Recently Santhya et al. (1999) has reported separation of galena from sphalerite by depressing galena in presence of Thiobacillus thioxidans. The use of heterotroph in bio-beneficiation is very new. Phalguni et al. (1996) reported calcium and iron removal from alumina using Paenibacillus polymyxa. Desiliconisation of calcite, alumina and iron oxide (Deo and Natarajan, 1997) and selective separation of silica and alumina from iron ore (Deo and Natarajan, 1998a) was reported using Paenibacillus polymyxa. Paenibacillus polymyxa also showed capability to degrade collectors like dodecyl amine, diamine, isopropyl xanthate and sodium oleate (Deo and Natarajan, 1998b), which can be used for stripping the residual collector from the mineral surface and can solve environmental problems associated with mineral processing industry. Excellent reviews (Bos et al.1999) and books (Mozes et al. 1991) are available on the microbial cell physico-chemical surface characterisation methods and the relevance of the tests on the microbial adhesion on substrates. Zeta-potential has been extensively used to understand the changes in surface property of cells along with its use to understand the surface composition (Rijnaarts et al. 1995) and to explain their adsorption behaviour on solid surfaces (van Loosdrecht et al. 1987b). Surface

1. Introduction Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001 hydrophobicity and surface free energy have also has been extensively used to understand the adhesion behaviour of microbial cells (van Loosdrecht 1987 a&b, Stenström 1989, chapter 12: Doyle and Rosenberg 1990). Physico-chemical characteristics of the cell surface has been correlated to the surface structure (Busscher et al. 1990, Stenström & Kjelleberg 1985), presence of additional proteins on the surface (Parker & Munn 1984), presence and chain length of mycolic acid (Bendinger et al. 1993) and its genetic dependence (Reid et al. 1999) In the present work, an attempt has been made to use both chemolithotrophs, which use sulfide minerals as their energy source, and heterotrophs, which use organic sugar as their energy source, in bio-flotation of sulfide minerals. There are both advantages and disadvantages associated with the use of chemolithotrophs and heterotrophs. Since chemolithotrophs derive energy by dissolving sulfide minerals, a special affinity towards the mineral surface is expected and could cause changes in mineral surface properties. But the growth kinetics of chemolithotrophs is slow, e. g., a fully-grown culture of T. ferrooxidans can be obtained only after 44 hours and the final cell densities achieved are low. On the other hand the growth kinetics of heterotrophs is fast, e.g., a fully-grown culture of P. polymyxa can be obtained in 8 hours. The other advantage of using heterotrophs apart from achieving high cell densities is that the amount of biopolymers generated by these bacteria is higher as compared to chemolithotrophs. Although heterotrophs facilitate generation of higher cell mass but since they derive energy from organic sugar which is present in the aqueous phase, there is no reason why they should attach themselves to the mineral surface and the fact that aseptic environment is required for the growth is a disadvantage for their use in mineral industry. A proper understanding of bio-beneficiation will lead to a proper control and prior prediction of mineral behaviour with respect to beneficiation. Prior prediction of the microbe-mineral interaction is possible by using extended DLVO approach (van Oss et al. 1986, Bos et al. 1999) or by calculation of free energy of adhesion (Bos et al. 1999) and the final surface-chemical properties of the mineral can be predicted only when the surface properties of the microbe are fully known (Fig. 1b). The adhesion behaviour of bacteria on minerals is thus followed by the thermodynamic and extended DLVO approaches after evaluating the necessary parameters experimentally in this work.

Chapter 2: Microbes and Minerals

2. Microbes and Minerals Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001 2.1 The cell Living single cells are divided in two broad groups, eukaryotic and prokaryotic, according to whether or not their genes are contained in a well-defined nucleus (Fig. 2.2). Living things are also classified in six kingdoms based on their structure. Within prokaryotes are the kingdoms of Monera (Eubacter) and Archea. Within eukaryotes are the kingdoms of Protista, Plantae, Fungae and Animalia. Prokaryotes can be simply described as molecules surrounded by membrane and cell wall, they lack characteristic eukaryotic subcellular membrane enclosed "organelles", but they contain cell membrane inside the cell wall. Prokaryotes are found in simple shapes- cocci (round), baccilli (rods) and spirilla (helical). Only bacteria and few species of blue-green algae belong to this group. Bacteria can be distinguished from blue-green algae in several ways, one of which is that blue-green algae evolve oxygen whereas bacteria do not. Oxygen is released by blue-green algae and higher plants as a by-product of photosynthesis, the process whereby light energy is converted to chemical energy. Although some bacteria are also photosynthetic, they use quite a different mechanism, including a different set of light capturing pigments. Bacterial never evolves oxygen nor do they have chlorophyll a green pigment common to all other photosynthetic systems. Bacteria have dimensions of a few µm, many bacteria have appendages in the form of flagella (Fig. 2.1). They are typically 0.01-0.02 µm's in diameter and up to 10 to 11 µm's' long. Although the flagella is a simple structure with parallel strands of protein but it imparts mobility to the cell. In addition to the flagella, certain bacteria have numerous projections called pilli or fimbriae with diameters of 0.01 µm or less and lengths up to one or two µm.

Figure 2.1 A typical bacterial cell

Bacteria can be classified using various criterions (Fig. 2.2). One way to classify is according to their response to the Gram staining procedure. In this procedure the fixed bacterial smear is subjected to four different reagents in the order listed: crystal violet (primary stain), iodine solution (mordant), alcohol (decolourising agent) and safranin (counter stain). The blue-violet colour reaction is caused by the crystal violet, the primary Gram-stain dye, complexing with the iodine mordent. Alcohol acts as a decolouriser, Gram-negative bacteria looses its primary stain whereas the Gram-positive bacteria retains the blue-violet coloration. Safranin (counter stain) stains the Gram-negative in red colour, but Gram-positive remain of blue-violet colour hence retaining the primary stain. The difference in staining response to Gram stain is related to the chemical and structural differences in bacterial cell walls, which is further explained in the next chapter.

5 2. Microbes and Minerals Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001

Cells

Prokaryotic Cells Eukaryotic Cells No well defined Nucleus for Genetic material contained in the Genetic material well defined nucleus

Some Blue Green Algae Cells from higher Bacteria Evolve O as a by product animals, plants and many 2 Does not evolve O of Photo-synthesis 2 microscopic organisms

Classification I Classification II According to difference in the According to their structure of cell wall and hence nutritional requirements their response to Gram staining Gram positive Heterotroph Thicker cell wall, Requires complex consists of multilayers organic molecules Paenibacillus polymyxa for nutrition, generally of peptidoglycan (Peptidoglycan >90 % Grown on Sucrose supplied by destruction of other cells of cell wall Autotrophs Gram negative Utilises CO2 from Much thinner cell air, reducing it to wall with the required organic < 20% peptidoglycan compounds

Chemoautotrophs Photoautotrophs Energy obtained from Energy required to reduce CO obtained Thiobacillus ferrooxidans oxidation of inorganic 2 compounds eg. Fe2+, S, from light Grown on Fe2+ and So S 2-, H S, NH , H via photosynthesis Thiobacillus thiooxidans 3 2 3 2 Grown on So

Chemolithoautotrophs Can grow in a strictly mineral environment in absence of light

Figure 2.2 Classification of cells in reference to the bacterial cells under investigation

The other way to distinguish bacteria is by their nutritional requirements. If a cell requires complex organic molecules of the kind that would normally be supplied only by destruction of other cells, it is called heterotrophic ("feeding on others"). If, however a cell can utilise the carbon dioxide CO2 from air, reducing it to the organic compounds needed, the cell is called an autotroph ("self feeder"). If the energy required for CO2 reduction is obtained from light via photosynthesis, the cell is photoautotrophic. If the energy is supplied by the oxidation of inorganic compounds 2+ 2- e.g. Fe , S, S3 , H2S, H2 and NH3 etc., the cell is chemoautotrophic (Dyson, 1978). But in the field of minerals bioprocessing the particular category of

6 2. Microbes and Minerals Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001 chemolithoautotrophs is of major importance, since they grow in a strictly mineral environment in the absence of light (Rossi, 1990)

Paenibacillus polymyxa Paenibacillus polymyxa is a spore forming, Gram-positive, heterotrophic facultative neutrophile, aerobic bacterium associated with oxide mineral deposits and uses organic sugar as energy source. It is motile with peritrichous flagella and occurs in rods that vary in size from 0.5 to 1 µm in width and 2 to 8 µm in length. It secretes exopolysaccharides, several proteins, enzymes and organic acids like acetic, formic, and oxalic acid. The old nomenclature of this bacterium was Bacillus polymyxa but it has been recently renamed (Ash et al., 1993). The extracellular polysaccharide (ECP) aids in biological uptake of metal ions necessary for metabolism and growth. P. polymyxa is a versatile organism; it produces catalase and hydrolyses starches. Most of the strains ferment arabinose, cellobiose, dextrine, galactose, glucose, inuline, lactose, levulose, mannitol, mannose, raffinose, salicin, sorbitol, starch, trehalose and xylose. It is also capable to ferment a variety of properly prepared grain mashes- corn and wheat. P. polymyxa is a facultative anaerobe and under aerobic conditions glucose is completely oxidised by O2 to CO2, but in limited supply of O2 glucose is partially oxidised to ethanol, butanediol, acetoin, acetic acid, lactic acid and formic acid. Such oxidation occurs in two stages, first glucose is oxidised to the end product, say acetic acid, by simultaneous reduction of the coenzyme NAD (Eq. 2.1). In the second step, the reduced coenzyme transfers their electrons to oxygen and gets regenerated (Eq. 2.2). Both these reactions are accompanied by generation of ATP, energy packet for the cell (Prescott and Dunn, 1959) C6H12O6+2H2O+4(ADP)+4NAD→2CH3COOH+2CO2+4(NAD)H2+4(ATP) (2.1) (NAD)H2 + ½O2 +2ADP → NAD + H2O + 2(ATP) (2.2) Extra cellular proteins or exoproteins are defined as the proteins present in the medium around the cell, having originated form the cell without any alteration to cell structure greater than that maximum compatible with the cell's normal processes of growth and reproduction. It has been found that Gram-positive organisms produce true exoproteins, while the Gram-negative bacteria do not. P. polymyxa produces multiform β-amylase with approximate molecular weight of 70, 56 and 42 kDa; it also produces 48 kDa α-amylase (Tokekawa et al. 1991).

Thiobacilli Thiobacilli are a common group of microorganisms in acidic mines and are active in the degradation of almost all sulfide minerals. Thiobacillus thiooxidans is a Gram negative, acidophilic, chemolithoautotrophic bacterium that utilises elemental sulphur or reduced sulphur compounds as substrates at temperature up to 40oC and pH values as low as 0.5. The most important of the Thiobacilli group is Thiobacillus ferrooxidans which is a motile, Gram negative, non spore forming and chemolithoautotrophic rod shaped (0.5 to 1.5 µm) bacterium (Brierley, 1978). During the growth, cell pass through three phases, namely, lag, log and stationary phase. The cells divide by binary division every 8 hours. Bacteria is a mesophile having optimum activity in temperature range of 25 to 30oC, being an acidophile it functions actively in pH range of 1 to 5 with an optimum pH range of 2-2.5. The redox potential corresponding to optimum bacterial activity is in the range of 750 to 850 mV, which depends on the ferrous to ferric ratio in the medium. CO2 is its sole source of carbon, which is converted to organic carbon by Carbon Benson cycle. Oxygen is required for

7 2. Microbes and Minerals Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001 the oxidation of reduced valence sulphur compounds (So etc.) to sulphate form and ferrous to ferric. The O2 and CO2 consumption by Thiobacillus ferrooxidans in the medium containing ferrous ions is about 183 to 187 times higher then the maximum amounts of O2 and CO2 soluble in the medium, therefore adequate supply of O2 and CO2 is essential (Natarajan, 1998; Rossi, 1990). Ferrous ion and sulphur oxidation takes place through the Thiobacillus ferrooxidans membrane/envelop. Ferrous sulphate oxidation takes place by the following reaction (Eq. 2.3). Energy of about 5.9 to 7.8 kcal/mol is available to the bacteria in the pH range of 1.5 to 3.0. The key enzymes involved in the Fe2+ oxidation are cytochrome A and coenzyme Q. A copper containing rusticyanin acts as the initial electron acceptor. 4FeSO4 + O2 + 2H2SO4 → 2Fe2(SO4)3 + 2H2O (2.3) Thiobacillus ferrooxidans and Thiobacillus thiooxidans oxidises inorganic sulphur compounds for its energy requirements. The oxygenation of elemental sulphur with the formation of sulphite is catalysed by sulphur oxidising enzyme; 2- sulphur is progressively converted to sulphite via thiosulphate (S2O3 ) and 2- thetrathionate (S4O6 ) intermediates. Thiosulphate can disproportionate into sulphite and sulphide. Alternatively, it may be oxidised to tetrathionate, which then is transformed to sulphite, and trithionate which in turn, gets converted to sulphite and thiosulphate. Oxidation of elemental sulphur takes the following route. S8 + GSH → GS8SH (2.4) Sulphur oxidising system GS8SH + O2 → → GS8SO2H (2.5) GS8SO2H + H2O → GS7SH + H2SO3 (2.6) Elemental sulphur exists most commonly in a ring structure having eight atoms in a molecule. It is attacked by a sulphydryl-containing agent resulting in an organic polysulfide. The sulphur oxidising enzyme catalyses the oxidation of the terminal atom which on hydrolysis gives sulphite. Sulphite is the central intermediate in sulphur oxidation reaction and it is through this compound that all the pathways pass in the formation of sulphate and ATP. Sulphite can thus be oxidised to sulphate either by a cytochrome-mediated oxidation or by adenosine phosphate. A cytochrome-mediated AMP- independent sulphite oxidising system has been studied for Thiobacillus thiooxidans (Natarajan, 1998). The first of these pathways is- 2- ADS reductase SO3 + AMP → → APS (2.7) ADP sulphurylase 2- APS → → SO4 + ADP (2.8) ADP →Adenylate Kinase→ ½ AMP + ½ ADP (2.9)

2.2 Bacterial strains: Their growth and adaptation

Paenibacillus polymyxa Pure strains of Paenibacillus polymyxa NCIM 2539 and CCUG 26013 (Type strain DSM 365) are subcultured on a sucrose rich Bromfield medium (0.5 g/l H2PO4, 1 g/l (NH4)2SO4, 0.2 g/l MgSO4.7H2O, 0.15 g/l yeast extract, 5 g/l sucrose and pH of 7.0) (Vishniac et al., 1974). The medium was autoclaved at 125oC for 20 minutes and then cooled. A 10 % active cell culture was added to the medium and incubated in a rotary shaker at 150 rpm at 300C. Cell count and pH measurements monitored the growth of P. polymyxa. Adaptation of P. polymyxa to sulphide minerals was performed by repeated subculturing of the bacteria in Bromfield medium and in presence of increasing pulp density of -38+5 µm mineral particles. The subculturing was initiated with only 0.1 %

8 2. Microbes and Minerals Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001 pulp density and then the pulp density was successfully increased during subsequent subculturing. At each new step, the inoculum from the previous lower pulp density was introduced into the medium containing higher pulp density. In the present study, P. polymyxa has been adapted to 5 wt% Galena, 5 wt% Sphalerite, 0.5 wt% Chalcopyrite and 0.35 wt% Pyrite.

Thiobacillus ferrooxidans Thiobacillus ferrooxidans isolated from Chitradurga pyrite mines, India were used in this study. T. ferrooxidans was cultured and maintained in 9K medium (3 g/l (NH4)2SO4, 0.5 g/l MgSO4.7H2O, 0.5 g/l K2HPO4, 0.1 g/l KCl, 44.5 g/l FeSO4.7H2O and pH of 2.0) given by Silvermann and Lundgren (1959). 9K - (without Ferrous sulphate) is prepared in flask I with 700 ml of distilled water and 2 ml of 10N H2SO4, flask II contains ferrous sulphate dissolved in 300 ml of distilled water with 0.3 ml of o 10N H2SO4. Flask I is autoclaved at 125 C for 20 minutes. Contents of flask II is filtered through previously autoclaved millipore filter in order to remove any contaminants. The filtrate of flask II is mixed with contents of flask I and inoculated with 10% v/v of T.f. culture and incubated in a rotary shaker at 30oC at 150 rpm. Thiobacillus ferrooxidans were also grown in presence of elemental sulphur (5 wt%) or pyrite (5 wt%) or chalcopyrite (5 wt%). Before the use of pyrite and chalcopyrite, they were washed in dilute sulphuric acid of pH 2.0 and the dried powders were then sterilised at 15 lbs pressure for 30 min. Sulphur was sterilised for 15 min at 5-6 lbs pressure. Oxidation of ferrous ion in the presence of bacteria was monitored by measuring the content of ferrous, total iron (Fe2+ + Fe3+), pH, and cell density with time. Similarly, the growth in presence of solid substrates (sulphur, pyrite and chalcopyrite) was monitored by measuring ferrous and ferric ions, Eh and cell density. The ferrous ions in the liquid medium were monitored using 0-phenanthroline-method (Vogel, 1961). The ferric ion content was assayed after reducing it to ferrous iron by hydroxyammonium hydrochloride. The Eh was noted using a platinum electrode with Ag/AgCl reference (186 mV at 35oC) and the cell numbers in the liquid phase were enumerated using a Bürker counting chamber under a phase contrast microscope. This clearly shows that it was possible to adapt T. ferrooxidans towards much higher pulp density of sulfide minerals (chalcopyrite and pyrite) than P. polymyxa. The reason of which is the fact that T. ferrooxidans is associated with the sulphide mineral ores i.e. it is found as a native organism in sulphide ore bodies and hence have higher tolerance towards these minerals. The chemical changes on the microbial surface can be envisaged in its behaviour indirectly

Thiobacillus thiooxidans Pure strain of DSM 9463 isolated from a bioreactor in Australia is used for the study. The bacteria is grown in the MS-medium containing 2.00 g/l (NH4)2SO4, 0.25 g/l MgSO47 H2O, 0.10 g/l K2HPO4, 0.1 g/l KCl, the pH of medium is adjusted to 3.5 with 1N Sulphuric acid prior to autoclaving. Before inoculation 5 g/l of sterile powdered sulphur is added in the medium. Sterile sulphur is produced by taking sulphur powder in a capped test tube, putting few drops of water in it and putting it in boiling water for 1-2 hours, this procedure is repeated for 3 days consecutively. A pure 10 % v/v of an active inoculum is added to the MS-medium and incubated at 30oC at 150 rpm. Measuring the pH and bacterial cell count in the broth monitors the growth of the bacteria.

9 2. Microbes and Minerals Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001 Cell harvesting Thiobacillus thiooxidans cell grown on sulphur and Thiobacillus ferrooxidans cells grown on ferrous iron, sulphur, pyrite and chalcopyrite were filtered through Whatman filter paper to remove cells from the suspended solid materials. The liquid containing the cells was then centrifuged at 15,000 rpm for 10 min and the cell pellet was obtained. It was washed thrice in dilute sulphuric acid (pH 2) in order to remove any trapped ions Paenibacillus polymyxa containing broth was centrifuged at 15,000 rpm for 20 min to obtain the cell pellet, which was re-suspended in neutral, deionized water and centrifuged in order to remove the metabolic products from the cell surface Some of the centrifuged and re-suspended cells were stored, as it is for use in zeta potential measurements. Some portion was filtered through the Millipore filter for contact angle measurements and some portion of the pellet was freeze-dried so as to record the XPS, FT-IR and FT-Raman spectra.

Cell counting. Bürker counting chamber During the usual laboratory experiments, cells were counted by using hemocytometer (Bürker counting chamber), as this is fast and convenient. Bürker counting chamber is a special microscope slide with 0.65x0.65 mm square subdivided in 13x13 squares and each square 0.02 mm deep, having a volume of 0.0025 mm2. The number of bacterial cells present in each small square represents number of cells present in 5x10-8 ml of suspension. The numbers of bacteria were counted for at least 10 squares and the average was multiplied with a factor of 2x107 in order to attain the number of cells per ml of the cell suspension.

Serial dilution plate count A 1 ml from each stock solution was serially diluted by a factor of 1/10 in test tubes containing 9 ml of saline (0.85% NaCl) for 8 times so that the last one comprise a total dilution of 10-8. A 0.1 ml each from 10-5, 10- 6, 10-7 and 10-8 dilution test tubes was spread on sterilised petri dishes with solidified nutrient agar (8 % nutrient medium, 12% agar). These petri dishes were incubated at 370C for one day and after the growth of cell colonies, they were counted for the number of the cells. From the dilution factor, the original cell count was obtained in the stock solutions (Aneja, 1996).

Cell count- using spectrophotometer Bacterial population can be determined by measuring turbidity or optical density of the bacterial suspension using UV-Visible spectrophotometer. Since turbidity is directly proportional to the number of cells, this property is used as an indicator for bacterial concentration. The cells suspended in the suspension interrupt the passage of light allowing less light to reach the photoelectric cell and the amount of light transmitted through the suspension is measured as percentage transmission or %T. The turbidity for cell suspension is measured at 400 nm against clear water as reference. This method is also very fast and convenient as compared to the serial plate counting method.

10 2. Microbes and Minerals Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001

1011 Serial Plate counting Vs Bürker Counting Chamber Serial Plate counting Vs Turbidity 101

1010

100

9 10

10-1

108

10-2 (Absorbtion units) Turbidity

(Cells/ml) Bürker Counting Chamber 107 106 107 108 109 Serial Plate counting (Cells/ml) Figure 2.3 Calibration curve for bacterial cell counting. Obtained using Paenibacillus polymyxa NCIM 2539

Calibration Curve Since an overestimation of the actual cells was conceivable in Bürker counting chamber method, the cell numbers were reported based on plate count. To this end a comparative calibration curve was made between the enumerated cell count using Bürker counting chamber and Enumeration counting method (serial dilution plate count). Since the turbidity of the cell suspension is just an indicator of the cell concentration, actual cell count can be estimated only if there is a calibration curve between the turbidity and serial plate count for a range of dilute and dense cell suspensions. A concentrated solution of bacterial cells was prepared by dispersing centrifuged cell pellet in deionized water. A series of cell density stock solutions from 1011 to 107 cells/ml were made by diluting the concentrated solution. Cell density (cells/ml) was determined 3 times independently, using each Bürker counting chamber, turbidity and serial plate count. The calibration curve constructed using P. polymyxa NCIM 2539 is shown in Fig. 2.3 and the counts are reported in Table 2.1, this calibration curve was constructed in the later part of the study and is different from the one reported in Sharma and Hanumantha Rao, 1999, because it was based on single measurements. Since the turbidity of the bacterial cell suspension depends on the bacterial cell size and shape apart from their number therefore, the curve between serial plate count and turbidity is valid only for P. polymyxa NCIM 2539, whereas the curve between Bürker count and serial plate count is a general curve valid for a wide range of motile bacterial cells. The large amount of error for both Bürker counting and serial plate count, but very low error in turbidity measurements is seen in Fig. 2.3 and Table 2.1. The reason for high error in the counting methods is due to the fact that these methods use high dilutions to bring the cell number low enough to be countable, hence the presence or absence of one cell at 107 times dilution of 1 ml results in an error of 107 cells/ml. It is clear from Fig. 2.3 that after about 108 cell/ml (Serial plate count) there is a definite change in the slope of both the line correlating serial plate count to Bürker count and turbidity. The curve also delineates the fact that an over estimation of 10 times for low cell densities (<3x108 cells/ml) and 20 times for high cell densities 11 2. Microbes and Minerals Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001 (>3x108 cells/ml) of the actual cell count is inherent in the Bürker counting chamber method. Table 2.1 Cell densities and errors involved in different counting methods (Data points for Fig. 2.3)

Bürker Error Serial plate Error Turbidity Error Count count (Absorption units) (cell/ml) (cell/ml) 3,66x1010 2,14x1010 1,03x109 2,52x108 0,62852 1,08x10-3 1,28x1010 1,35x109 6,00x108 3,61x108 0,32538 9,39x10-4 2,38x109 8,63x108 3,26x108 3,79x108 0,06338 3,49x10-4 1,03x109 6,22x108 9,33x107 2,08x107 0,02996 1,67x10-4 5,05x108 1,11x108 2,50x107 1,03x107 0,01024 6,23x10-4 1,52x108 6,51x107 6,00x106 1,00x106 0,00358 2,39x10-4 1,80x107 7,12x106 5,50x105 7,00x105 Very low -

2.3 Minerals and reagents

Pure crystalline mineral samples were purchased from Gregory, Bottley & Lloyd Ltd., London having chemical composition pyrite (Fe-27.9%, S-53.5%, Cu- 0.04%) and chalcopyrite (Cu-29.8%, Fe-27.9%, S-31.9%). The samples were crushed and finely wet ground in a ceramic ball mill. The ground material was wet-sieved and the −106+38 µm fraction was collected. The −38 µm fraction was microsieved using ultrasonic bath to obtain −5 µm material. These three size fractions of the minerals were dried in an oven at 50oC for 2 days and stored at 4oC in plastic bags. Table 2.2 Lists the surface areas for various mineral size fractions and the test in which they were used. The collector potassium isopropyl xanthate was from Hoechst AG, Germany. Analytical grade 4-methyl-2-pentanol (MIBC frother) was obtained from Merck- Schuchardt. All other reagents were procured from KEBO Lab, Sweden.

Table 2.2 Physical properties of the mineral samples.

Size Fractions Surface Area (m2/g) Test in which (µm) used Pyrite Chalcopyrite -5 2.64 4.166 Electrokinetic measurements +5 -38 0.192 0.7543 Adaptation +38 -106 0.0831 0.362 Flotation

Paper I Selected by the conference organisers for publication in "Hydrometallurgy" journal Presented at International Biohydrometallurgy Symposium, IBS-2001, Sept. 16-19, 2001, Ouro Preto, Minas Gerais, Brazil, Published in Chapter: Analytical techniques applied to biohydrometallurgy of Biohydrometallurgy "Fundamentals, Technology and Sustainable Development", Eds. Ciminelli, V.S.T. and Garcia, O. Jr., Elsevier, Amsterdam, 2001

2. Microbes and Minerals: Paper I Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001

Chapter 3: Surface Characterisation

3. Surface Characterisation Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001

Adhesion of microbial cells depends upon electrostatic, van der Waals and acid/base interactions with the solid substrate. All these interactions are function of the solid and microbial surface properties like, surface charge, surface hydrophobicity, the acid-base and van der Waals components of surface energy (Fig. 3.1). Microbial adhesion involves Alteration of mineral surface chemistry after microbial adhesion depends upon the bacterial surface characteristics van Electrostatic Acid-base der waals interactions Interactions interactions

Depends upon Electrostatic interaction Spectroscopy

Mineral and Microbial surface charge Zeta-potential

Rithenium red staining for Polysaccharides Mineral and Microbial surface composition XPS Spectroscopy

FT-Raman Spectroscopy FT-IR Spectroscopy

Mineral and Microbial hydrophobicity Adhesion to hydrocarbons BATH,MATS

Contact Angle Salt aggregation test Adhesion to hydrophobic surfaces Hydrophobic interaction Surface Energy spectroscopy evaluation

Figure 3.1 Microbial surface characterisation relevant to microbial adhesion in general and biobeneficiation. 3.1 Bacterial cell surface structure (Hammond et al., 1984)

The bacterial cell surface is composed of the cell envelopes, cell wall, cell membrane and surface appendages like pilli, fimbriae etc. The bacterial cell wall is a unique structure, which surrounds the cell membrane. Although not present in every bacterial species, the cell wall is very important as a cellular component. Structurally the cell wall is necessary for: 23

3. Surface Characterisation Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001

• Maintaining the cells characteristic shape. • Counteracting the effect of osmotic pressure. Inside the bacterial cell there is high solute concentration and because of the outside low solute concentration, water tends to flow inside the cell. A great turgor pressure of the order of 5 bar (Dinsdale and Walsby, 1972), and potentially 20-25 bar (Mitchell and Moyle, 1956), is exerted on the cell wall. And hence the cell wall keeps the cell from bursting. • Providing a rigid platform for surface appendages-flagella, fimbriae and pilli. • Providing attachment sites for bacteriophages e.g. Teichoic acid acts as attachment site for viruses. A single structural unit, such as the thick peptidoglycan-containing cell walls of Gram-positive bacteria can impart all these properties. However in many cases complete cell envelops consists of several layers with specialised functions, for example in Gram-negative bacteria the mechanical strength is provided by a peptidoglycan but the outer membrane, overlaying peptidoglycan, is responsible for molecular sieving. The presence of firmly bound capsules, glycocalyces and S-layer crystalline protein arrays, outside the other components of cell wall, may add to the complexity of the layered structure of the bacterial cell surface. The overall physico- chemical properties of the cell surface are an amalgam of the properties of these individual components, which will exert their influence only over a certain distance within and beyond the cell surface.

Teichoic acid or Group specific S-Layer polysaccharide

Protein intercalated

Fimbriae into wall

Peptidoglycan Anionic Mesh

polymers e.g. Lipoteichoic

acid

Periplasmic Space

Cell Membrane

Cytoplasm Membrane Protein

Figure 3.2. Typical Gram-positive cell wall

The distinction between Gram-positive and Gram-negative bacteria, although is based upon the ability of the bacteria to take up the crystal-violet dye, in fact is due to the presence and absence of the outer membrane; the terms Firmicute and

3. Surface Characterisation Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001

Gracilicute have been coined to indicate these two major classes of bacteria (Gibbons and Murry, 1978).

Gram-positive (Firmicutes) cell wall (Hancock, 1991) The main constituent of the Gram-positive cell wall is the thick layer of peptidoglycan, which can be upto 40 layers thick constituting at least 40%, sometimes upto 90% of the cell wall. Peptodiglycan layer can makeup about 10% of the total cell volume. The rest of cell wall is composed of anionic polymers (e.g. Teichoic acid and Teichuronic acid) that are covalently linked to the peptodiglycan.

Peptidoglycan is the stress-bearing component of the cell wall. This consists of glycan chains of alternating N-acetylglucosamine (NAG) and N- acetylmuramic (NAM) acid residues, which are cross linked by short peptide chains (Fig. 3.3 a&b) generally composed of four amino acids L-alanin, D- alanine, D-glutamic acid and diamino pimelic acid (DPA). The peptidoglycan layers are negatively charged

Anionic wall polymers: The most commonly occurring types of anionic polymers are teichoic acids and uronic acid-containing polysaccharides usually called teichuronic acids. Teichoic acid (Fig. 3.3c) are strongly acidic linear polymers of alditol phosphates (glycerol phosphate, ribitol phosphate or mannitol phosphate) or sugar phosphates (usually N-acetylglucosamine phosphate or N-acetylgalactosamine phosphate) in which the phosphate groups connect adjacent alditols or sugars by phosphodiester linkages. The alditol residues are frequently substituted with a sugar; occasionally a sugar alternates with alditol phosphate residue in the main polymer chain. The type of teichoic acid is species specific. Teichoic acid are attached to muramic acid residues in peptidoglycan by a "linkage unit" that usually consists of about three glycerophosphate residues linked to a disaccharide of N- acetylhexosamine. The main teichoic acid is attached to the terminal glycerophosphate unit and the disaccharide is in turn attached to C-6 of the muramic acid by a sugar-1-phosphodiester linkage. The anionic nature of teichoic acid is due to its charged phosphate groups. Charge Characteristics of Teichoic Acid: The phosphate groups in the polymers are fully ionised above pH 2.5, so a typical teichoic acid of 40 repeating units would contain 40 negatively charged phosphates per molecule. The teichoic acid frequently contains amino acid in very alkali-labile ester linkage to alditol residues in the polymer. In most Gram-positive bacterial D-alanine is found, but L- lysine is common in teichoic acid of some bacteria. These contribute one, or in case of lysin two, positive charges per substituted alditol so that the teichoic acid may contain almost as many positively charged group as negatively charges ones. Teichuronic Acid do not conform to any particular structural pattern and are best regarded as any covalently attached acidic polysaccharide with a regular repeating unit in which the anionic function is a carboxyl group provided by uronic acid residue. Their mode of attachment to the peptidoglycan may also vary: direct 25

3. Surface Characterisation Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001

phosphodiester linkage between the reducing end of the polysaccharide and muramic acid, intervention of an N-acetylhexosaminephosphate between the polysaccharide terminus and muramic acid, and direct glycosidic linkage to peptidoglycan have all been described.

(a) (c)

(b)

One Glycan chain

Figure 3.3 (a) Chemical structure of Peptidoglycan, (b) Cartoon

of the peptidoglycan mesh (c) Teichoic acid.

Amphiphilic Lipoteichoic acid These "microampliphiles", originally termed "membrane teichoic acids" before their full structure was determined, originate as membrane-linked molecules since they are assembled by donation of glycerophosphate units from the membrane phospholipid, phosphatidylglycerol, to another membrane glycolipid acceptors. They thus contain polyglycerophosphate chain, often partially glycosylated and alanylated, attached through a terminal phosphodiester to a diglyceride or, more commonly, a glycolipid.

Gram-negative (Gracilicute) cell wall (Hancock, 1991) The cell wall have a more complicated structure and is much thinner, being comprised of only 20% peptidoglycan as compared to the Gram-positive cell wall. Though teichoic acid is 26

3. Surface Characterisation Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001 absent from Gram-negative bacterial surface but it has two unique regions, which surround the cytoplasmic membrane (or cell membrane): The periplasmic space and the outer membrane containing lipopolysaccharide (LPS) layer. The periplasmic space separates the outer plasma membrane from the peptidoglycan layer. It contains proteins, which destroy potentially dangerous foreign matter present in this space. The outer membrane is located adjacent to the exterior peptidoglycan layer. It is a phospholipid bilayer construction similar to that in the cell membrane and is attached to the peptidoglycan by lipoproteins. Lipopolysaccharide layer is present on the outside of the outer membrane, the lipid portion of the LPS contains a toxic substance, called Lipid A, which is responsible for all the pathogenic affects associated with the harmful Gram-negative bacteria.

S-Layer O-antigen Lipopolysaccharides side chain Porin

Lipid A Outer Membrane

Braun's Lipoproteins Peptidoglycan Mesh Periplasmic Space

Cell Membrane

Membrane Protein Cytoplasm

Figure 3.4 Typical Gram-negative cell wall

The Outer Membrane The typical feature of the cell walls of Gram- negative bacteria is the highly organised outer membrane (OM) structure in which an asymmetric bilayer of phospholipid and lipopolysaccharide constitutes a permeability barrier with specific hydrophilic diffusion pores, formed of aggregates of integral "Porin" proteins, connecting the periplasm of the cell to the external environment. The bilayer thus has a hydrophobic core resembling that of a conventional plasma membrane, though much less fluid due to the predominance of saturated fatty acids in LPS and an extremely hydrophilic outer surface provided by the polysaccharide chains (O-antigen) of the LPS

3. Surface Characterisation Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001

Lipopolysaccharides (LPS) are composed of two parts, Lipid A and the polysaccharide chain (Fig. 3.5) that reaches out into the environment. Lipid A is a derivative of 2 NAG units with upto 7 fatty acids connected to it that anchor the LPS in the membrane. Attached to the lipid A is the conserved core polysaccharide that contains KDO (3-deoxy-D- mannooctulosonic acid), heptose, glucose and glucosamine sugars. The rest of the polysaccharide contains repeated sugar units and this is called O-antigen. The O-antigen gets its name from the fact that it is exposed to the outer environment and the host often raises antibodies to this structure, whereas the bacteria protects itself from the hosts defences by varying the make-up of the O-antigen. LPS confers a negative charge and also repels hydrophobic molecules since it is itself Figure 3.5 Structure of highly hydrophilic. Lipopolysaccharide (LPS) Surface Proteins Although the LPS Abe; abequose, Rha; L- molecules are tightly packed on the surface - rhamnose, Gal;Galactose, about 3.5 million molecules per cell surface area Glc; glucose, Hep; L- of 6 µm2 in E.coli (Nikaido and Vaara, 1987)- the glycerol-D-mannoheptose, O-antigen chains are not the only molecules KDO; 3-deoxy-D- exposed at the surface. mannooctulosonic acid Proteins make up roughly half the weight of the outer membrane. Some of the surface-exposed outer membrane proteins (OMP), such as pore-forming Porin protein, span the bilayer region of the membrane and may interact with the pepitidoglycan in the periplasmic region that lies between the inner surface of the outer membrane bilayer and the outer surface of the cytoplasmic membrane. Several other OMPs are low molecular weight lipoproteins, with acylated N-termini. Quantitatively the most significant of these is the murein lipoprotein (Braun, 1975), about the third of the molecules are covalently linked to the peptide moiety of peptidiglycan by their C-terminal while the rest occur as free lipoproteins. Where the outer membrane LPS O-antigen chains form the outer surface of the cell, extremely hydrophilic. However, in some cases very hydrophobic surfaces has been detected and attributed to the presence of proteins outside the LPS layer. Many strains of Serratia marcescens adhere avidly to oil droplets and to hydrophobic plastic surfaces due to the presence of a hydrophobic outer surface protein "serraphobin" (Bar-Ness and Rosenberg, 1989).

3. Surface Characterisation Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001

Surface components found in both Firmicutes and Gracilicutes: (Hancock, 1991) S-layer protein arrays are found in a wide range of Gram-positive and Gram-negative bacteria. S-layers are monomolecular arrays composed of a single protein or glycoprotein species and are the most common outermost cell envelope component of prokaryotic organisms. Many strains of Bacillus, Lactobacillu and Clostridium contain large amounts of these non-covalently bound protein (upto 50% of the cell wall weight in B. sphaericus and B. brevis, which do not contain teichoic acid) in the form of crystalline arrays, exhibiting oblique, square or hexagonal lattice symmetry, of globular protein sub-units forming continuous sheets around the cell surface. An S-layer usually consists only one or two types of polypeptides, often glycosylated, arranged in a monomolecular or bimolecular later a few nanometer thick, with the center-to-center spacing of, most commonly, between 8 to 15 nm. Isolated S-layer proteins have tendency of self- reassembly. In Gram-positive bacteria the S-layer lattice is attached to a rigid wall matrix (Fig. 3.6), which is composed mainly of peptidoglycan and accessory cell wall polymers most importantly teichuronic acid (Sleytr, et al., 2001). In Gram-negative bacterial cell envelopes S-layers are linked to the LPS component of the outer membrane. The presence of protein S-layer in Gram- negative bacteria renders the cell surface much more hydrophobic by at least partly masking the extremely hydrophilic lipopolysaccharide surface.

Figure 3.6 Schematic drawing of different S-layer lattice types: Oblique (p1 and p2), square (p4) or hexagonal (p3, p6) lattice symmetry. Taken from Sleytr et al. 2001. Capsules and Gycocalyces (Hancock, 1991) A wide range of bacteria possess capsules or other extracellular slime layer or glycocalyces usually consisting of polysaccharides. The capsular and glycocalyx polysaccharides are usually anionic, obtaining their negative charges from polymer repeating units containing uronic acid residues. An increasing number of cases are being found in which N-acetylaminouronic acids occur. However, phosphate-containing capsular

3. Surface Characterisation Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001

polymers, similar in composition to teichoic acid but of higher molecular weight, occur in several genera of Gram-negative bacteria. Capsules exist in a wide range of thickness, varying between different strains of a single species, and different stage of growth. In Gram-positive bacteria the capsular material adheres quite firmly to the peptidiglycan layer and can usually be removed only by chemical or mechanical treatments capable of breaking covalent bonds in large macromolecules. In Gram-negative bacteria, the capsular polymers can be extracted by treatment with detergents or phenol. The capsular polysaccharides of several Gram-negative bacteria have been found to have their reducing-terminal sugar covalently bonded to a phospholipid, suggesting that they may be attached to the outer membrane.

Cell surface appendages (Hancock, 1991)

Fimbriae and conjugative Pili Usage of these terms is confused. Pili are the appendages involved in conjugal transfer of genetic material and fimbriae are adhesive in nature. Both types of appendages are found predominantly in Gram-negative bacteria, though fimbriae have also been described on oral species of Gram-positive Sterptococcus and Actinomyces. Both fimbriae and pili are made up of organised rod-like arrays of protein sub- units, which are strain specific. Conjugative pili are substantially thicker and longer (up to 20 µm) than fimbriae, but since only 1 to 10 conjugative pili are usually present per cell their effect on surface contact may be limited. Fimbriae occur in much larger numbers, in E.coli 300-400 type I fimbriae (Diameter ~ 7 nm, heterogeneous in length) are present, fairly evenly distributed over the cell surface and projecting stiffly and approximately radially. On the cell of typical surface area 7.2 µm2 bearing 400 fimbriae, they would be on an average 150 nm apart, would not affect the approach of small molecules to cell surface but will certainly regulate the contact with large particles and surfaces.

Fibrils this term has been applied to another form of cell appendage that is distinct in morphology from fimbriae and pili, being shorter (usually < 200 nm) and having no measured width, as they usually appear in clusters. The clusters may be distributed over the whole cell surface, but are sometimes found as localised lateral or polar tufts. All the fibrils whose structure has been studied in detail have proved to be glycoproteins, this clearly distinguishes them from fimbriae.

Flagella are the longest cell appendages, extending up to 20 µm from the cell surface in some bacteria. In some bacteria only about 10 flagella are found per cell, however some bacteria possess large number of flagella and in this case, due to their extreme rigidity and great length, they are likely to effect the closeness with which the cell can approach a surface.

3.2 Bacterial Cell Surface properties and its Characterisation

Charge Characteristics (James, 1991) The cell surface of all micoorganisms are anionic as demonstrated by particle electrophoresis and interaction with cationic compounds. The negative charges are provided by phosphate, carboxylate and, less

3. Surface Characterisation Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001 commonly, by sulphate groups in the cell wall and capsular macromolecules. The positive charge is due to the presence of amino groups on some cell wall constituents. The net charge on the cell wall is a function of the amounts of positively and negatively charged groups in the range of cell wall components has been described in the previous section. Although there is a large variation in the size and difference in the charge-determining groups between different species of cells and strains of bacteria, nevertheless the mobility values are all of the same order of magnitude. Under the controlled condition of growth and measurement, cells of a given strain have reproducible mobility value; the value is not constant for all the cells in the population but in general shows a Gaussian distribution about a mean. Electrophoresis: The most common method to measure the charge characteristics of a particle is the electrophoresis. Particles in suspension acquire a surface charge either by the adsorption of ions (e.g., mineral particles, air bubbles, oil droplets) or by ionisation of surface charged groups (e.g., microbial cells), in some systems both these mechanisms may be operative. The system comprising electrolyte and particles in suspension is electrically neutral; however, each particle is much larger than a simple ion and so the particle charge in many times greater than that of an ion. At any boundary between two phases the intermolecular forces will be different from those in interior of either of the phase. In consequence the concentration of any mobile species is likely to differ in this region from those in the bulk phases; surface forces may orient any dipolar molecule in the region. This result in the establishment of an electrical double layer at the surface, one side of which carries a positive charge and the other an equal negative charge. Zeta- potential, which is experimentally measurable, is the potential at the plane of shear, i.e., the plane at which the phases move relative to one another on application of electric field. The zeta-potentials measurements of cells and sulfide minerals (-5µm) were made with a Laser Zee Meter (Pen Kem Inc., Model 501) equipped with a video system after 60 min conditioning at a specified pH value. The cell zeta- potentials were measured with a bacterial population of about 2x106 cells/ml and for mineral samples, a concentration of 1 g/l was used. All the measurements were made at constant ionic strength of 0.001 M. In some cases mineral samples were pre-conditioned with sufficient amount of bacterial cells in order to attain a bacterial monolayer before measuring the zeta-potential.

The variation of mobility with the pH of the suspension medium, at constant ionic strength, permits the characterisation of cell surfaces. The simplest pH-mobility is one in which there is only one ionogenic group e.g. anionic group (say carboxyl group of Glucuornic acid on the capsule of Klebsiella aerogenes). In this type there is little, if any charge at low pH values; increasing the pH of the suspension medium results in a dissociation of the weak acid group (pK value of the surface 2.8) leading to the increase in the negative value of the mobility to a final plateau value of mobility, usually at about pH 5. Cells of E. coli with a pK of 2.9 (Davies et al., 1956) and cells of Rhizobium with pK 2.3 to 2.5 (Marshall, 1967) have an exclusive carboxyl surface. The charge carried by five variant strains of Mycobacterium bovis BCG, M. microti and M. phlei is attributed to phosphodiester groups (pK 3.2-3.5) (Hardham, 1980; Hardham and James, 1981)

3. Surface Characterisation Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001

Other bacteria exhibit more complex pH-mobility curves due to the contribution of two or more ionogenic groups. Cells with mixed amino-carboxyl- phosphate surfaces are positively (or slightly negatively) charged at low pH values; the + positive charge is attributed to the presence -NH3 groups. The cell becomes progressively negative as the pH of the suspension medium increases, attaining a plateau value at about pH 6 (Douglas and Shaw, 1958; Few et al. 1960; Hill et al., 1963; Mozes et al., 1988). At higher pH values (> 9) there is a further increase in the negative mobility due to the loss of the proton on the amino group; residual charge is due to the fully dissociated anionic groups. Isoelectric point (IEP) is that point in the pH-mobility curve where the particle (cell) has zero mobility. This point remains the same even is the ionic strength of the suspension is changed. IEP of the bacterium is determined by the balance between charging of anionic and cationic acid/base groups on the cell surface. Table III shows the acid/base groups involved in imparting charge to the bacterial cell surface (James, 1991). Table IV lists the individual ionogenic polymers present on the bacterial surface and their IEP.

Table 3.1 Acid/base couples imparting charge to the bacterial cell surface (James, 1991) Groups Exist as pKa Located on Phosphate Phosphodiester bridges Teichoic acid on − Gram + bacteria R-O-HPO2-O-R/R-O-PO2 -O-R − R-H2PO4/R-HPO4 2.1 Phospholipid on Gram − bacteria − 2− Protonated R-HPO4 / R-PO4 7.2 Phosphate − Carboxyl COOH/COO 4≤pKa≤5 Protein or Peptidoglycan − COOH/COO 2.8 Polysaccharide + Ammonium R-NH3 /R-NH2 9≤pKa≤9.8 Protein or peptidoglycan

Table 3.2 Bacterial surface polymers and their electrokinetic characteristics (Rijnaarts et al., 1995)

Polymer Charged due to IEP Polysaccharide Phosphate and Carboxyl groups ≤ 2.8 Protein Ammonium and carboxyl groups > 4.0 Peptidoglycan Contains one ammonium group per three > 3.8 carboxyl groups Rijnaarts et al., 1995 used the IEP as an indicator for the presence of some or the other surface polymers. The cells are divided in three categories cell with IEP ≤ 2, 2 < IEP ≤ 2.8 and IEP > 3.2. IEP ≤ 2 can only result from the presence of phosphate groups (van der Mei et al. 1988a; 1988b). In Gram-positive bacteria this can happen due to the phosphate group on the cell wall teichoic acid and, though contributing less due to the greater depth, phosphodiester bridges that covalently bind the mycolic acid- arabogalactan molecule to peptidiglycan muramic acid (NAM).

3. Surface Characterisation Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001

In Gram-negative bacteria this may be due to the phosphate groups associated to the lipopolysaccharides on the outer membrane. 2 < IEP ≤ 2.8 has been shown to result from the predominance of cell wall glucornic acids or other polysaccharide-associated carboxyl groups (James, 1991) The IEP of Gram-negative bacteria in this range may be caused by the carboxyl groups associated with polysaccharide part of the LPS of the outer membrane. IEP > 3.2 of the bacterial cells is difficult to interpret. They reflect mixed contribution of protein- or peptidoglycan- associated carboxyl or ammonium group and may in principle result from a combination of ammonium containing polymers (proteins) and low pKa anionic polysaccharides containing phosphate and/or carboxyl group. High IEP Gram-positive bacterial surface is found to be coated by uncharged long hydrocarbon tails of mycolic acid, which is a fatty acid an ingredient of glycolipid (Bendinger et al., 1993) High IEP Gram negative strains indicate that the outer membrane LPS is covered with a non-polysaccharide layer, such as proteins (Bendinger et al., 1993; Nikaido and Vaara, 1985; De Flaun et al., 1990; Irvin et al., 1990) which may be contributed from the S-layer (Sleytr and Messner, 1988)

Attempts has been made to correlate the elemental analysis of the surface of freeze dried cells (XPS) with electrokinetic properties of hydrated cells in suspension (van Haecht et al. 1982). For a range of bacteria and yeast the mobility measured at pH 4 in water, becomes progressively more negative as the surface phosphorous concentration increases, finally attaining a plateau value (Amory and Rouxhet, 1988; Amory et al. 1988; Mozes et al., 1988,1989). Phosphate, presumably in the form of phosphodiester, is therefore assumed to be predominant contributor to the total charge density. This kind of correlation of freeze dried surface analysis to hydrated cell electrokinetic property can be misleading as the XPS measures at the depth of 5 nm but the ionogenic groups only within 1.5-3 nm contribute to the charge.

Electrokinetic behaviour of T.f. and P.p. The bacterial cell surface is charged due to the presence of functional groups such as carboxyl (- COOH), amino (-NH2) and hydroxyl (-OH), originating from the cell wall components of lipopolysaccharides, lipoprotein and bacterial surface proteins. And the iso-electric point (iep) of a bacterium reflects a balance between the anionic and cationic acid-base groups. The zeta potentials of the three types of T. ferrooxidans cells grown in ferrous ions, elemental sulphur, pyrite and chalcopyrite mineral as a function of pH are shown in Fig. 3.7(a). The ferrous grown cells exhibited an IEP at about pH 2.0 and above which, they are negatively charged. The IEP's of sulphur, pyrite and chalcopyrite are in between the pHs of 3.0 and 3.5. These results are in agreement with the studies reported earlier (Amaro et al., 1991; Blake et al., 1994; Ohmura et al., 1993). An IEP < pH 2.0 for Fe2+ grown T.f., which is a Gram-negative bacteria, may be because of the phosphate groups associated with the lipopolysaccharides on the outer membrane. (Rijnaarts et al., 1995). Gram- negative bacteria with an IEP greater than or equal to pH 3.2 (i.e. sulphur,

3. Surface Characterisation Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001

pyrite and chalcopyrite grown T.f.) have cell walls prevalent with protein molecules rather than polysaccharides.

5 25 (a) (b) Unadapted P.polymyxa 20 Pyrite-adapted P.polymyxa 0 15 Chalcopyrite-adapted P.polymyxa 23456789 Galena-adapted P.polymyxa 10 Sphalerite-adapted P.polymyxa -5 pH 5 Chalcopyrite-adapted P.polymyxa old 0 234567891011 -10 -5 pH -10 -15 -15 -20 Zeta-potential (mV) -20

Zeta-potential (mV) Zeta-potential -25 ++ Fe grown T.ferrooxidans -30 -25 Sulfur-adapted T.ferrooxidans Pyrite-adapted T.ferrooxidans -35 Chalcopyrite-adapted T.ferrooxidans -40 -30 Figure 3.7 Zeta-potential behaviour of microbial cells (a)Thiobacillus ferrooxidans,

(b) Paenibacillus polymyxa

Using sodium dodecyl suphate-polyacrylamide gel electrophoresis for the carbohydrate containing bands Blake et al., 1994 confirmed earlier reports that the lipopolysaccharides found on Fe2+ grown T.f. is different from the one found on So grown T.f., the pyrite grown T.f. had all the carbohydrate bands present for both Fe2+ and So grown T.f. The zeta-potentials of T. f. illustrate that the substrate grown cell surfaces contains different lipopolysaccharides and higher protein content than the ferrous grown cells. When the ferrous ions are deprived, there is a necessity for the bacteria to get adsorbed on the substrates in order to obtain energy for their survival and growth. So it has to produce necessary polymers to adhere on the solid surface out of which elemental sulphur is very hydrophobic. The zeta-potentials of the five types of P. polymyxa as a function of pH are shown in Fig. 3.7(b). The zeta-potential of unadapted, pyrite-, chalcopyrite-, galena- and sphalerite-adapted P. polymyxa are negative for the entire pH range and exhibit an iep of about pH 2. Which means that the zeta- potential behaviour of P.polymyxa did not change after adaptation. Being a Gram-positive bacterium, the reason for IEP<2 can be the phosphate groups present on the cell wall teichoic acid. However, adaptation is not a very reproducible process as microbial cells are living creatures and they can react to the change in environment in more that one way. The old chalcopyrite adapted P. polymyxa strain had an iep of pH ~4 and above which it was negatively charged but. This strain was later lost because of contamination and the adaptation of P.p. did not yield the same electrokinetic property.

Surface hydrophobicity (van der Mei et al., 1991) Bacterial hydrophobicity is a term used to describe the hydrophobic properties conferred on bacterial cells by their outermost cell surface. In physical chemistry, the implications of the words "hydrophobic" or "hydrophilic" seldom go beyond the macroscopic wetting behaviour of a surface by water in air. Thus teflon and paraffin wax are said to be hydrophobic since water drop does not spread and clean glass is said to be hydrophilic as the water droplet spreads on it readily. Hydrophobicity does not necessarily associate with repulsion between water and the surface. Even between water and hydrocarbon 34

3. Surface Characterisation Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001 attractive interactions exist (van der Waals), which are however smaller than the attraction for water to itself. This lack of attraction between water and a surface forms the basis of hydrophobicity.

Hydrophobic bacterial surfaces, or domain on the surface, could be produced by a range of known surface components, though these have been identified in few cases. Protein are the most likely candidates in many cases (Hancock, 1991). The hydrophobicity of many Staphylococci (Gram-positive) has been attributed o cell wall proteins. Evidence for this comes from observations of the masking of hydrophobicity by capsules (Hogt et al., 1982; 1986) and from studies of the effect of treatment with proeolytic enzyme. Similarly the presence of a cell surface virulence protein in Aeromonas salmonicida has been shown to confer hydrophobicity (Parker and Munn, 1984) Lipoteichoic acids (LTAs) have also been implicated in hydrophobic interactions of Gram-positive bacteria, particularly with Streptococci. LTA released into the cell wall from its initial site on the surface of cytoplasmic membrane surface might adopt a conformation, in which ionic interactions between its polyglycerolphosphate chain and cell wall components orient the LTA with its lipid portion towards the outer surface, conferring hydrophobic properties. Apart from LTA, major amounts of lipids could also render hydrophobicity to the surface for actinomycetes bacteria, particularly Mycobacterium, Corynebacterium and Nccardia. The Gram-positive and hydrophobic Mycobacterium phlei has been used in beneficiation of coal by flotation and flocculation (Raichur et al., 1996). The structure of which has been described by Smith et al., 1991 and the hydrophobicity has been attributed to the presence of 30-60% of lipids in the cell wall (Minneken et al., 1982)

Assessment of bacterial cell surface hydrophobicity: (van der Mei et al., 1991; Doyle and Rosenberg, 1990) The task of describing the hydrophobicity of microbial cell surface is daunting and much more complicated that in case of solid surface. There is no universal definition of bacterial hydrophobicity and generally distinction between more or less hydrophobic strains has been made on the basis of • High or low water contact angle • High or low adsorption to hydrocarbon (solvent) droplets (BATH and MATS tests) • High or low adsorption to octyl- or phenyl-sepharose (Hydrophobic interaction spectroscopy) • High or low amounts of salt required for aggregation (Salt aggregation test) • High or low adhesion to hydrophobic surfaces

Contact angle measurements Contact angle measurements are often used to obtain an indication of the hydrophobicity of solid surfaces. The contact angle expresses the balance between the liquid-vapour, solid-vapour and solid-liquid surface tensions and hence gives wonderful opportunity to quantify either of them. Contact angles were measured with the help of FIBRO 1100 DAT dynamic absorption tester. Polar liquids like water and formamide

3. Surface Characterisation Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001 and apolar liquids like α-bromonapthalene and methylene iodide were used to measure the contact angles. The surface energy components of the liquids are shown in Table II. The contact angle data was later used in evaluation of bacterial cells surface energies.

Bacterial cell contact angle. The contact angle of bacterial cells is measured by producing a uniform layer of cells on agar (van Oss et al. 1975) or the bacterial lawn is deposited on membrane filters as proposed by van Oss et al., 1975; Busscher et al., 1984 and van der Mei et al. 1987. The membrane technique is preferred over the agar techniques as the cells are less apt to detachment in the liquid phase, secondly by depositing the bacterial lawns on the membrane they can be dried of that only bound water is present on the bacterial surface. The membrane method is described in detail by Busscher et al, 1984 and van der Mei et al., 1991; 1998. The bacterial substrate for measuring contact angles are prepared by depositing bacterial cells, suspended in water, on cellulose triacetate filter (preferable pore diameter of 0.45 µm) by applying negative pressure. The bacterial lawns are deposited to a density of 108 cells/mm2 (Busscher et al. 1984) or approximately 50 layers of bacteria (van der Mei et al. 1991), 800-900 layers (Sharma et al. 2001). To establish constant moisture content the filters with bacteria are placed in a petri dish on the surface of a layer of 1% (wt./vol.) agar in water containing 10% (vol./vol.) glycerol. The filters are left in the petri dish till they can be used for contact angle measurement. This serves two purposed- Agar acts as a moisture buffer for about 3-5 hours and it does not allow the filter to dry, secondly the moisture content in all the filters with bacterial lawns are brought to the same level before they are dried under controlled conditions (the moisture content left after the filtration process may not be the same). The filter with bacterial lawns is cut in strips of appropriate width (about 1 cm) and fixed on the sample holder with the help of double-sided adhesive tape. The bacterial lawns are allowed to air-dry till a physiologically relevant state (van Oss et al. 1975) is achieved, where only bound water is present on the bacterial surface. This physiologically relevant state is characterised by attainment of a plateau region in the water contact Vs drying time curves, this state lasts for 30-60 mins (van der Mei et al., 1998). The contact angle behaviour after the drop has been positioned on the bacterial lawn is different for polar and apolar liquids. For the apolar liquids contact angle along with the drop volume, height and base diameter stays constant with time under the period of monitoring the drop i.e. 30 seconds. But the contact angle changes with time for polar liquids (shown in Fig. 3.8). There are three different regions in the contact angle Vs time curve. In the beginning the contact angle drops till about 0.1 to 0.3 seconds this happened because the drop spreads on the surface as can be inferred from the fact that the drop volume stays constant, the drop height decreases and the base diameter increases.

3. Surface Characterisation Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001

90 6 Stage I Stage II Stage III Spreading Equilibrium Absorption Contact angle 80 Drop Volume (µl) Drop Height (mm) Base diameter (mm) 70 4 ) o 60

40 2

Contact Angle(

20 0 0,1 1 10 100 Time (Seconds)

Figure 3.8 A typical curve for polar liquids-contact angle Vs time lapsed after positioning of drop over the bacterial lawns. Measured by the FIBRO DAT 1100 dynamic absorption tester. After this stage the drop comes in equilibrium where there is no change in drop contact angel, volume, height or base diameter. The equilibrium stage lasts till about 1-3 seconds. Again after some time the contact angle starts decreasing this is because the polar liquid wets the bacterial lawns and absorption of the liquid takes place. This stage is characterised by steep decrease of volume and height; the drop keeps on spreading as seen from the increase in the drop diameter. The relative spans of the three stages depend on the bacterial lawn thickness and probably on the bacterial strain hydrophobicity. Sharma et al. 2001 reported that 800-900 bacterial layers were required to get a reasonably long equilibrium stage for wild and sulfide mineral adapted Paenibacillus polymyxa. If too less bacterial layers are taken then the spreading can directly merge with the absorption stage and hence no equilibrium stage is available to measure the contact angle value. Thought, other authors have also observed this decrease in contact angle after positioning of the drop (Sar, 1987) but they have considered it be an anomaly and have proposed extrapolation of the curve to t=0 in order to account for the stability problems. In our view the initial drop in contact angle takes place for the drop to attain equilibrium and van der Mei et al., 1991 are also of the same opinion that it takes about 5-7 seconds for the contact angle equilibrium. The contact angle is measured using four standard liquids- water, formamide, α-Bromonapthalene and Methyleneiodide, to fully characterise the bacterial cell surface energy. The liquids along with their surface energy parameters are listed in Table 3.3.

3. Surface Characterisation Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001

Table 3.3 Surface energies (mJ/m2) as obtained from literature (van der Mei et al. 1998, Chapter XIII van Oss 1994, Bellon-Fountaine et al. 1990, Bellon-fontaine 1996, Owens and Wendt 1969)

− Liquid γTotal γd/γLW γp/γAB γ+ γ Water (H2O) 72,8 21,8 51 25,5 25,5 Formamide (CH3NO) 58 39 19 2,3 39,6 α-Bromonapthalene (C10H7Br) 44,4 44,4 ≈0 <0,1 <0,1 Methyleneiodide (CH2I2) 50,8 50,8 ≈0 <0,1 <0,1 Hexane (C6H14) 18,4 18,4 0 0 0 n-Hexadecane (C16H34) 27,7 27,7 0 0 0 Chloroform (CHCl3) 27,2 27,2 0 3,8 0

ADSA-CD Neumann and co-workers has been using the Axisymmetric Drop Shape Analysis-Contact Diameter (ADSA-CD) technique to measure the contact angles on bacterial lawns - Lin et al. 1999; Drumm et al. 1989 and Duncan-Hewitt et al. 1989. ADSA-CD is a modified version of ADSA-P, which was developed by Rotenberg and implemented by Skinner et al. 1989. This technique was initially developed to measure low contact angles. The technique requires the contact diameter, the drop volume and the liquid surface tension, the density difference across the liquid-liquid interface, and the gravitational constant as inputs to calculate the contact angle by means of numerical integration of the Laplace equation of capillarity. As the contact angle decreases, the profile of sessile drop becomes increasingly flat about the apex, and the accuracy of directly methods, such as goniometry, is adversely affected. The lack of curvature in the profile also presents a problem for all methods that rely on the profile of a drop to determine the contact angle. The success of drop profile methods is dependent on the existence of comparable surface tension and gravitational effects. For very flat drops, the effect of gravity dominates, and the surface tension has negligible effect on the shape of the interface. Thus, using the shape of the interface to determine the surface tension is not effective method for such experimental situations. Therefore, the ADSA-P technique and goniometry are not suitable. ADSA-CD circumvents this problem by utilising top view of the drop instead of the side view. Essentially, the contact angle is computed by numerically minimising the difference between the volume of the drop, as predicted by Laplace equation of capillarity and the experimentally measured volume. Although, ADSA-CD was originally developed to measure the contact angles of very flat drops but since it uses the top view of the drop, it is found to be very useful for measurement of drops on non-ideal surfaces, which are relatively rough and heterogeneous. It is practically impossible to form axisymmetric drop on such surfaces. The irregularities in the three-phase contact line are averaged to an average diameter of the drop, by a least-square fit of a circle to the experimentally measured points along the three-phase boundary and then an average contact angle is determined. All these facts has made ADSA-CD methodology particularly useful for biological materials e.g.

3. Surface Characterisation Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001

layers of bacterial cells which are necessarily rough and absorb water and other liquids so that the drops sink into the layer of cells, in addition the hydrophilic, bacterial layers produce small time dependent contact angles.

Table 3.4 Contact angles measured with different liquids on Sulphide minerals and five different Paenibacillus polymyxa (NCIM 2539)

Surface Contact Angle θ(o) Water Formamide Diiodomethane α-Bromonapthalene Unadapted P. polymyxa 40.81 56 66.68 51.84 Pyrite-adapted P. polymyxa 19.85 18.42 71.8 56.66 Chalcopyrite-adapted P. 15.85 21.05 70.6 55.6 polymyxa Galena-adapted P. polymyxa 17.55 19.37 51 48.3 Sphalerite-adapted P. 29.97 27.63 69.94 57 polymyxa Pyrite mineral 70 50,35 35,2 27,5 chalcopyrite mineral 70 57,16 48,83 Asymmetric drop

The contact angle measurements were performed on unadapted and mineral-adapted P. polymyxa cells. The contact angles measured with the four liquids are shown in Table VI. The water contact angles show that mineral- adapted cells are more hydrophilic as compared to the unadapted cells, which have a contact angle of 42o. Among the mineral-adapted cells chalcopyrite- adapted cells are the most hydrophilic with a contact angle of 16o and sphalerite-adapted cells are most hydrophobic with a contact angle of 30o.

Mineral surfaces contact angle. Pure solid mineral crystals were cut in order to attain small pieces with flat surfaces. These mineral pieces were polished successively on finer and finer polishing paper with water as a medium. The final paper used for polishing was #4000. Mineral pieces were mounted on the sample holder with the help of plasticine clay and than the contact angle were measured. In between measurements the mineral surfaces were freshly polished with paper #4000 and dipped in ethanol in order to remove water and prevent any oxidation of the surface, contact angle was measured within 5-10 minutes of polishing. The contact angles measured with the four standard liquids are presented in Table 3.4.

Surface Energy Evaluation: Various different approaches are available in literature to evaluate the solid surface energy from contact angle data, which are listed in Table 3.5. The listed approaches fall in two broad categories. Fowkes, Geometric mean (GM) and LW-AB approaches divide the total surface energy into different components and hence represent the solid- liquid interfacial tension in terms of the components of solid and liquid surface energies. But the Equation of state (ES) approach asserts that the surface energy is indivisible and hence represents the solid-liquid interfacial energy in terms of solid and liquid surface energies. The third column of Table 3.5 shows the way solid-liquid interfacial energy is expressed in terms of the solid and liquid surface energies. The fourth column shows the final expression, obtained by replacing the γsl

3. Surface Characterisation Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001 termfrom third column into Young equation, which is to be solved to arrive at the solid surface energy values. Though the Fowkes approach divides the total surface energy in dispersion and hydrogen bond (or polar) components, it considers only the dispersion interactions taking place between the liquid and solid and hence estimates only the dispersion energy component of the bacterial cell surface. This approach needs contact angle with only one liquid, which has to be apolar e.g. methylene iodide or α-bromonapthalene. Similar to Fowkes approach the GM divides the total surface energy in dispersion and hydrogen bond (or polar) components, hence gives rise to the final equation with two unknowns - γ d γ h s and s . So the contact angle data from two liquids of apolar and polar are needed. For this approach the water (or formamide) and methylene iodide (or α-bromonapthalene) contact angles are used. ES needs only one contact angle to evaluate the solid surface energy; contact angles with all the four liquids are used to determine the total surface energy. LW-AB approach divides the surface energy in Lifshitz-van der Waals LW AB (γs -apolar) and Acid-base (γs -polar) components. It further divides the + − acid-base component in electron accepting (γs ) and electron donating (γs ) components. The final expression obtained using LW-AB approach has three LW + − unknowns- γs , γs and γs . This equation has three unknowns and needs contact angle data with three liquids out of which two must be polar in nature. Therefore water, formamide are used as polar liquids and one of the two- apolar liquid (methylene iodide or α-bromonapthalene) contact angles is used for the calculations.

3. Surface Characterisation Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001

Table 3.5 Thermodynamic approaches used to evaluate the bacterial cell surface energies

Division of total Interfacial free energy Equation solved to evaluate components of total surface energy Approach γ surfaceγ energy γ of solid surfaceθ γ γ Fowkes approach = d + γ h = + − γ d γ d γ  γ d  γd sl s l 2 s l Only θ cos = −1 + 2 d  l  (Fowkes 1964, γ s  γ  Fowkes et al. 1980) γ γ  l  γ 2 ES No division γ ()− ()0.015γ − 2.00 γ + + Neumann, et = s l = sv sv γ lv lv γsl cos γ γ al., 1974 γ 1− 0.015 γ γ ()0.015 γ −1 γ s l θ lv sv lv γ β γ Li et al., 1989 γ γ − ()−γ 2 γ β γ = + − γ lv sv θ sv − ()−γ 2 sl lv sv γ 2 lv sv e cos = −1 + 2 e lv sv γ γ γ lv β γ γ Kwok, 1998 = + − ( − ()− γ 2 ) β γ sl lv sv 2 lv sv 1 1 lv sv θ= − + sv ()− ()− γ 2 cos 1 γ 2 γ 1 1 lv sv γ γ lv γ γ GM d h d d γ h h γ = + γ γ = γ + γ − 2 − 2 γ  d  γ  γ h  (Owens and sl s l s l s l 1+ cos = 2 d  l  + 2 h  l  Wendt, 1969) sθ  s  γ  γ γ γ lv lv γ γ     LW-AB = LW + γ AB 2 γ γ = ( LW − γγLW )()+ = ( LW γ LW ) (van Oss et al. γ γ sl s l γ 1 cos γl γ2 s l 1987;1988) AB = γ +γ − 2 + − γ + − + − γ − + + − γ − + + 2( + γ )− 2( − γ ) + ( + γ ) s l s l s l s l 2 s l s l γd-Dispersion (apolar) component of surface energy, γh- Hydrogen bonding (polar) component of surface energy, γLW- Lifshitz-van der Waals − (apolar) component, γABAcid base (polar) component, γ+ - Electron-accepting parameter, γ - Electron-donating parameter 2 2 2 2 β= 0.000115 (m /mJ) ; β1=0.0001057 (m /mJ)

3. Surface Characterisation Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphate Minerals, P.K. Sharma, 2001 Table 3.6 Surface free energies and components of surface free energy of unadapted and sulfide mineral-adapted Paenibacillus polymyxa. All values in mJ/m2

Approaches/P.p Unadapted Pyrite-adapted Chalcopyrite- Galena- Sphalerite- Strain adapted adapted adapted Fowkes γd 29.05 26.625 27.18 30.78 26.47 GM γd 29.05 26.63 27.18 30.78 26.47 γh 29.58 39.43 40.67 37.17 38.69 γTotal 58.63 66.06 67.85 67.95 65.16 ES γTotal 57.56 68.8 70.29 69.79 64.45 LW-AB γLW 28.62 26.65 27.15 30.78 24.68 γ+ 0.001 4.73 6.78 2.94 3.27 γ− 59.13 49.84 45.36 52.59 49.39 γAB 0.48 30.7 35.07 24.90 25.44 γTotal 29.1 57.35 62.22 55.68 50.12 γd-Dispersion (apolar) component of surface energy, γh- Hydrogen bonding (polar) component of surface energy, γLW- Lifshitz-van der Waals (apolar) component, − γABAcid base (polar) component, γ+ - Electron-accepting parameter, γ Electron- donating parameter

Adhesion to Hydrocarbons/Solvents: Bacterial adhesion to hydrocarbons test (BATH) was originally described by Rosenberg (1980), where a turbid, aqueous suspension of washed microbial cells is mixed by vortexing in the presence of a test liquid hydrocarbon under well controlled conditions in a glass test tube and the percentage of cells adhering to the hydrocarbon is considered the measure of hydrophobicity. Microbial adhesion to solvents (MATS) test is described by Bellon-fontaine (1996), where instead of using apolar hydrocarbons mono-polar solvents are used to probe the specific acid-base characteristics of the microbial cell. In the present study liquids used were hexane (BATH), hexadecane and chloroform (MATS). It can be seen from Table VI that the total surface energy and the dispersion component (or Lifshitz van der Waals component) of hexadecane and chloroform is almost the same but chloroform have a slight electron-accepting character (γ+). 5 ml of cell suspension containing 5x109 cells/ml was taken in a test tube and slowly about 0.84 ml of organic solvent was added so as to maintain the cells/solvent ratio of 1.2:0.2 according to Bellon-fontaine (1996). The test tube was vortexed for 90 (or 120) seconds at a vortexing speed of 7 on Heidolph REAX 2000 vortex. After which, it was allowed to stand for 15 minutes, then the aqueous phase was carefully sucked and the absorbance (turbidity) was measured at 400 nm on Perkin Elmer UV-Visible spectrophotometer- Lambda 2S. The percentage adherence to the organic phase was calculated using the following relation where Ai is absorbance of the cell suspension and Af is absorbance after the test.

3. Surface Characterisation Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001

A =  − f  × %Adherene 1  100 (3.1)  Ai  A higher adherence to hydrocarbon means that more cells have transferred to the organic phase from the aqueous phase or organic-aqueous interface, and hence they have less affinity towards the aqueous phase. The affinity of bacterial cells to solvent can be quantified by the thermodynamic free energy of adhesion (∆Gadh) parameter. The free energy of adhesion can be divided into Lifshitz van der Waals component and acid/base component. ∆ = ∆ LW + ∆ AB Gadh Gadh Gadh (3.2) ∆ LW = − ( γ LW − γ LW )( γ LW − γ LW ) Gadh 2 bv wv sv wv (3.3) ∆ AB = + ( γ + − γ + )( γ − − γ − )− ( γ + − γ + )( γ − − γ − ) Gadh 2 bv sv bv sv 2 bv wv bv wv . − ( γ + − γ + )( γ − − γ − ) 2 sv wv sv wv (3.4)

Where, γbv is bacterial surface energy, γsv is solvent surface energy and γwv is water surface energy. The Lifshitz van der Waals component quantifies the dispersion interaction at the bacteria-water and solvent-water interfaces. The acid/base free energy of adhesion component (E. 3.4) contains three terms each quantifying the acid/base interactions at bacteria-solvent, bacteria-water and solvent-water interfaces respectively. Percentage adherence of unadapted and sulfide mineral-adapted P. polymyxa to hexane, hexadecane and chloroform is shown in Fig. 3.9a. Hexane and hexadecane are entirely apolar liquids but chloroform has electron-accepting character. Both hexane and hexadecane are apolar liquids where, hexane has lower surface energy then hexadecane. Higher bacterial adherence is observed for hexane as compared to hexadecane, which is inconsistent with the deductions drawn from the free energy of adhesion calculations (Fig. 3.9b). But the adhesion of all the cells to chloroform is in agreement with the more negative free energy of adhesion (Fig. 3.9b)

-40 80 -35 74 70 -30 e 60 2 c -25 n 0 e 5 47 36 r -20

e 0 -23 -17 -21 h 4 -21 44 37 37

d 43 28 -15

A 30

37 -10 % 20 25 -9 -8

-12 of Adhesion energy Free mJ/m 19 -13 10 -8 0 0 19 13 -8 -11 -12 Hexane 15 Chloroform Unadapted Unadapted Chloroform 6 Pyrite-adapted Pyrite-adapted Hexadecane Chalcopyrite-adapted Chalcopyrite-adapted Galena-adapted Hexadecane Galena-adapted Hexane Sphalerite-adapted Sphalerite-adapted Figure 3.9 Adhesion of unadapted and sulfide mineral-adapted Paenibacillus polymyxa (NCIM 2539) strains to organic solvents experimentally measured by MATS test and their free energy of adhesion to the solvents evaluated theoretically.

3. Surface Characterisation Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001

This phenomenon can be understood from the point of view of electron accepting and donating characters of bacterial cells and solvent. The bacterial cells have very high electron donating character (Table 3.6) and chloroform has electron-accepting character. Therefore, bacterial cells have higher affinity towards chloroform than hexadecane. Adhesion is affected by the presence of both electron-accepting and donating characters on both bacterial cells and the substrate (solvent in this case), like characters repel and unlike characters attract each other. Unadapted P.p. have negligible electron- accepting character (γ+=0.001) whereas mineral-adapted P.p. have higher electron accepting character (Table 3.6). Therefore unadapted P.p. adheres to chloroform more than the mineral-adapted P.p. This affect is absent for hexane and hexadecane as there are no polar interactions.

Kinetic BATH Test: The original BATH test has been criticised for an insufficient quantitative control of the experimental parameters. To avoid this Lichtenberg et al., 1985 proposed the kinetic BATH test. In this test the optical density of the bacteria suspension (log[At/Aox100]) is determined as a function of increasing vortexing time, the slope of each of these lines (called R min-1, removal coefficient) is plotted as a function of increasing hydrocarbon/water volume ratio (VH/VW). Higher the slope of the R Vs VH/VW, more hydrophobic is the bacterial cell surface

Hydrophobic Interaction Chromatography (HIC) measures the adhesion of cells to columns of Sepharose beads that have been made hydrophobic (Smyth et al., 1978), e.g. by covalent binding of octyl- or phenyl residues. Cells suspended in a buffer are applied to the columns and subsequently the percentage of cells eluted from the columns by various agents is determined by cell concentration measurements (cell count, turbidity, radioactivity etc.). Control experiment is performed where cells are eluted from unsubstituted Sepharose. Data is expressed as Hydrophobic Index (HI). A hydrophobic strain yields high HI values, close to 1.

HI= (% Control Eluted − % Octyl eluted) / ((% Control Eluted) (3.5)

Salt Aggregation Test (SAT) is a simple technique by which one can infer the hydrophobicity of bacterial cell surfaces based on salt induced aggregation (Lindahl et al., 1981). Hydrophobic cell tend to aggregate at lower salt concentrations than do hydrophilic cell due to the interplay of electrostatic and van der Waals forces between individual cells. Basically in the test, a set of dilutions of ammonium sulphate is made in sodium phosphate buffer pH 6.8 (0.002 M) over the range of 4.0 to 0 M. A small volume of bacterial suspension positioned on a microscope slide is then mixed with an equal volume of salt solution during 2 minutes. Subsequently the minimal salt concentration (SAT) yielding visible aggregation is determined by comparing the aggregation in a salt-phosphate solution with the aggregation in phosphate buffer (negative control). Since visual observation is involved therefore, it is advised to test a wide range of bacterial cell densities.

3. Surface Characterisation Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001

Other methods used for determination of hydrophobicity of bacterial cells are- Hydrophobic partitioning in aqueous two-phase systems, adhesion to hydrophobic surfaces.

Bacterial cell surface Composition Intermolecular forces- electrostatic, Van der Waals and hydrophilic/hydrophobic determine the behaviour of the bacterial cell at the interface (adhesion, flocculation, and flotation). As we have already seen that both the charge and hydrophobic characteristics of the bacterial cells depends on the molecular constituents of the cell surface. Therefore, determination of both the elemental (XPS) and molecular (FT-IR, FT-Raman spectroscopy) surface composition is important.

Table 3.7 The peak assignments for the FT-IR and FT-Raman spectra.

Wave Number cm-1 Assignments(Naumann et al. 1996, Bellamy 1975, FT-IR FT-Raman Twardowski and Anzerbacher 1994) 3284 3277 NH stretching of proteins (Amide A) 3071 3065 Amide B vibration 2957 (CH3) stretching 2930 2916 CH stretching in fatty acids 2878 (CH3) stretching of methyl 2852 (CH2) stretching in fatty acids 1653 1665 Amide I (C = O stretching in protein) of α-helical structure 1637 Amide I (C = O stretching in protein)of β-pleated sheet structure 1541 Amide II (N-H bending in protein) 1455 1454 Asymmetric -CH deformation of C-CH3 group 1406 Symmetric stretching of carboxylate group 1378 Asymmetric -CH deformation(bending) for C-CH3 group 1331 1337 -CH deformation (bending) for -CH-group - 1236 Phosphate band (PO2 stretching) in phosphodiesters 1082 C-O-C, C-O, C-O-P and P-O-P vibrations of polysaccharides

Infrared Spectroscopy (DRIFT and FT-Raman) All atoms contain a positive core surrounded by negatively charged electrons that form bonds with the neighbouring atoms. Each molecular system can be visualised as an oscillating dipole. The dipole can be either permanent or induced. A change in dipole moment is responsible for the infrared activity, as infrared radiation is absorbed when the dipole moment changes. The dipole moment of a molecule changes on account of vibrations that occur in the molecule. The vibrations fall into basic categories of stretching, bending and rotational. Rotational vibrations are confined mainly to gases. Stretching vibration involves a continuous change in the inter-atomic distance along the axis of the bond whereas; bending vibrations are characterised by a change in the angle between two bonds. Infrared spectroscopy of microbial cells can give information about the type of polymers present on the cell surface. The bands,

3. Surface Characterisation Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001 which can be found in the spectra of bacterial cells, are summarised in Table 3.7. The diffused reflectance infrared fourier transform spectra (DRIFT) of the samples were recorded using a Bruker Fourier transform spectrometer with diffuse reflectance attachment. The radiation was measured with a MCT nitrogen cooled detector against a non-absorbing KBr matrix, used as a reference. The samples for diffuse reflectance were prepared by dispersing 12% of the sample in KBr. Typical measurement time while recording the spectra was about 5 min (100 scans) at a resolution of 4 cm-1. The FT-Raman spectra for solid pellets were recorded using a Perkin- Elmer 1700. The samples were excited with 500 mW of unpolarized intensity- stabilized 1064 nm radiation from a Spectron SL 301 Series Nd:YAG laser. The spectra were registered at 4 cm-1 resolution using InGaAs detector, about 300 scans were required to get good noise to signal ratio. Since the quantity of bacterial cell samples was small and so it was diluted with KBr and the pure KBr spectrum was later subtracted. FT-IR has been extensively used in two different ways: • DRIFT and transmission (KBr pellet) methods are used to characterise the bacterial cell surface • ATR (Attenuated total reflection) is used in order to monitor the real time biofilm growth on a surface. The molecular surface of oral Streptococci has been characterised by van der Mei et al., 1989 a;b and correlated with the XPS elemental analysis data. It was concluded that transmission infrared spectroscopy of freeze-dried bacterial cells yields surface-sensitive information comparable to XPS; this was based on the following observations. • Higher N/C surface concentration ratio from XPS, which is indicative of higher surface proteins is concurrent with the higher Amide II/CH (CH chosen at 2930 cm-1) absorption bands in FT-IR, though this correlation holds for Gram positive bacteria. • Negative correlation exists between O/C surface concentration ratio and Amide I/CH absorption bands, because oxygen is involved in many bonds other than amide e.g. in phosphate and sugar. • Positive relation exists between the Amide I/CH and the fraction of carbon atoms involved in the amide bond [O=C(NH)−]

Infrared spectroscopy has also been extensively used to characterise the bacterial cells of relevance to the bio-beneficiation area. Since the bacterial cells have to survive and flourish in adverse environments like very low pH (1.5 to 2), high metal concentration in pulp and heavy agitation. Therefore it is tried to adapt the bacteria in a particular harsh environment by repeated subculturing. After the bacterial cells have adapted to a particular environment, infrared spectroscopy becomes an important tool to characterise the changes in the molecular composition at the bacterial cell surface.

3. Surface Characterisation Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001

1,4 1655 1535 (b) 1236 2956 1451 1,2 1389 1079 3071 2934 3301 2875 1,0

0,8

A 0,6 Sulfur grown T.f. 0,4 Pyrite grown T.f. 0,2 Ferrous iron grown T.f. 0,0 4000 3500 3000 2500 2000 1500 1000 Wave Number (cm-1) Figure 3.10(a) FT-IR Spectra of Figure 3.10(b) FT-Raman spectra of Thiobacillus ferrooxidans cells grown in Thiobacillus ferrooxidans cells grown in different conditions different conditions

FT-IR spectroscopy was used in order to understand the surface groups present on T. ferrooxidans and P. polymyxa. Spectra for both the bacterial cells are presented in (Sharma et al. 1999; Sharma and Hanumantha Rao, 1999; Sharma et al., 2001) The diffuse reflectance FT-IR spectra of air-dried T. ferrooxidans cells grown in different conditions is shown in Fig. 3.10(a). The bands are assigned according to Naumann et al. (1996), Bellamy (1975) and Twardowski and Anzerbacher (1994). The characteristic peaks at 2957, 2923, 2852, 1455, 1378 and 1331 cm-1 are due to the alkyl groups present on the cell surface. While the peaks at 3284, 3071, 1656, 1637 and 1544 cm-1 are assigned to the amide A,B,I (α helical protein), I (β helical protein) and II vibrations respectively. The peaks at 1235 and 1071 cm-1 are related to the polysaccharides present on the cell surface. Therefore the presence/prominence of these peaks indicates presence/prominence of respective compounds on the cell surface. The solid substrate (elemental sulphur and pyrite) grown T. ferrooxidans cells have higher amount of protein on their surface as compared to the soluble ferrous ions grown cells. Since protein molecules are hydrophobic, it can be speculated that solid substrate grown T. ferrooxidans are more hydrophobic than the soluble iron grown cells.

FT-Raman spectra showed (Fig. 3.10(b)) some new bands at 1339, 1122, 1001 and 973 cm-1 wavelengths. Band at 1340 cm-1 is assigned to -CH deformation for -CH- group. The band at 1317 cm-1 characterises the N-H vibrations of amide III peptide bond of proteins. The C-C vibrations in secondary structure of proteins are associated with the bands at 1001 and 973 cm-1. Amide I (~ 3285 cm-1) band is found in both FT-IR and FT-Raman, but as expected amide II (1535 cm-1) is found only in FT-IR and amide III (1317 cm -1) only in FT-Raman. Thus, the FT-IR and FT-Raman spectra obtained showed the presence of CH, CH2, CH3, NH, NH2, NH3, COOH and CONH groups on the surface of all the grown cells. As revealed from the spectra, the intensity of all the peaks are higher in solid grown cells than in the soluble ferrous ions grown cells. The groups obtained are the groups present in any protein molecule. The solid substrate grown cells have higher amount of protein than in the soluble ferrous ions grown cells on the cell surface.

3. Surface Characterisation Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001

X-Ray Photoelectron Spectroscopy (XPS) (Rouxhet and Genet, 1991; van der Mei et al., 2000) involves irradiation of the sample of whole cells, freeze dried beforehand, by an X-ray beam that induces ejection of electrons (called photoelectrons) from the sample surface. The kinetic energy, Ek, of the emitted electrons is analysed, and their binding energy in the sample is determined. Each peak of the recorded spectrum is characteristic of an element (C, O, N, P etc). Though the X-rays penetrate deep in the sample (~1 µm) and electrons are emitted at the entire depth of X-ray penetration, but due to the inelastic collision only the electrons emitted from the 5 nm surface layer escape from the surface and observed. Hence the information from XPS concerns the outermost molecular layers and leads to an elemental analysis of the surface. A detailed examination of the spectrum (peak shift and peak fitting) makes it possible to investigate the way the atom are bound i.e. the chemical functional groups present on the surface. The kinetic energy of the emitted electrons is the difference between the energy of the incident X-rays and the work required to extract the electron from the orbit, called the Binding Energy. The spectrum obtained from XPS is the plot between the number of electrons emitted Vs the binding energy i.e. number of electrons emitted with a particular binding energy. The main XPS peaks are due to electrons that are ejected by the Kα photons and do not suffer inelastic collision between their ejection from the atom and their detection. For microorganisms the following peaks are usually observed on a wide scan spectrum O1s, N1s, C1s, P2s, P2p, O2s. The C1s line is sensitive to the chemical state of the carbon, following components are observed • Carbon bound only to carbon and hydrogen, as in aliphatic chains, set at 285 eV to calibrate the spectra • Carbon singly bound to one oxygen (C−O) as in sugars or one nitrogen (C−N), near 286.4 eV • Carbon involved in one double bond (C=O) or two single bonds with oxygen including amide functions , near 287.9 eV • Carbon of carboxyl (−COOH) and ester (R−COOR) groups, near 289.0 eV The literature values for the binding energies of different O1s components do not always agree, but he main O1s observed for microorganisms are • Oxygen (OH) in C−OH groups found on sugars, near 532.7 eV • Oxygen one double bond with carbon (C=O) or two single bonds (C−O−C, C−O−N or C−O−P), near 531.1 eV The nitrogen N1s peak appears near 400 eV due to amide and neutral amino groups; this may sometime be accompanied by a weak component at binding energy about 2 eV higher, which is attributed to protonated amine. XPS spectra for bacterial cells were obtained by harvesting them in their stationary phase by centrifuging and then freeze-drying. The spectra were recorded with an AXIS Ultra (Kratos) electron spectrometer with monochromatized microspot X-ray Al source with sample cooling. The − vacuum in the sample analysis chamber during measurement was 10 8 Torr. The value of 285.0 eV was adopted as the standard C 1s binding energy for calibrating the spectra. 48

3. Surface Characterisation Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001

Table 3.8 Characteristics of the XPS peaks for bacterial cell surface

Line Liquid Nitrogen Cooled Warmed Gram + Gram − T.f. (So) P.p. T.f. (Fe2+) T.t. T.f. (So) C1s C−(H,C), Eb 285 285 285 285 285 FWHM* 1.2 1.19 1.32 1.31 1.13 Atomic Conc. (%) 18.44 15.71 26.9 23.91 25.99

C−OH, Eb 286.5 286.6 286.6 286.7 286.6 FWHM 1.15 1.41 1.46 1.4 1.24 Atomic Conc. (%) 32.59 30.59 27.22 30.19 30.23 C=O, Eb 288.1 288.2 288.2 288.2 288.2 FWHM 1.15 1.25 1.45 1.4 1.16 Atomic Conc. (%) 11.54 7.13 9.25 8.6 9.05 COOH, Eb 289.2 289.6 289.3 289 289.5 FWHM 1.25 1.25 1.47 1.55 0.93 Atomic Conc. (%) 0.77 0.66 1.16 1.39 0.76 O1s O−R, Eb 531.4 531.7 531.7 531.8 531.5 FWHM 1.35 1.6 1.47 1.66 1.28 Atomic Conc. (%) 4.58 3.11 4.2 4.41 2.72

O−H, Eb 532.9 532.9 533 533.1 533 FWHM 1.45 1.69 1.71 1.56 1.48 Atomic Conc. (%) 26.13 34.66 22.44 24.37 25.81 N1s ≡N (Amide), Eb 400.1 400.2 400.3 400.2 400.2 FWHM 1.3 1.29 1.46 1.39 1.24 Atomic Conc. (%) 5.16 1.49 4.7 2.18 2.5 ≡N+−H (Protonated Amide), Eb 401.6 402.2 402.1 401.9 401.9 FWHM 1.5 1.64 1.49 1.82 1.61 Atomic Conc. (%) 0.33 1.15 1.07 1.12 0.82 S2p 3/2 o S (Elemental), Eb 163.8 163.8 163.7 FWHM 0.71 0.7 1.02 Atomic Conc. (%) 2.35 3.12 1.48 2− −SO4 , Eb 169.1 168.8 168.8 168.8 FWHM 1.19 1.57 1.58 1.32 Atomic Conc. (%) 2.65 0.73 0.73 0.65 Fe2p 3/2 FeO, Eb 709.8 FWHM Atomic Conc. (%) 0.34 FeSO4, Eb 711.6 FWHM Atomic Conc. (%) 1.06 P2p 2− PO4 , Eb 133.7 FWHM 1.55 Atomic Conc. (%) 0.13 * Width at the half maximum

3. Surface Characterisation Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001

The characteristics of O1s, N1s, C1s, P2p 3/2, S2p 3/2 and Fe2p 3/2 peaks for nitrogen cooled Paenibacullus polymya (P.p.), Thiobacillus thiooxidans (T.t.), Thiobacillus ferrooxidans Fe2+ grown (T.f. Fe), Thiobacillus ferrooxidans So grown (T.f. So) and warmed Thiobacillus ferrooxidans So grown (T.f. So) are presented in Table 3.8. The spectra for warmed Thiobacillus ferrooxidans So was taken after leaving the sample in the spectrometer overnight where the sample was still under vacuum but because of the lack of liquid nitrogen it warmed up to room temperature. Table 3.8 presents the elemental composition ratios for the aforementioned bacterial cells.

Table 3.9 The elemental composition ratios for the different bacterial cells

Liquid Nitrogen Cooled Warmed Atomic Gram + Gram − T.f. (So) Concentrations (%) P.p. T.f. (Fe2+) T.t. T.f. (So) Total C 63,34 54,09 64,53 64,09 66,03 Total O 30,71 37,77 26,64 28,78 28,53 Total N 5,49 2,64 5,77 3,3 3,32 C bound to O & N 44,9 38,38 37,63 40,18 40,04 O/C 0,485 0,698 0,413 0,449 0,432 N/C 0,087 0,049 0,089 0,051 0,05 (C bound to O & N) / Total C 0,709 0,71 0,583 0,627 0,606

0,7 P.p. T.f. (S) T.f. (Fe) 0,6 T.f. (S) warmed T. t. 0,5

0,4

0,3

e 0,2 in o L (C bound to O or N) / C or N) O to bound (C 45 0,1 0,1 0,2 0,3 0,4 0,5 0,6 0,7 O/C + N/C

Figure 3.11 Fraction of carbon bound to oxygen and nitrogen as a function of O/C + N/C surface concentration ratios for the bacteria, showing fairly good internal consistency of the XPS data.

Figure 3.11 shows the fraction of carbon bound to oxygen and nitrogen as a function of O/C + N/C surface concentration ratios for the bacteria. Though all the points do not exactly fall on the 45o line but still they follow the same trend and hence we can conclude a fairly good internal consistency of the obtained XPS data. The S lines for T.t., T.f. (Fe) and T.f. (S) indicate that on T.f. (Fe) the sulphur is only present in the sulphate form coming from the FeSO47H2O added as the constituent of growth medium. Whereas elemental sulphur and

3. Surface Characterisation Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001 sulphate are both present on the T.t. and T.f. (S) cells, the presence of elemental sulphur is confirmed by the reduction of the intensity of the 163.8 eV peak for the warmed T.f. (S) sample as compared to the nitrogen cooled T.f. (S). Fe both in the form of FeO and FeSO4 is found only on the surface of T.f.(Fe) cells. The presence of P line in P.p. maybe attributed to the presence of Teichoic acid on these Gram positive cells. Phosphorous was below the detection limit for the other cells. Comparing the N/C ratio for T.f. cells grown on Fe and S (Table 3.9) suggests a higher amount of amide groups (proteins) on the surface of elemental sulphur grown T.f. which, is in agreement with the FT-IR results and the fact that the cells have to attach themselves on the hydrophobic S particles in order to derive energy. For the same reason the N/C is higher for T.t. cells which are also grown on So.

Paper II International Journal of Mineral Processing, 62 (2001) 3-25

3. Surface Characterisation: Paper II Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001

Paper III Sent to International Journal of Mineral processing

3. Surface Characterisation: Paper III Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001

Surface Characterisation of bacterial cells Relevant to Mineral Industry

P.K. Sharma, K. Hanumantha Rao Division of Mineral Processing, Luleå University of Technology SE-97187 Luleå, Sweden

ABSTRACT Thiobacilli group of bacteria is widely used in mineral processing industry in bioleaching and biobeneficiation operations. Now a days Paenibacillus polymyxa has also found application in the biobeneficiation studies. In the present study, physico- chemical characterisation of Thiobacillus ferrooxidans grown in Fe ions and elemental S, Thiobacillus thiooxidans and Paenibacillus polymyxa bacterial cell surfaces is carried out. Surface hydrophobicity has been evaluated using the microbial adhesion to solvents test, contact angle measurement and surface energy evaluation. The surface composition of the bacterial cell surfaces is determined using X-ray Photoelectron Spectroscopy (XPS) and Diffused Reflectance Infrared Fourier Transform (DRIFT) spectroscopy.

INTRODUCTION The biological processes relevant to mineral industry are bioleaching and biobeneficiation. Bioleaching can be defined as a hydrometallurgical dissolution process assisted by microorganisms for the recovery of metals from their ores/concentrates. Major activity has been in bioleaching of sulphide minerals and chemolithoautotrophic bacteria have been used for the bio-leaching process. Over the past three decades bioleaching has come a long way and is now economically competitive, many processes have been commercialised and are in use. Whereas, biobeneficiation is relatively a new area and a new term which, has recently has been defined as “bio-beneficiation involves the selective removal of undesired mineral constituents from an ore through interaction with microorganisms, enriching the solid residue with respect to the desired mineral phase” (1). Direct and indirect mechanisms are reported to be involved in the oxidation of sulfide minerals and the contribution of each mechanism is still unknown. The direct mechanism concerns the adhesion of bacteria with the surface of sulphide minerals inducing the oxidation process (2). The ferric ions generated by the bacterial oxidation of sulphides provoke further oxidation of sulphides, recognising the indirect mechanism. The Thiobacillus ferrooxidans (T.f.) is proclaimed to adhere on mineral surfaces by hydrophilic (3) and hydrophobic (4,5) interactions. Pyrite oxidation by T. f. is thought to involve the regeneration of ferric ion in an indirect mechanism where protein-bound ferric ions in the cell envelopes oxidises free ferrous ions when the cell is free swimming (6,7). The rusticyanin protein present in T. f. is thought to function as an adhesive for the attachment of the cell to pyrite (8). Chalcocite oxidation is caused by the attached bacteria on the mineral surface rather than by the free- swimming bacteria (9). Recently it has been revealed that a sulphur binding protein, flagellar protein of 40-kDa, in T.f. incites the binding of cells to elemental sulphur by a chemical bond (10). The adhesion of bacteria to the sulfide minerals results in the 77

3. Surface Characterisation: Paper III Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001 modification of surface properties to that of the bacterial cell surfaces (4,11). The bacterium surface properties depend on the growth conditions (4,12,13). Components on bacterial surface play an important role in adhesion (14, 15). Sulphur grown cells were found to be more hydrophobic than the iron grown cells due to the synthesis of a proteinaceous compound on the surface. In the biobeneficiation process, selective adhesion of microbial cells on the mineral surface is a pre-requisite for the separation of mineral mixtures. Biobeneficiation has shown to work in desulphurisation coal using T.f. (16,17,18) and M. phlei (19,20,21), separation of mixtures of antimony and mercury sulphides using T.f. (22) and desiliconisation of calcite, alumina and iron oxide has been shown in presence of Paenibacillus polymyxa (P.p.)(23,24,25). Microbial adhesion on mineral surface depends on the physico-chemical characteristics of the microbial cell surface- charge characteristics, hydrophobicity and the chemical composition (26,27). Adaptation of bacterium towards a harsh environment e.g. low pH, high metal ion concentrations etc. also leads to the change in microbial surface properties (26). Thiobacilli group of bacteria i.e. Thiobacillus thiooxidans and Thiobacillus ferrooxidans, both Fe++ and So grown, are characterised for their surface properties in the present study. Since there has been some biobeneficiation work performed with Paenibacillus polymyxa therefore this bacteria is also characterised for its surface properties.

EXPERIMENTAL METHODS

Bacterial Strain

Thiobacillus ferrooxidans isolated from Chitradurga pyrite mines, India were used in this study. T. ferrooxidans was cultured and maintained in 9K medium (3 g/l (NH4)2SO4, 0.5 g/l MgSO4.7H2O, 0.5 g/l K2HPO4, 0.1 g/l KCl, 44.5 g/l FeSO4.7H2O and pH of 2.0) given by Silvermann and Lundgren (28). 9K - (without Ferrous sulphate) is prepared in flask I with 700 ml of distilled water and 2 ml of 10N H2SO4, flask II contains ferrous sulphate dissolved in 300 ml of distilled water with 0.3 ml of o 10N H2SO4. Flask I is autoclaved at 125 C for 20 minutes. Contents of flask II is filtered through previously autoclaved millipore filter in order to remove any contaminants. The filtrate of flask II is mixed with contents of flask I and inoculated with 10% v/v of T.f. culture and incubated in a rotary shaker at 30oC at 150 rpm. Thiobacillus ferrooxidans were also grown in presence of elemental sulphur (5 wt%) or pyrite (5 wt%) or chalcopyrite (5 wt%). Before the use of pyrite and chalcopyrite, they were washed in dilute sulphuric acid of pH 2.0 and the dried powders were then sterilised at 15 lbs pressure for 30 min. Sulphur was sterilised for 15 min at 5-6 lbs pressure. Pure strain of Thiobacillus thiooxidans, DSM 9463 isolated from a bioreactor in Australia is used for the study. The bacteria is grown in the MS-medium containing 2.00 g/l (NH4)2SO4, 0.25 g/l MgSO47 H2O, 0.10 g/l K2HPO4, 0.1 g/l KCl, the pH of medium is adjusted to 3.5 with 1N Sulphuric acid prior to autoclaving. Before inoculation 5 g/l of sterile powdered sulphur is added in the medium. Sterile sulphur is produced by taking sulphur powder in a capped test tube, putting few drops of water in it and putting it in boiling water for 1-2 hours, this procedure is repeated for 3 days consecutively. A pure 10 % v/v of an active inoculum is added to the MS-medium and

3. Surface Characterisation: Paper III Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001 incubated at 30oC at 150 rpm. Measuring the pH and bacterial cell count in the broth monitors the growth of the bacteria. Pure strains of Paenibacillus polymyxa CCUG 26013 (Type strain DSM 365) are subcultured on a sucrose rich Bromfield medium (0.5 g/l H2PO4, 1 g/l (NH4)2SO4, 0.2 g/l MgSO4.7H2O, 0.15 g/l yeast extract, 5 g/l sucrose and pH of 7.0) (Vishniac et al., 1974). The medium was autoclaved at 125oC for 20 minutes and then cooled. A 10 % active cell culture was added to the medium and incubated in a rotary shaker at 150 rpm at 300C. Cell count and pH measurements monitored the growth of P. polymyxa.

Cell Harvesting

The cells were harvested just at the beginning of the stationary phase of their growth. Cells grown in presence of sulfide mineral were filtered to separate cells from the suspended solid materials. The liquid containing the cells was then centrifuged at 11,000 rpm for 30 min and the cell pellet was obtained. It was washed twice in deionized water in order to remove any trapped ions and the concentrated cells were stored at room temperature for use in interaction with minerals and zeta-potential measurements. Fresh cell mass was generated everyday for experimentation. Some portion of the pellet was freeze dried at XPS and FT-IR spectroscopy. Some of the cell pellet was re-suspended in water in order to perform MATS test and to filter through Millipore filter to produce bacteria lawns for contact angle measurements.

Diffused Reflectance Infrared Fourier Transform spectroscopy (DRIFT)

The infrared spectra of the samples were recorded using a Perkin Elmer Fourier transform spectrometer with diffuse reflectance attachment. The radiation was measured with a MCT nitrogen cooled detector against a non-absorbing KBr matrix, used as a reference. The samples for diffuse reflectance were prepared by dispersing 15% of the freeze dried bacterial mass in KBr. A typical measurement was done with 200 scans at a resolution of 4 cm-1.

X-Ray Photoelectron Spectroscopy (XPS)

XPS spectra for bacterial cells were obtained by harvesting them in their stationary phase by centrifuging and then freeze-drying. The spectra were recorded with an AXIS Ultra (Kratos) electron spectrometer with monochromatized microspot X-ray Al source with sample cooling. The vacuum in the sample analysis chamber − during measurement was 10 8 Torr. The value of 285.0 eV was adopted as the standard C 1s binding energy for calibrating the spectra.

For microorganisms the following peaks are usually observed on a wide scan spectrum O1s, N1s, C1s, P2s, P2p, O2s (29). The C1s line is sensitive to the chemical state of the carbon, following components are observed • Carbon bound only to carbon and hydrogen, as in aliphatic chains, set at 285 eV to calibrate the spectra • Carbon singly bound to one oxygen (C−O) as in sugars or one nitrogen (C−N), near 286.4 eV

3. Surface Characterisation: Paper III Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001

• Carbon involved in one double bond (C=O) or two single bonds with oxygen including amide functions , near 287.9 eV • Carbon of carboxyl (−COOH) and ester (R−COOR) groups, near 289.0 eV

The literature values for the binding energies of different O1s components do not always agree, but he main O1s observed for microorganisms are

• Oxygen (OH) in C−OH groups found on sugars, near 532.7 eV • Oxygen one double bond with carbon (C=O) or two single bonds (C−O−C, C−O−N or C−O−P), near 531.1 eV The nitrogen N1s peak appears near 400 eV due to amide and neutral amino groups; this may sometime be accompanied by a weak component at binding energy about 2 eV higher, which is attributed to protonated amine.

Contact Angle Measurements

Contact angles were measured on 800-900 bacterial layer thick lawns with the help of FIBRO 1100 DAT dynamic absorption tester. The bacterial lawns were obtained by filtering a thick cell suspension through millipore filter (30,31). The bacterial suspension was enough to completely distribute over the filter during the process of filtration in order to achieve a homogeneous thickness of the lawn. The millipore filters were placed on 1% agar plate (10% vol./vol. glycerol in water) for about 15-30 minutes for homogenisation of water content in the lawn. The filter was then cut in square pieces and mounted on the sample holder plate with the help of double sided tape and allowed to air dry for 30-60 minutes so that only bound water is present on the cell surface (32) and then the contact angle was measured. Although the aforementioned method suggests about 60-70 layer thick lawns, the layer was found to be too thin to achieve an equilibrium state (region where the angle stays constant for some time) while measuring with polar liquids. Polar liquids of water and formamide, and apolar liquids of α-bromonapthalene and methylene iodide were used to measure the contact angles. From the measured contact angles and following the approaches described earlier, the bacterial cells surface free energy is calculated.

Surface Free Energies

The surface energies of the bacterial lawns were evaluated by Fowkes, Geometric mean (GM), Equation of state (ES) and Lifshitz-van der Waals Acid/Base (LW-AB) approaches described in detail elsewhere (27,33). The contact angles obtained with the four liquids, i.e., water, formamide, α-bromonapthalene and methylene iodide, were used to evaluate the surface free energies.

Adhesion to Hydrocarbons

This test was performed to incorporate both the original BATH (bacterial adhesion to hydrocarbons) test as described by Rosenberg (34) and MATH (Microbial adhesion to solvents) test described by Bellon-fontaine (35). The organic liquids used were hexane (BATH), hexadecane and chloroform (MATS). It can be seen from Table 2 that the total surface energy and the dispersion component (or Lifshitz van der 80

3. Surface Characterisation: Paper III Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001

Waals component) of hexadecane and chloroform is almost the same but chloroform have a slight electron-accepting character (γ+). 5 ml of cell suspension containing 5x109 cells/ml was taken in a test tube and slowly about 0.84 ml of organic solvent was added so as to maintain the cells/solvent ratio of 1.2:0.2 according to Bellon-fontaine (35). The test tube was vortexed for 120 seconds at a vortexing speed of 7 on Heidolph REAX 2000 vortex. After which, it was allowed to stand for 15 minutes, then the aqueous phase was carefully sucked and the absorbance (turbidity) was measured at 400 nm on Perkin Elmer UV-Visible spectrophotometer- Lambda 2S. The percentage adherence to the organic phase was calculated using the following relation where Ai is absorbance of the cell suspension and Af is absorbance after the test. A =  − f  × %Adherene 1  100 (1)  Ai  A higher adherence to hydrocarbon means that more cells have transferred to the organic phase from the aqueous phase or organic-aqueous interface, and hence they have less affinity towards the aqueous phase.

Results and Discussion

Surface Composition

DRIFT spectroscopy was used in order to understand the surface groups present on T.f., T.t. and P.p. The diffuse reflectance FT-IR spectra of air-dried T. ferrooxidans cells grown in different conditions is shown in Fig. 1. The bands are assigned according to (36,37,38). The characteristic peaks marked are CH, CH2-CH3 band at 2930 cm-1, Am I, amide I band (C=O stretch in proteins) at 1653 cm-1 and the Am II, amide II band (N-H bending in protein) at 1541 cm-1. The peaks at 1235 and 1071 cm-1 are related to the polysaccharides present on the cell surface. Table I presents the corrected peak areas for the CH, Am I and Am II peaks along with the AmI/CH and AmII/CH peak ratios. The corrected peak area for CH has been taken between 2950 to 2900 cm-1, for AmI the area is taken between 1715 to 1585 cm-1 and for AmII the area is taken between 1580 to 1485 cm-1. Therefore the presence/prominence of these peaks indicates presence/prominence of respective compounds on the cell surface. The solid substrate (elemental sulphur and pyrite) grown T. ferrooxidans cells have higher amount of protein on their surface as compared to the soluble ferrous ions grown cells. Since protein molecules are hydrophobic, it can be speculated that solid substrate grown T. ferrooxidans are more hydrophobic than the soluble iron grown cells. P.p. is a Gram-positive bacteria whereas the other bacteria are Gram-negative and hence have different cell surface structure. Peptidoglycan dominating the P.p. cell surface and the phospholipid bilayer present on the other three bacterial cell surfaces. Even with the differences in the surface structure it can be seen that the solid substrate grown cells (T.t. and T.f. S) have higher protein content on their surface as compared to the liquid substrate grown cell (P.p. and T.f. Fe) evident from the AmII/CH peak ratios. For T.f. we can see that when it is adapted to solid sulphur medium than it has to become more hydrophobic, by secreting more proteins on its surface, to attach itself

3. Surface Characterisation: Paper III Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001 on the hydrophobic sulphur particle. This is evident from the higher AmII/CH ratios for both T.t. and T.f. S Am I CH Am II T.t. 1,5

T.f. Fe 1,0 T.f. S

0,5

P.p.

Absorbance Units (A) 0,0

4000 3500 3000 2500 2000 1500 1000 Wave Number (cm-1) Figure 1. Diffused Reflectance Fourier Transform Spectra (DRIFT) of freeze dries Thiobacillus ferrooxidans- Fe++ grown (T.f. Fe) and So grown (T.f. S), Thiobacillus thiooxidans DSM 9463 (T.t.) and Paenibacillus polymyxa CCUG 26013 (P.p.)

Table I Corrected peak areas and peak ratios for the characteristic peaks from DRIFT spectra of Fig. 1

Corrected Peak Gram + Gram − areas P.p. T.f. (Fe2+) T.f. (So) T.t. CH 1,36 1,32 2,05 2,03 Am II 8,53 9,01 14,35 15,27 Am I 34,83 29,17 33,44 33,94 Peak Ratios Am II/CH 6,29 6,83 7 7,5 Am I/CH 25,68 22,1 16,32 16,7

X-Ray Photoelectron Spectroscopy (XPS) The characteristics of O1s, N1s, C1s, P2p 3/2, S2p 3/2 and Fe2p 3/2 peaks for nitrogen cooled Paenibacullus polymya (P.p.), Thiobacillus thiooxidans (T.t.), Thiobacillus ferrooxidans Fe2+ grown (T.f. Fe), Thiobacillus ferrooxidans So grown (T.f. So) and warmed Thiobacillus ferrooxidans So grown (T.f. So) are presented in Table II. The spectra for warmed Thiobacillus ferrooxidans So was taken after leaving the sample in the spectrometer overnight where the sample was still under vacuum but because of the lack of liquid nitrogen it warmed up to room temperature. Table III presents the elemental composition ratios for the aforementioned bacterial cells. Figure 2 shows the fraction of carbon bound to oxygen and nitrogen as a function of O/C + N/C surface concentration ratios for the bacteria. For good internal consistency the points must lie on the line with a slope of 45o, passing through zero (39). Though all the points do not exactly fall on the 45o line but still they follow the same trend and hence we can conclude a fairly good internal consistency of the obtained XPS data.

3. Surface Characterisation: Paper III Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001

Table II Characteristics of the XPS peaks for bacterial cell surface

The S lines for T.t., T.f. (Fe) and T.f. (S) indicate that on T.f. (Fe) the sulphur is only present in the sulphate form coming from the FeSO47H2O added as the

3. Surface Characterisation: Paper III Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001 constituent of growth medium. Whereas elemental sulphur and sulphate are both present on the T.t. and T.f. (S) cells, the presence of elemental sulphur is confirmed by the reduction of the intensity of the 163.8 eV peak for the warmed T.f. (S) sample as compared to the nitrogen cooled T.f. (S). Fe both in the form of FeO and FeSO4 is found only on the surface of T.f.(Fe) cells. The presence of P line in P.p. maybe attributed to the presence of Teichoic acid on these Gram positive cells. Phosphorous was below the detection limit for the other cells. Comparing the N/C ratio for T.f. cells grown on Fe and S (Table III) suggests a higher amount of amide groups (proteins) on the surface of elemental sulphur grown T.f. which, is in agreement with the FT-IR results and the fact that the cells have to attach themselves on the hydrophobic S particles in order to derive energy. For the same reason the N/C is higher for T.t. cells which are also grown on So.

Table III Elemental composition ratios for the different bacterial cells

Liquid Nitrogen Cooled Warmed Atomic Gram + Gram − T.f. (So) Concentrations (%) P.p. T.f. (Fe2+) T.f. (So) T.t. Total C 63,34 54,09 64,09 64,53 66,03 Total O 30,71 37,77 28,78 26,64 28,53 Total N 5,49 2,64 3,3 5,77 3,32 C bound to O & N 44,9 38,38 40,18 37,63 40,04 O/C 0,485 0,698 0,449 0,413 0,432 N/C 0,087 0,049 0,051 0,089 0,05 (C bound to O & N) / Total C 0,709 0,71 0,627 0,583 0,606

0,7 P.p. T.f. (S) T.f. (Fe) 0,6 T.f. (S) warmed T. t. 0,5

0,4

0,3

e 0,2 in o L (C bound to O or N) / C 45 0,1 0,1 0,2 0,3 0,4 0,5 0,6 0,7 O/C + N/C

Figure 2 Fraction of carbon bound to oxygen and nitrogen as a function of O/C + N/C surface concentration ratios for the bacteria, showing fairly good internal consistency of the XPS data.

3. Surface Characterisation: Paper III Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001

Correlation between DRIFT and XPS spectroscopic results The N/C ratio indicative of the presence of amide groups from XPS and hence presence of proteins on the bacterial cell surface has a good correlation with the AmII/CH peak ratios, another indicative of presence of proteins, from DRIFT, on the cell surface. This correlation is observed for Thiobacilli group of bacteria which are Gram-negative (Fig. 3a). The point for P.p. does not follow this correlation, the reason could be the fact that P.p. is Gram-positive. AmI/CH which is indicative of the C=O bonds, in DRIFT, present on the cell surface does not show any correlation with the O/C or (O in C=O)/C ratios from XPS (Fig. 3b).

P.p. (Gram-positive) (a) O / C peak ratio (b) O in C = O bond 0,09 10 8 0,08 6 0,07 4 0,06 2 data spectral XPS 0,05 0 N/ C peak ratio from XPS 6,2 6,4 6,6 6,8 7,0 7,2 7,4 7,6 16 18 20 22 24 26 AmII / CH peak ratio from DRIFT AmI / CH peak ratio from DRIFT

Figure 3. Correlation between the XPS and DRIFT spectroscopic data

98 99 100

e 80

n e r 60 e 60

h 58

d A

27 % 34 20 30

0 form 8 9 Chloro 14 4 xane T.f. (Fe) He T.f. (S) cane T.t. (D xade P.p SM 9463) He . (CCUG 2 6013) Figure 4. Adhesion of different bacterial cells to organic solvents experimentally measured using MATS test

Surface Hydrophobicity

MATS test The adhesion of Thiobacilli group of bacteria along with P.p.(CCUG 26013) to solvents is shown in Fig. 4. The solvents used have different properties, Hexane and Hexadecane are apolar liquids with dispersion component of surface energy of 85

3. Surface Characterisation: Paper III Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001

18,4 and 27,7 mJ/m2. Chloroform on the hand is monopolar with electron-accepting characteristic (3,8 mJ/m2), it has dispersion component of surface energy as 27,2 mJ/m2. All the bacterial cells have higher attachment to chloroform than hexadecane even though they have the same total surface energy. This can be attributed to the slight electron-accepting characteristic of chloroform, and all the bacterial cells have relatively high electron-donating characteristic (Table IV). The Thiobacilli group of bacteria has much higher attachment to the chloroform and hexane drops as compared to P.p. Whereas P.p. shows higher attachment to Hexadecane drops.

Contact Angle and Surface Energy evaluation

Table IV enlists the contact angles measured by different liquids and the surface energies evaluated using different approaches on the Thiobacilli group of bacteria. Water contact angle on T.t. could not be measured, as the drop was not stable, therefore only the dispersion component of the surface energy could be evaluated for T.t. in the present study.

Table IV Contact angle values and Surface free energies and components of surface free energy for bacterial cell surfaces.

Contact angle T.f. (Fe) T.f. (S) T.t. liquids Water 37,5 25,1 - Formamide 40,4 23,1 18,5 Methyleneiodide 44,0 45,0 45,0 α-Bromonapthalene 39,8 51,5 44,0 Approaches Surface energy (mJ/m2) Fowkes (Using α- Bromonapthalene) γd 34,7 29,2 32,8 GM γd 34,7 29,2 32,8 γh 28,0 38,2 - γTotal 62,7 67,4 - ES γTotal 60,5 66,8 - LW-AB γLW 34,7 29,2 32,8 γ+ 0,38 3,33 - γ− 47,2 48,0 - γAB 8,56 25,3 - γTotal 43,26 54,5 - γd-Dispersion (apolar) component of surface energy, γh- Hydrogen bonding (polar) component of surface energy, γLW- Lifshitz-van der Waals (apolar) component, − γABAcid base (polar) component, γ+ - Electron-accepting parameter, γ Electron- donating parameter

Both GM and ES approaches evaluate similar total surface energy values. But these approaches are not internally consistent because the total surface energy varied when the liquid contact angle used was changed, here the values with water are reported. 86

3. Surface Characterisation: Paper III Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001

The total surface energy values evaluated by LW-AB approach is totally different than the values evaluated by the ES and GM approaches.

Conclusions: The XPS and DRIFT spectroscopic characterisation of the bacterial cells showed that the solid substrate grown cells had higher protein secretions on their surface. The AmII/CH from DRIFT and the N/C from XPS had an increasing correlation for Thiobacilli group of bacteria.

Acknowledgements: The authors wish to thank Dr. A. V. Shchukarev, Department of Inorganic Chemistry, Umeå University, Umeå, Sweden for the help in XPS measurements.

References:

1. K.A. Natarajan, Microbes minerals and environment, Geological survey of India, 1998 2. C.L. Brierley, Bacterial leaching. Crit. Rev. Microbiol. 6 (1978) 207 3. N. Ohmura, K. Kitamura, and H. Saiki, Appl. Environ. Microbiol. 59 (1993) 4044 4. P. Devasia, K.A. Natarajan, D.N. Sathyanarayana, and G. Ramananda Rao, Appl. Environ. Microbiol. 59 (1993) 4051 5. J.A. Solari, G. Huerta, B. Escobar, T. Vargas, R. Badilla-Ohlbaum, and J. Rubio, Coll. Surf. 69 (1992) 159 6. M. Boon, G.S. Hansford and J.J. Heijnen in T. Vargas, C.A. Jerez, J.W. Weirtz, H. Toledo, (Eds.), Biohydrometallurgical processing-Vol. 1, Universidad de Chile, Santiago, Chile, 1995 7. A. Scippers, P. Jozsa, and W. Sand, Appl. Environ. Microbiol. 62, No. 9 (1996) 3424 8. N. Ohmura and R. Blake, Int. Biohydrometallurgy Symp., Sydney, Australia, (1997) PB1.1 9. H.M. Lizama, C.C. Abraham, and R.G. Frew, Int. Biohydrometallurgy Symp., Sydney, Australia (1997) M 4.2.1 10. N. Ohmura, T. Katsuyuki, J. Koizumi, and H. Saiki, J. Bacteriol. 178 (1996) 5776 11. R.C. Blake, E.A. Shute, and G.T. Howard, Appl. Environ. Microbiol. 60 (1994) 3349 12. A.M. Amaro, D. Chamorro, M.R. Seeger, R. Arredendo, I. Peirano, and C.A. Jerez, J. Bacteriol. 173 (1991) 910 13. C.A. Jerez, M. Seeger, and A.M. Amaro, FEMS Microbiol Lett., 98 (1992) 29 14. R.T. Irvin in Microbial cell surface hydrophobicity, R.J. Doyle and M. Rosenberg, eds.,. Am. Soc. Microbiology, Washington, D.C., 1990 15. K.C. Marshal, In Interfaces in microbial ecology,. Harvard University Press, Cambridge, Massachusetts, (1976) 27 16. Y.A. Attia, M. Elzeky, M. Ismail, Int. J. Min. Proc., 37 (1993) 61 17. Atkins, A.S., Townsley, C.C., Al-Ameen, S.I., 1987, Application of a biological sulphur depressant to the desulphurisation of coal in froth flotation, MINPREP 87, 5 18. Attia, Y.A., Elzeky, M., Biosurface modification in the separation of pyrite from coal by froth flotation, In: Processing and utilization of high sulphur coals, Eds. Attia, Y.A., Elsevier, Amsterdam, 1985 19. R.W. Smith, M. Misra, J. Dubel, Miner. Engg., 4 (1991) 1127 20. M. Misra, R.W. Smith, J. Dubel, S. Chen, Miner. Metal. Proc., 10 (1993) 20 21. A.M. Raichur, M. Misra, K. Bukka, R.W. Smith, Colloid. Surf. B, 8 (1996) 13

3. Surface Characterisation: Paper III Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001

22. N.N. Lyalikova, L.L. Lyubavina, in R.W. Lawrence, R.M.R. Branion, H.G. Ebner (Eds.) Fundamentals and applications of biohydrometallurgy, Elsevier, Amsterdam, 1986 23. N. Deo, K.A. Natarajan, Minera. Engg., 10 (1997) 1339 24. N. Deo, K.A. Natarajan, Int. J. Miner. Process., Vol. 55 (1998) 41 25. N. Deo, K.A. Natarajan, Minerals Engg., 11 (1998) 717 26. P.K. Sharma, K. Hanumantha Rao, K.S.E. Forssberg, K.A. Natarajan, Int. J. Miner. Proc. 62 (2001) 3 27. P.K. Sharma, Licenciate Thesis, Luleå University of Technology, Luleå, Sweden, 1999 28. M.P. Silverman, D.G. Lundgren, J. Bacter., 77 (1959) 642 29. P.G. Rouxhet, M.J. Genet in N. Mozes, P.S. Handley, H.J. Busscher, P.G. Rouxhet (Eds.) Microbial cell surface analysis, VCH, New York, 1991 30. H.J. Busscher, A.H. Weerkamp, H.C. van der Mei H.C., A.W.J. van pelt, H.P. de jong, J. Arends, Appl. Environ. Microbiol., 48 (1984) 980 31. H.C. van der Mei, M. Rosenberg, H.J. Busscher, in N. Mozes, P.S. Handley, H.J. Busscher, P.G. Rouxhet (Eds.) , Microbial cell surface analysis, VCH, New York, 1991 32. H.C. van der Mei, R. Bos, H.J. Busscher, Coll. Surf. B: Biointerfaces, 11 (1998) 213 33. P.K. Sharma, K. Hanumantha Rao, Submitted to Adv. Colloid. Inter. Sci., 2001 34. M. Rosenberg, D. Gutnick, E. Rosenberg, FEMS Microbiol. Lett., 9 (1980) 29 35. M.N. Bellon-Fontaine, J. Rault, C.J. van Oss, Colloid Surf. B: Biointerfaces, 7 (1996) 47-53. 36. L.J. Bellamy, The Infrared spectra of complex molecules, Vol I, IIIrd edition, Chapman & Hall, London, 1975 37. D. Naumann, C.P. Schultz, D. Helm, D., in H.H. Mantsch, D. Chapman (Eds.) Infrared spectroscopy of Biomolecules, Wiley-Liss Inc., 1996 38. J. Twardowski, P. Anzenbacher, 1994. Raman and IR spectroscopy in biology and biochemistry, Ellis Horwood, London and Polish Scientific Publishers, Warsaw, 1994 39. H.C. van der Mei, J. de Vries, H.J. Busscher, Surf. Sci. Report, 39 (2000) 1

Paper IV Submitted to Advances in Colloid and Interface Science

3. Surface Characterisation: Paper IV Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001

Analysis of Different Approaches for Evaluation of Surface Energy of Microbial Cells by Contact Angle Goniometry

P.K.Sharma, K. Hanumantha Rao Division of Mineral Processing, Luleå University of Technology, SE-97187, LULEÅ, SWEDEN

Abstract: Microbial adhesion on solid substrate is important in various fields of science. Mineral-microbe interactions alter the surface chemistry of the minerals and the adhesion of the bacterial cells to mineral surface is a prerequisite in several biobeneficiation processes. Apart from the surface charge and hydrophobic or hydrophilic character of the bacterial cells, the surface energy is a very important parameter influencing their adhesion on solid surfaces. There were many thermodynamic approaches in the literature to evaluate the cells surface energy. Although contact angle measurements with different liquids with known surface tension forms the basis in the calculation of the value of surface energy of solids, the results are different depending on the approach followed. In the present study, the surface energy of 140 bacterial and 7 yeast cell surfaces has been studied following Fowkes, Equation of state, Geometric mean and Lifshitz-van der Waals acid-base (LW-AB) approaches. Two independent issues were addressed separately in our analysis. At first, the surface energy and the different components of the surface energy for microbial cells surface are examined. Secondly the different approaches are evaluated for their internal consistency, similarities and dissimilarities. The Lifshitz-van der Waals component of surface energy for most of the microbial cells is realised to be about 40mJ/m2 ±10%. Equation of state and Geometric mean approaches do not possess any internal consistency and yield different results. The internal consistency of the LW-AB approach could be checked only by varying the apolar liquid and it evaluates coherent surface energy parameters by doing so. The electron-donor surface energy component remains exactly the same with the change of apolar liquid. This parameter could differentiate between the Gram-positive and Gram-negative bacterial cells. Gram-negative bacterial cells having higher electron-donor parameter had lower nitrogen, oxygen and phosphorous content on their cell surfaces. Among the four approaches, LW-AB was found to give the most consistent results. This approach provides more detailed information about the microbial cell surface and the electron-donor parameter differentiates different type of cell surfaces.

Introduction: The adhesion of microbial cells on solid surfaces and then the formation of biofilms is important in many diverse areas - biocorrosion [1-5], biofouling [2,6-8], biodeterioration [9,10], opthalmology [11], odontology [12,13], thrombosis of biomaterial implants [14] and biobeneficiation [15,16]. The separation of minerals in biobeneficiation, which comprises bioflotation and bioflocculation as sub-processes, is governed by selective adhesion of microbial

3. Surface Characterisation: Paper IV Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001 cells on mineral surface (Fig. 1). In order to fully understand and control the biobeneficiation processes, it is important to understand the adhesion process. The attachment of microbial cells on mineral surface is influenced by several properties, for example, surface charge, surface hydrophobicity, presence and configuration of surface polymers. Any theory that attempts to explain the bacterial adhesion must incorporate all these parameters. In general, the bacterial adhesion can be illustrated by the surface thermodynamics and by the extended DLVO theory of calculating the interaction energy between cells and substrate as a function of separation distance [15,17]. These methods accommodate Lifshitz van der Waals interactions, electrostatic interactions and hydrophobic/hydrophilic force interactions. These interactions are very well understood and formulated in mathematical equations. The most important input, in aforementioned calculations, is the surface energy and its different components (polar, apolar, electron donating and electron accepting) of the bacterial cell surface and the solid substrate. Although, the solid surface tensions (energy) can be estimated using different independent approaches, i.e., direct force measurement, contact angles, capillary penetration into columns of powders, sedimentation of particles, solidification front interactions with particles, gradient theory, Lifshitz theory of van der Waals forces and theory of molecular interactions, the contact angle is believed to be the simplest and hence widely used. In the literature various different approaches were mentioned which makes it possible to evaluate the solid surface tension using measured contact angles by liquids with known or pre-characterised surface energy parameters. Depending on the theoretical basis of the approaches, contact angles with one or more than one liquids are required on the solid surface for which surface energy is required. Three major approaches namely- Equation of state, Geometric mean and Lifshitz-van der Waals/ Acid-base, respectively needs contact angles with 1, 2 and 3 liquids (Fig. 1). Fig. 2 chronologically presents the development of the field of theoretical evaluation of solid surface energy from contact angle goniometry. About 200 years ago, Thomas Young [18] proposed contact angle of a liquid as mechanical equilibrium of the drop resting on a plane solid surface at the three-phase boundary. The three forces at the interface are the surface tensions at liquid-vapour interface, γlv, solid-liquid interface, γsl, and solid-vapour interface, γsv, which in equilibrium gives the following relation (Fig. 3a). γ θ = γ − γ lv cossv sl (1) In 1937, Bangham and Razouk [19,20] pointed out the importance of not neglecting the adsorption of vapour on the surface of the solid phase in deriving the equilibrium relation concerning the contact angle. They suggested the following equation with the spreading pressure πe term (Fig. 3b): γ θ = γ − γ − π lv coss sl e (2) where πe is the reduction of γs resulting from vapour adsorption on the solid surface. Young's equation contains the known parameters of γlv and cosθ and it is understood that this equation provides the unknown solid surface energy, γsv. The only drawback being the unknown solid-liquid interfacial energy, γsl. If this parameter could be represented or expressed in terms of the solid and liquid surface energies then problem is solved. Three methods have come into existence to tackle this problem.

3. Surface Characterisation: Paper IV Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001

The first method is to express γsl in terms of solid and liquid surface energy using some mathematical formulation and then evaluating the unknown γsv. First attempts were made in 1898 where the solid-liquid interfacial energy was expressed in terms of a geometric mean of solid and liquid surface energies. This method is followed till date in the form of Equation of state approach for which the latest formulation has been published as recent as 2000. The use of second method dates back to 1952, where approximation of solid surface energy is done using the concept of critical contact angle, which is the surface tension that divides the liquids forming zero contact angles on the solid from those forming contact angle greater than zero. This concept is used extensively in the fields of paints, adhesives etc. In the third method, the total surface energy is divided into different components and then the solid-liquid interfacial energy is expressed in terms of solid and liquid surface energy components. Fowkes pioneered this approach in 1962 [21] and this method is still used in the form of Lifshitz-van der Waals/ Acid-base approach. The following section describes each approach successively in detail with the criticisms by various workers and clarifications provided by the proponent authors. Finally, an analysis has been presented for the various approaches for the microbial cells surface using the contact angle data from the work of van der Mei and co- workers [22]. Chemical composition and structural arrangement of microbial cell surface is very complex due to the presence of a large variety of chemical groups and surface appendages of different lengths. Bacterial species belonging to prokaryotic microorganisms can be divided into two different groups on the basis of Gram- staining reaction on the cell wall distinguishing Gram-positive and Gram-negative bacteria. The Gram-positive bacteria usually have a well-defined rigid cell wall composed of peptidoglycan and underlying phospholipid bilayer of the cytoplasmic membrane. The cell wall constitutes 60-70% of the weight of the cell wall. On the contrary, Gram-negative bacteria have very thin, 1-2 nm thick, peptodiglycan layer sandwiched between outer and inner cytoplasmic membranes. Surface appendages, often contacining proteins and lipoteichoic acids as well as polysaccharides rich capsules, [23] are found as the outer most material on the bacterial cell surface which probably determines the surface energy and the other surface properties. Yeast (Candida etc. species) represents a different class of microorganisms belonging to unicellular eukaryotics. The major difference is the size (3-10 µm compared to 1-2 µm diameter for bacteria). These organisms possess a thick cell wall consisting mainly manna, insoluble glucan, protein and small amount of chitin.

Thermodynamic Approaches

Berthelot´s Combining Rule Brethelot [24] used the geometric mean combining rule for the first time in obtaining the interfacial tension from the surface tensions of the two phases. The ij relationship was based on the way dispersion energy coefficients C6 can be written in ii jj terms of C6 and C6 in the treatment of London theory of dispersion forces: ij = ii jj C6 C6 C6 (3)

3. Surface Characterisation: Paper IV Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001

This relation forms the basis of the Berthelot combining rule (Eq. 4) where εij is the potential parameter (well depth) of unlike-pair interactions, εii and εjj are the potential energy parameters (well depth) of like-pair interactions [25]: ε ij = ε = ε ε 1 1 or ij ii jj (4) ()ε ε 2 ii jj This equation was written in terms of work of adhesion between 2 phases, (i.e. Wsl) and work of cohesion of the 2 phases (i.e., Wss and Wll). = Wsl WssWll (5)

Putting the relevant values i.e. Wss=2γsv, Wll=2γlv and Wsl=γlv+γsv-γsl in Eq. 5, and rearranging we get the final relation of Berthelot: γ = γ + γ − γ γ sl sv lv 2 sv lv (6)

Antonow´s Rule: In 1907 Antonow [26] related γSL in terms of γSV and γLV in a simple manner as in Eq. 7. There was no theoretical background behind this relationship according to Kwok and Neumann [25]. γ = γ − γ sl lv sv (7)

Zisman Approach: Zisman [26] has introduced the concept of critical surface tension γc as an empirical method of determining the “wettability” of solid surfaces by plotting the cosine of the contact angle θ versus the surface tensions of a series of liquids. The point at which the resulting curve intercepts the line at cosθ = 1 is called the critical surface tension γc. The γc is the surface tension that divides the liquids forming zero contact angle on the solid surface from those forming contact angle greater than zero. The liquids with surface tension γl below the γc value of the solid simply spread on the solid. This approach has been extensively used to determine the critical surface tension γc of various low energy solids and organic films deposited on high energy solids like glass and metals. In general, a rectilinear relation is established empirically between the cosine of the contact angle and the liquid surface tension, for each homologous series of organic liquids. Fig. 4a illustrates the results with the n-alkanes on polytetrafluoroethylene. Even when cosθ is plotted against γlv for a variety of nonhomologous liquids, the results fall close to a straight line or collect around it in a narrow band like shown in Fig. 4b. Certain low energy solids exhibit curvature of this band for liquids with surface tension above 50 dynes/cm2 (Fig. 4c), this results because weak hydrogen bonds form between the molecules of liquid and those in the solid. This is most likely to happen with liquids of high surface tension, because they are always hydrogen bond forming liquids (polar liquids). If critical surface tension is considered to give an indication of the surface tension of the solid then by using this method: 1) it is possible to obtain the total solid surface energy of an apolar solid by using series of homologous apolar liquids, e.g. n-alkanes, d 2) it is possible to find only the dispersion force component (γs ) of the total surface energy of a polar solid by using series of homologous apolar liquids, e.g. n- alkanes, and

3. Surface Characterisation: Paper IV Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001

3) deviation from rectilinear relation is observed when polar liquids are used on polar and apolar solids by using Zisman method. Hence it is not possible to determine any component of the solid surface tension by using polar liquids using Zisman method. In 1970 using the Good-Garifalco-Fowkes approach, Dann [28,29] demonstrated, it is expected to obtain different values of γc for a particular solid surface, depending upon what liquid series used in the determination of the critical angle by the Zisman method. He used homologous series of ethanol/water, mixed glycols and ASTM series of liquids prepared from mixtures of 2-ethoxyethanol and formamide on apolar, polar and mono-polar solids. The results were compared with the ones obtained by Zisman and co-workers using homologous series of n-alkanes. Considerable difference was found between the measured values of γc and the generally accepted values of Zisman. The deviation can be regarded as a difference between the plots between cosθ Vs γl for different homologous series of liquids on the same solid as shown in Fig. 5. The difference between the critical surface tension γc found by hydrocarbon series (A) and ethanol/water (B) series was explained using Good-Garifalco-Fowkes- d Young equation (Eq. 14). It was demonstrated that with some precaution γs values of the polymers can be accurately determined from contact angle measurements with standardised series of polar liquids by the use of conversion curves (Fig. 6). As an 2 example, let γs obtained by Ethanol/Water homologous series be 24 mJ/m . A vertical line is drawn from the point where a horizontal line at γs = 24 cuts the curve C. The d 2 point at which this vertical line cuts curve A gives the γs value, which is 33.5 mJ/m in this case. The deviation of the curve C, measured for ethanol/water from the curve B, which is theoretical ethanol/water, is attributed to the contributions due to polar interactions between the solid and the liquid. This was very clearly explained and proved by Owens and Wendt [30], by introducing the terms for polar contribution in interfacial energy in the Fowkes equation (Eq. 13).

Good and Garifalco approach: Accepting the Berthelot relation for the attractive constants between like molecules, Aaa and Abb, and between unlike molecules Aab, i.e., Aab = 1 1 (8) ()2 Aaa Abb the authors arrived at almost similar relationship as Brethelot but with a constant φ. Taking analogy from Eq. 8 they set up the corresponding ratio, involving the energies (or free energy) of adhesion and cohesion of two phases which was taken to equal a constant φ: ∆G a ()γ + γ − γ ab = a b ab = φ (9) ∆ c∆ c γ γ Ga Gb 2 a b ∆ a = γ − γ − γ Using free energy of adhesion Gab ab a b and free energy of cohesion ∆ c = γ Gn 2 n , the following relation was attained: γ = γ + γ − φ γ γ sl sv lv 2 sv lv (10) and φ was evaluated for different systems. The value of φ was found to be 1 for “regular” interfaces, i.e., systems for which the cohesive forces of the two phases and the adhesive forces across the interface are of the same type. 93

3. Surface Characterisation: Paper IV Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001

When the predominant forces within the separate phases are unlike, e.g., London-van der Waals vs metallic or ionic or dipolar, then low values of φ are expected. Clearly this applies to pairs such as non-hydrogen bonding in organic compound vs water; organic compound vs metal and salt vs metal. When there are specific interactions between the molecules forming the two phases, the energy of adhesion is greater than the value it should have in the absence of specific interactions.

Fowkes equation [21,31,32] This approach forms a basis of all the surface tension component approaches used today and the dispersion component of the total surface energy is still calculated by using this approach. Fowkes considered the surface tension (γ) to be a measure of the attractive force between surface layer and liquid phase, and that such forces and their contribution to the free energy are additive. Therefore, the surface tension of liquid metals, polar liquids, hydrocarbons, low energy solids and other solids is considered to be made up of independent additive terms. γ = γ d + γ h + γ m ...... (11) He attributed the γd term only to the London dispersion interactions because it has been shown for macroscopic condensed systems in aqueous media that out of the three electrodynamic interactions only London´s dispersion interaction is predominant [33,34]. γh is due to hydrogen bonding and γm due to metallic bonding etc. The intermolecular attractions, which cause surface tension, arise from a variety of well-known intermolecular forces. Most of these forces, such as metallic bonding and hydrogen bonding, are a function of specific chemical nature. On the other hand, London dispersion forces exist in all types of matters and always give an attractive force between adjacent atoms or molecules, no matter how dissimilar their nature may be. The London dispersion forces arise from the interaction of fluctuating electronic dipoles with the induced dipoles in neighbouring atoms or molecules. The effect of fluctuating dipoles cancel out, but not that of the induced dipoles. These dispersion forces contribute to the cohesion in all substances and are independent of other intermolecular forces, but their magnitude depends on the type of material and density. Therefore, γd term includes only the London dispersion force contribution and, Keesom and Debye force contributions are included in the γh term. The interface between two phases with only dispersion force interactions is composed of two monolayers as indicated in Fig. 7. At the interface, the adjacent layers of dissimilar molecules are in a different force field than the bulk phase and consequently, the molecules or atoms in these layers have a different pressure, intermolecular spacing and chemical potential. If the molecule in one of these monolayers are less strongly attracted by the adjacent phase than by its bulk phase, the molecules in the interfacial layer have an increased intermolecular distance and are in tension. However, if the attraction by the adjacent phase is greater than that of the bulk phase, the molecules of the interfacial monolayer have a shorter intermolecular distance and are under two-dimensional pressure. The measured tension of the interface is always the sum of the tensions in the two interfacial monolayers. The surface monolayer of phase 1 has a tension γ1 resulting from the unopposed attraction of the bulk liquid. An interfacial monolayer of phase 1 is attracted by its bulk in the identical manner, but this attraction is opposed by the

3. Surface Characterisation: Paper IV Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001 attraction of phase 2. When the interacting forces are entirely dispersion forces (such as in between saturated hydrocarbons and water or mercury) then the decrease in tension in the interfacial monolayer of phase 1 resulting from the presence of phase 2 γ dγ d is 1 2 . Consequently the tension in the interfacial monolayer of phase 1 is γ − γ dγ d γ − γ dγ d 1 1 2 and in the interfacial monolayer of phase 2 is 2 1 2 which leads to the equation γ = γ + γ − γ dγ d 12 1 2 1 2 (12) By measuring the interfacial tension at the saturated hydrocarbon/water d interface and using the above equation, Fowkes calculated γ Water to be 21.8±0.7 dynes/cm2, since only dispersive interactions between water and hydrocarbons exists. d 2 Similarly he arrived at γ Hg=200±7 dynes/cm by using saturated hydrocarbon/mercury interface. Since mercury and water interacts only by dispersion forces, Fowkes was able to predict the interfacial tension to be 425±4 dynes/cm2 which agrees well with the experimental value of 426±4 dynes/cm2. This demonstrates the usefulness of geometric mean approach and helpful for both the liquid-liquid and liquid-solid interfaces At the solid-liquid interface the Fowkes relation looks as follows γ = γ + γ − γ dγ d sl s l 2 s l (13) Combining this with the Young´s equation (Eq. 2), we get an equation of the form:  γ d  π cosθ = −1 + 2 γ d  l  − e (14) s  γ  γ  l  l γ d π θ l If the spreading pressure term, e, is neglected then a plot of cos vs γ gives a l θ γ d straight line (Fig. 8) with the origin at cos = -1 and slope 2 s . Since the origin is fixed, one contact angle measurement is sufficient to determine the dispersion force component of the surface energy of the solid (γs). The spreading pressure term, πe, can be assumed zero only for the system where high energy liquids are brought in contact with low energy solids. The basic reason for this assumption is that all theoretical and experimental evidence predicts that adsorption of high-energy materials cannot reduce the surface energy of a low energy material. For example, adsorbing water never reduces the surface tension of a liquid hydrocarbon. The fact that a given liquid has a contact angle greater than zero degrees on a given low energy solid asserts that the liquid possesses a higher energy and therefore πe should be zero. This holds true only for the solids interacting by dispersion forces only. It does not apply for the high-energy solids such as metals, graphite; water does not wet these solids but it does absorb and produce appreciable πe. Zettlemoyer [35] corroborated the Fowkes approach of using geometric mean for estimating the interfacial forces for hydrophobic solids, but points out an interesting work by his co-worker Lavelle, who used arithmetic mean of the dispersion force attraction to estimate the magnitude of the interaction between dissimilar materials. Zettlemoyer agrees that “ the arithmetic mean approach does not have a great scientific basis for its uses as the geometric mean has”. Lavelle used the following equation and got some interesting results.

3. Surface Characterisation: Paper IV Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001

γ = γ + γ − γ d + γ d 12 1 2 ( 1 2 ) (15) γ d + γ d The surface energy of the two phases diminishes by 1 2 at the interface due to 2 the attractive dispersion interactions. The internal consistency was demonstrated by 2 d calculating water/mercury interfacial tension of 426 ergs/cm and the γ water to be 22 ergs/cm2 which were in agreement with the value found by geometric mean. However d 2 γ Hg was found to be 108 ergs/cm instead of 200 by this averaging technique. Based on just thermodynamic point of view there are some objections on the usage of geometric mean to combine the dispersion force contribution of the two phases to arrive at the interfacial energy. Lyklema [36] put forward two reservations - whether it is thermodynamically allowable to split the interfacial tensions in components and secondly, if geometric mean is the most appropriate mathematical form to combine them? The first objection stems from the fact that the interfacial tensions are Helmholtz energies, whereas Fowkes (Eq. 13) approach treats them as energies. The entropy contributions are totally neglected and when entropy contributions are combined, they do not follow the geometric mean law but go with the logarithm of the composition. α If only the energetic contribution, Ua , to γ is considered, the geometric average is a very acceptable choice because dispersion forces prevail obeying Berthelot's rule very well. Keeping this in mind if the energetic and entropic contributions of the work of adhesion are separated, then = ∆ σ − ∆ σ Wadh adhU a T Sa (16) ∆ σ = σ ,s + σ ,l − σ ,sl adhU a U a U a U a (17) ∆ σ = σ ,s + σ ,l − σ ,sl adh Sa Sa Sa Sa (18) with a good approximation 1 ∆ σ ≈ ()σ ,s σ ,l 2 adhU a 2 U a,d U a,d (19) and therefore the solid-liquid interfacial energy should be 1 γ = γ + γ − ∆ σ − ()σ ,s σ ,l 2 sl s l T adh Sa 2 U a,d U a,d (20) α where Ua,d stands for the dispersion part of the corresponding surface tensions. The purely energetic distribution contributions are independent of the temperature and the entropic term now accounts for the temperature dependence. The Fowkes relation (Eq. 13) has a term of spreading pressure, which is often neglected, but the present approach (Eq. 20) that term is not necessary as it is already accounted for in the equation. Here the T∆S term cannot be neglected as it accounts for about 20-30% of the Helmholtz energy. Lyklema (2000) also emphasises that there is no physical ground that this geometric mean law is applicable to the acid-base or hydrogen bond interactions, which has been done by various workers and presented in the following sections. Except the equation of state approache the Fowkes relation is used in all the surface tension component approaches to determine the dispersion (sometimes termed as “apolar” and “Lifshitz van der Waals”) component of the total surface energy. Since this equation takes only apolar interaction into account the contact angle data with an apolar liquid (e.g. Methyleneiodide, α-bromonapthalene) must be used as this liquid would only have dispersion / van der Waals interaction with the bacterial cell surface.

3. Surface Characterisation: Paper IV Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001

Evaluation of polar component of the solid surface energy, neglecting spreading pressure Tamai and co-workers [37] introduced a term “I” quantifying the interaction energy of the non-dispersive forces at the interface in the in Fowkes eq. (13): γ = γ + γ − γ dγ d − sl s l 2 s l I sl (21) They used two-liquid system to measure the water contact angle on the solid. Here the solid was immersed in a water-saturated hydrocarbon, the water drop was formed on the solid-hydrocarbon interface and then the contact angle was measured (Fig. 9). This was done in order to have minimum effect of the spreading pressure of water on the solid. The Youngs equation becomes γ = γ + γ θ sh sw wh cos (22) using Tamai´s equation (16) we get γ = γ + γ − γ dγ d − sh s h 2 s h I sh (23) γ = γ + γ − γ dγ d − sw s w 2 s w I sw (24)

Combining Eq.s 17, 18 and 19 and because Ish=0, we get γ − γ dγ d = γ − γ dγ d − + γ θ h 2 s h w 2 s w I sw hw cos (25) Inthis equation we have 2 unknowns and hence contact angle data of water on solid is required in presence of two different hydrocarbons as the third phase. The polar component of surface energy is not limited to dipole interactions but includes all of the non-dispersive forces such as hydrogen bonding. In fact, in condensed phases, dipole interaction are small and Fowkes [34] has concluded that "polarity" as measured by dipole moments is not significant factor in intermolecular interactions in liquids and solids. If the ionization potentials of the two phases are assumed to be equal and polar component is assumed to be dominated by dipole- dipole interactions, one obtains the geometric mean equation [30,38]. On the other hand, if the polarizability of the two phases are assumed to be equal (and assuming that the polar component has the same form as the dispersive component), one obtains the harmonic mean equation [39,40]. Owens and Wendt [30] proposed the division of the total surface energy of a solid or liquid in two components-dispersion force component and hydrogen bonding d h d h component (γl=γl +γl and γs=γs +γs ). The interaction energy of the nondispersive forces at the interface was quantified and included as geometric mean of the nondispersive components of solid and liquid. The equation proposed was the extension of the equation proposed by Fowkes (Eq. 12). γ = γ + γ − γ dγ d − γ hγ h sl sv lv 2 s l 2 s l (26) This can be alternatively expressed as 2 2 γ = ( γ d − γ d ) + ( γ h − γ h ) sl s l s l (27) h h The geometric mean combination of γs and γl was used even though the fact that hydrogen bonding interactions are more specific in nature. The main reason was that the authors believed that hydrogen bonding is similar to the dipole-dipole interactions that take the form of a geometric mean. Using the Young's equation and neglecting the spreading pressure (1), the following expression is obtained:

3. Surface Characterisation: Paper IV Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001

 γ d   γ h  1+ cosθ = 2 γ d  l  + 2 γ h  l  (28) s  γ  s  γ   lv   lv  γ d γ h Since this equation have two unknowns, s and s , the contact angle data from two liquids are needed. In general the liquids used are water and methyleneiodide. Owens and Wendt evaluated the surface energy of many polymers and then compared them to the values obtained by Zisman using the critical surface tension approach. A fairly good agreement was observed between the values from both the approaches. The differences, if any, were explained using the following arguments: According to Zisman the critical surface tension is the value of γlv at the intercept of the plot cosθ Vs γlv with the horizontal line, cosθ =1, i.e. when γlv=γc then cosθ=1 putting this in the Young´s equation (1), we get θγ = γ = γ − γ cos lv (1) c sv sl (29)

Although many workers have been inclined to identify γc with γsv, Zisman has been careful to point out that γc is symbatic with, but not necessarily equal to the solid surface free energy because it is not certain that γsl and π=0 when θ=0. In fact it has been shown that γsl is usually not equal to zero when θ=0. Combining eq. 23 and 25 we get 2 2 γ = γ − ( γ d − γ d ) + ( γ h − γ h )  (30) c s  s l s l  From eq. 30 it is apparent that γs - γc≥0. The following four cases follow should be recognised:

h h I. Use of Nonpolar liquid (γl =0) to determine γc of nonpolar solids (γs =0). Here h h d d γs = γl = 0 and γl = γl = γc substituting these in eq. 30 we get γs = γs = γc i.e. d the critical surface tension gives the dispersion surface energy (γs ) of the solid, which is equal to γs. h h h II. Use of Nonpolar liquid (γl = 0) to determine γc of polar solids (γs ≠ 0). Here γl d d = 0 and γl = γl = γc substituting this in equation 30 we again get γs = γc ≠ γs i.e. the critical surface tension gives only the dispersion part of the total surface free energy of the solid. h d h III. Use of polar liquid to determine γc of nonpolar solids (γs =0). Here γl = γl + γl 2 d  h d d  and γs = γs . Substituting these in eq. 30 we get γ = γ − γ + ( γ − γ ) c s  l s l  this means that the use of polar liquids to determine γc of nonpolar solids leads to a value considerably less than γs. h h d h IV. Use of polar liquid (γl ≠0) to determine γc of polar solids (γs ≠0). Here γl=γl +γl d h and γs=γs +γs . Substituting these values in eq. 30 we get 2 2 γ = γ − ( γ h − γ h ) + ( γ d − γ d )  which means that the use of polar c s  s l s l  liquids to determine γc of polar solids leads to a value considerably less than γs.

Wu [39,40] proposed the harmonic mean to combine the polar and dispersion components of the solid and liquid surface energies in order to obtain the solid-liquid interfacial energy and proposed the following expression (31):

3. Surface Characterisation: Paper IV Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001

 γ dγ d γ pγ p  γ = γ + γ − 4 l s + l s (31) sl s l γ d + γ d γ p + γ p   l s l s  On combining this to the Young´s equation, the following relation is obtained: 4  γ dγ d γ pγ p  1 + cosθ = l s + l s (32) γ γ d + γ d γ p + γ p  lv  l s l s  Similar to the Geometric mean approach, the contact angle data with two liquids are required in order to obtain the polar and dispersion components of the solids surface energy. Dalal [41] performed a comparative study of the two approaches, Geometric mean and Harmonic mean, on 12 common polymers using the published data with 6 liquids. It is found that the total surface energy of the solid obtained by the two methods are generally quite close, and neither of the two conceptually different equations is clearly incompatible with the available experimental data. However, the more widely used Geometric mean approach is preferable because it consistently fits the data better. Since Geometric mean approach is widely used and is found to be consistent, we have used only the geometric mean in the analysis. Dalal, 1987 developed a method for the best-fit solution of simultaneous equations, which are obtained when more than two contact angle liquids are used. This method is developed for both the Geometric mean and Harmonic mean systems. Later during the analysis of the Geometric mean approach the aforementioned minimisation method has not been used but least square method is used to solve the over-determined systems of equation. Since the contact angle data with 4 liquids is available for each microbial surface, the following matrix has been solved using least square method by Matlab 5.3. ()+ θ  1 cos W   γ d γ p     W W  2  ()1 + cosθ   γ d γ p  γ d   F   F F  s  = 2 (33) d p p ()1 + cosθ   γ γ  γ  M M M  s   2   γ d γ p  ()+ θ   Br Br   1 cos Br   2 

Equation of state approach [42,43] Considering the surface thermodynamics of a two component three-phase solid- liquid-vapour system and assuming ideal solid i.e. smooth, homogeneous, rigid with no appreciable vapour pressure, Ward and Neumann [44] showed that an equation-of- state type relation exists between the solid-vapour, the solid-liquid, and the liquid- vapour interfacial tensions. The solid-liquid interfacial tension is an unique function of liquid and solid surface tensions. The Gibbs-Duhem equation for the three interfaces can be written as γ = − sv − Γ sv µ d sv S1 dT 2(1)d 2 (34) γ = − sl − Γ sv µ d sl S1 dT 2(1)d 2 (35) γ = − lv − Γsv µ d lv S1 dT 2 d 2 (36) sv where, S1 is the surface entropy of the solid-vapour interface T is the absolute temperature µ2 is the chemical potential of the liquid components

3. Surface Characterisation: Paper IV Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001

sv Γ2(1) ´is the surface excess concentration of component 2 (liq.) at solid liquid interface

The above equations indicate that each of the surface tensions is a function of T & µ2, i.e., γ = γ µ sv sv (T, 2 ) (37) γ = γ µ sl sl (T, 2 ) (38) γ = γ µ lv lv (T, 2 ) (39) Therefore, there are 3 equations in terms of two variables and hence, any one of them can be expressed in terms of the other two, i.e., γ = γ γ sv f ( lv , sv ) (40) In 1989 Li and co-workers [45] gave another proof of the existence of equation of state by using modified phase rule for capillary system with curved surface. Classical Gibbs phase rule states, F=C+2-P (41) where, F is degree of freedom, C is independent chemical components and P is number of phases. This equation is not universally applicable. If the concentration of any set of components can be related by an equilibrium constant, then the number of degrees of freedom is reduced by one. Also explicit in the derivation of Eq. 41 is the assumption that if the temperature and pressure in one phase is known, they are determined in all other phases. Naturally having temperature and pressure the same in all phases satisfy this condition. If a system contains curved interfaces, such as sessile drop on a substrate, then the pressure will not be the same in all phases. The change in pressure between the two phases can be calculated by Laplace equation: Pα − P β = γ αβ J αβ (42) αβ αβ where γ is the interfacial tension and J the curvature. The curvature must be known to calculate the pressure difference across the interface. This apparently introduces a new degree of freedom for each curved interface, the curvature of the interface, and hence that the Gibbs phase rule has to be modified. But the system must have negligible boundary effects, i.e., all boundaries must be thermally conducting, deformable and permeable to all components, no chemical reaction must occur and volume is the only work ordinate (W=PdV). These conditions are not satisfied by the system under consideration (Fig. 1) therefore, a different form of phase rule has to be used to determine the number of independent intensive variables or degree of freedom. Essentially the number of degrees of freedom or variance of any composite system is evaluated by subtracting the equilibrium constraint equations from the variables used to describe the composite system. For a multicomponent, multiphase system in equilibrium if P if the total no. of bulk or volume phases with P independent chemical components. Each bulk phase α α α α α α α can be described by variables T , P , x 1, x 2, …..x r-1, where x i for i= 1,2,3,…..,P-1 is the bulk mole fraction of the ith component in the phase α. Consequently, for all P phases the required number of intensive variables will be P(C+1). Since these coexisting phases are all in equilibrium, the intensive variables are constrained to satisfy thermal, mechanical and chemical equilibrium conditions Thermal Equilibrium

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α β T =T = …….. =Tn P-1 equations Chemical equilibrium α β n µ i=µ I=………..=µ I C(P-1) equations Mechanical equilibrium are of three types, I. Laplace equations, α β αβ P -P =γαβJ (43) Where, α and β represent adjacent bulk phases separated by a curved liquid- αβ fluid interface and J is the mean curvature of the αβ interface. If the interface αβ α β is planer, i.e., J =0, then this equation reduces to P =P the equilibrium conditions used in the original derivation of Gibbs phase rule. II. Young equation, III. Neumann triangle relations, γ γ θ = ()γ 2 − ()γ 2 − ()γ 2 2 12 23 cos 12 23 13 (44) Therefore, the total constraint equations equal to (P-1)+C(P-1)+N (45) α β where, N is the total number of distinct P =P type relations. Therefore, the surface system phase rule is F=P(C+1)-[(P-1)+C(P-1)+N] (45a) F=C+1-N (46) Applying this to the system in consideration (Fig. 2a) where we have 3 bulk phases, 3 surface phases, 2 components(C=2) and if the solid phase is isotropic i.e. Ps=Pv (N=1) so F=2. This means that any two of the intensive variables describing the system can independently vary and any other variable is than function of the other γ = γ γ two, i.e., an equation of the form (40) sv f ( lv , sv ) . Li [45] applied the modified phase rule to liquid-liquid lens fluid system consisting of three bulk fluid phases of different densities as shown in Fig. 10a. Along with the three bulk phases, there are also three surface phases. If all the surfaces are curved, the surface mechanical equilibrium conditions will be P3-P1=γ31J31 (47) P3-P2=γ32J32 (48) P2-P1=γ21J21 (49) α β There are no pressure equality relations of the type P =P since all the interfaces are curved. As a consequence, N=0 and the phase rule (46) yields F=C+1. For a two component system the degrees of freedom are 3 and the equation of state of the form 40 does not exist. Only under very special conditions shown in Fig. 9b where the interface between fluid 1 and liquid 2 is planar the degrees of freedom are 2 and the relation of the type 40 may exist. This is because J12=0 and the mechanical equilibrium condition given in Eq. 49 is replaced by a pressure equality relation α β P =P and hence the phase rule yields F=C. For the two component system the degrees of freedom come out to be 2. Even without having an explicit formulation of this equation of state and just with the knowledge of its existence, a test for determining whether adsorption at the solid-vapour interface occurs, was proposed in the aforementioned paper [44]. If a system is considered where adsorption at the solid-liquid interface is absent then the solid-vapour interfacial tension, γsv, is independent of vapour in contact and has a value of γso. The surface tension of the solid-liquid interface, γsl, is given by γ o = ()γ γ sl f so , lv (50)

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o where γ sl denotes the solid-liquid interfacial tension in the absence of adsorption, o Substitution γ sl in the Young equation (Eq. 1) we get γ − ()γ γ = γ θ so f so , lv lv cos (51) If now succession of liquids are brought in contact with the solid, and if there is no φ()γ = γ θ adsorption and temperature is maintained same in each case, then lv lv cos . That is, if there is no adsorption of the vapour of any liquids, then from Young equation and existence of equation of state one concludes that there must be a correlation between γlv and γlvcosθ, i.e. a plot of γlv vs γlvcosθ will give a smooth curve as shown in Fig. 11. The difference between the Gibbs phase rule (Eq. 41) and the reformulated phase rule (Eq. 46) for the capillary systems is the term for the number of phases. For the Gibbs phase rule, the number of phases is equal to that of bulk phase, the interfacial phase is not a phase in the same sense. For the reformulated phase rule; the number of phases is not taken into account as long as all the interfaces are significantly curved. For every interface insignificantly curved, the mean curvature is no longer a variable, and the number of degrees of freedom is actually reduced by 1. Li [45] suggested the moderately curved interface to be a radius of 1mm. Neumann [42] developed the first form of equation of state which, allowed the determination of surface tension of low-energy solids from a single contact angle formed by a liquid which is chemically inert with respect to the solid and its surface tension is known. Experimental values obtained by measurement of contact angles with a series of liquids on 8 different low energy solids are plotted on a graph γlvcosθ Vs γlv. If there is no adsorption of vapour taking place at the solid-liquid interface then the points follow smooth curves (Fig. 11). Two general conclusions were drawn from the plots (second conclusion was more a hypothesis) 1. γlv decreases as γlvcosθ increases, this is true from the Young equation when γsv is constant. ()γ θ d lv cos θ γ θ γ o 2. The slope γ is zero at =0 i.e. at lvcos = sv, hence a line at 45 is d lv plotted. γ θ = γ 2 + γ + A second order polynomial of the type lv cos a lv b lv c is fitted to the o d()γ cosθ data. The intercept of the fitted curve to the 45 line and the slope lv γ at d lv γ 2 + ()− γ + = the point of intersection are determined by solving a lv b 1 lv c 0 and then ()γ θ γ d lv cos = γ + putting the value of lv (point of intersection) into γ 2a lv b . The d lv average limiting angle of inclination calculated from the average slope of 8 solids came out to be 0.1±4.2, here the standard deviation includes the value of zero and hence the aforementioned hypothesis is accepted. d()γ cosθ lim lv = 0 (52) θ →0 γ d lv

Since γsv is assumed to be constant d()γ cosθ d()γ − γ dγ lv = sv sl = − sl (53) γ γ γ d lv d lv d lv

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dγ which, implies that lim sl = 0 . (54) θ →0 γ d lv

From the fact that γsv decreases as γlv decreases and from Eq. 54 it is concluded that γsl has its minima when θ=0. From the knowledge of liquid-liquid interface where lower limit of interfacial tension between two liquids in equilibrium is zero, it was concluded that γsl has zero as its minimum value. γ = γ * = lim sl sl 0 (55) θ →0 The formulation of the equation of state was empirical curve fit to contact angle data in terms of the Girifalco and Good interaction parameter [46]. γ + γ − γ φ = sv lv sl (56) γ γ 2 lv sv The method was as follows * 1. γsv is constant and γsl =0, so γsv is determined from the plots by using γ = γ = γ * sv lim lv lv (57) θ →0

2. Using this value of γsv and experimental values of γlv and cosθ, value of γsl is obtained as a function of θ from the Young equation (Eq. 1) 3. Using values of γsv, γlv and γsl, the interaction parameter, φ is evaluated 4. φ vs γsl is plotted for the 8 solids (Fig. 12), as can be seen from the plots that φ is a γ φ = αγ + β linear function of sl and so straight lines of the type sl were fitted to the plots. 5. Values of the two constants were found from the curve fitting and were found to be dφ α = = −0.0075m2 / mJ & β = 1.000 (58) γ d sl Hence the equation of state is obtained ()γ − γ 2 γ = sv lv (59) sl − γ γ 1 0.015 sv lv Combining this to Young equation, we get ()0.015γ − 2.00 γ + γ + γ cosθ = sv sv lv lv (60) γ ()γ γ − lv 0.015 sv lv 1

Li and Neumann [47] gave another form of this equation of state using a totally different approach. Agreeing and continuing the Berthelots´s geometric mean combining rule for the attractive constant in van der Waals equation of state, the work of adhesion, Wsl, is taken as a geometric mean of the work of cohesion of the solid, Wss, and liquid, Wll, i.e. = Wsl WssWll (61) = γ = γ where, Wss 2 sv ;Wll 2 lv (62) = γ γ = γ + γ − γ hence, Wsl 2sv lv sv lv sl (63) γ = γ + γ − γ γ or, sl sv lv 2 sv lv (64)

This equation works only when γsv values are very close to the value of γlv, because the function form is the result of Berthelot´s geometric mean combining rule,

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= ≈ Wsl WssWll , which is valid only when Wss Wll. To modify this combining rule, Good and Girifalco introduced the interaction parameter (1957) = φ Wsl WssWll (65) where φ is found to be below 0, this means that the geometric mean combining rule over estimates the value of Wsl. In general the geometric mean combining rule is applicable to bulk phases also. In theory of intermolecular interaction and the theory of mixtures the combining rule is used to evaluate the parameters of unlike-pair interactions in terms of the like pair interactions. Berthelot´s geometric mean combining rule (Eq. 66) is only a useful approximation. ε = ε ε ij ii jj (66)

where, εij is the energy parameter of unlike-pair interaction and εii & εjj are energy parameters for like-pair interactions. By London´s theory of dispersion forces it has been shown [48] that geometric mean combining rule is applicable only for similar molecules, because implicit in this rule is the condition that the two energy parameters of the like-pair interaction must be very close to each other i.e. εii≈ εjj. However, for the interaction between two very dissimilar molecules of the material, where there is an apparent difference between εii and εjj, it has been demonstrated that the geometric mean combining rule generally overestimates the strength of the unlike-pair interactions. In the study of mixtures commonly a factor like, (1-kij) is introduced in the combining rule i.e. ε = ()− ε ε ij 1kij ii jj (67)

where kij is an empirical parameter quantifying deviation from the geometric mean. The factor should decrease with the difference, (εii-εjj), and be equal to 1 when (εii-εjj) is zero. Based on this Li and Neumann [47] introduced a modified combining rule −α ()ε −ε 2 ε = ε ε ii jj ij ii jj e (68)

where, α is an empirical parameter and the square of (εii-εjj) is used rather than the difference it self for taking into account the symmetry of the combining rule. Correspondingly, for the case of large differences between Wll and Wss ; γlv and γsv, the combining rule for the work of adhesion of a solid-liquid pair is written as: −α ()− 2 = Wll Wss Wsl WssWll e (69) Introducing values from equations 62 and 63 we get −β ()γ −γ 2 γ = γ + γ − γ γ sv lv sl sv lv 2 sv lv e (70) Combining this with the Young equation (Eq. 1) we get

γ −β ()γ −γ 2 cosθ = −1 + 2 sv e sv lv (71) γ lv

For the set of γlv and θ data obtained by different liquids on the same solid, the constants β and the surface energy of solid-γsv can be determined by least-square analysis method. While, starting with arbitrary values of γsv and β, iterative procedure is used to estimate those values of γsv and β which best fit the experimental data.

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The value of β=0.000115 (m2/mJ)2 was obtained [47] by fitting Eq. 71 to the data presented in Fig. 11 and used by Neumann [42] in the derivation of the first form of the equation of state (i.e. Eq. 59). But later a value of β=0.0001247 (m2/mJ)2 was obtained [49] by fitting Eq. 71 to accurate contact angle data obtained on very smooth and homogeneous solid surfaces of polyethylene terephthalate (PET), fluorinated ethyl propylene (FEP) and glass coated with fluoropolymer FC-721. The contact angle were measured by ADSA-P (Asymmetric drop shape analysis-Profile) and capillary rise techniques. Kwok, [50]; Kwok and Neumann [25,51] modified the Berthelot´s rule by 2 introducing another modifying factor of the form 1-κ1(εii-εjj) which is a decreasing function of the difference (εii-εjj) and is equal to one when εii=εjj. Here κ1 is an unknown constant. Therefore after modification the Berthelot´s combining rule Eq. 66 becomes ε = ( − κ ()ε − ε 2 ) ε ε ij 1 1 ii jj ii jj (72)

The square of the difference (εii-εjj), rather than the difference itself is used which reflects the symmetry of this combining rule. Since free energy is directly proportional to the energy parameter ε, then for the cases of large differences, i.e., Wll-Wss or γlv-γsv the combining rule for the energy of adhesion of a solid liquid pair is written as = ( − α ()− 2 ) Wsl 1 1 Wll Wss WllWss (73)

where, Wll=2γlv and Wss=2γsv = ( − β ()γ − γ 2 ) γ γ Wsl 2 1 1 lv sv lv sv (74)

where, β1=4α1 γ = γ + γ − γ γ ( − β ()γ − γ 2 ) sl lv sv 2 lv sv 1 1 lv sv (75) Combining Eq. 75 with Youngs equation Eq. 1 we arrive at γ cosθ = −1 + 2 sv ()1 − β ()γ − γ 2 (76) γ 1 lv sv lv

A value of β=0.0001057 (m2/mJ)2 was obtained by using the same least square method (as used by Li and Neumann [47]) to fit the contact angle data obtained by various liquids on 15 homogeneous and smooth solids. The contact angles were measured using both ADSA-P and capillary rise techniques. The solids and techniques used for generating the data were Fluoropolymers- FC-721 on mica by capillary rise, FC-722 on mica by ADSA-P, FC-722 on silicon wafer by ADSA-P, FC-725 on silicon wafer by ADSA-P, Teflon by capillary rise, Hexatriacontane by capillary rise, cholestery acetate by capillary rise, poly(propane- alt-N-(n-hexyl)maleimide) by ADSA-P, poly(n-butyl methacrylate) by ADSA-P, polystyrene by ADSA-P, poly(styrene-(hexyl/10-carboxydecyl 90:10)-maleimide) by ADSA-P, poly(methyl methacrylate/n-butyl methacrylate) by ADSA-P, poly(propene- alt-N-(n-propyl)maleimide) by ADSA-P, poly(methyl methacrylate) by ADSA-P and poly(propene-alt-N-methyl maleimide) by ADSA-P. More detailed data can be found in Table 4 of Kwok and Neumann [25].

Objections on Equation of state approach:

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The initial formulation of equation of state [42] was critically analysed by Van de Ven [52] and some objections were presented. Before the formulation of equation of state [42] some basic assumptions were made, γ = γ γ 1. There exists a relation of the form sv f ( lv , sv ) [44]. (Theoretical assumption) 2. For all non-spreading liquids (θ > 0), as the contact angle approaches zero + * (θ→0 ), γsl approaches a minimum value γsl which depends only on the properties of the solid (Experimental assumption) 3. For θ > 0, plot of the Goods interaction parameter, φ, (Eq. 56) as a function of γsl yield an indeterminate series of straight lines with slopes and intercepts * determined by the choice of γsl (or γsv) (Experimental assumption) 4. Among all conceivable liquids there exists at least one for which γsl=0 equivalently (Goods interaction parameter) φ=1 (Philosophical assumption)

* From the second and fourth assumptions, it was concluded [42] that γsl =0 i.e. γ = γ * = lim+ sl sl 0 (77a) θ →0 φ = φ * = lim+ 1 (77b) θ →0 but the authors point out that this does not logically follow from assumptions 2 and 4. There are many spreading liquids (θ = 0) for which no information about γsl exists because Youngs equation (Eq. 1) does not apply. The γsl for spreading liquids can be * less than the γsl , therefore no conclusion about the absolute minima of γsl for all the liquids can be made by considering only the experimental evidence for non-spreading liquids. It cannot be assumed that the absolute minima of γsl for the spreading liquids + * as θ→0 is zero i.e. γsl =0, in other words the minimum value of γsl can be negative. In the same paper the plots of φ vs γsl, Fig. 13 (Fig.3 of [42]) with lines passing through φ=1 and γsl=0 were accepted but the other lines which had points for which φ>1 or when φ*<1 were discarded. This means that the applicability of the equation of 2 state was within a short range of φ or only when γsl + γsl ≤ 130 ergs/cm which is not satisfied by some systems like blood cells in aqueous media. It is implicit from assumption 4 that γsl<0 is impossible, thus zero is taken as the lowest possible value. But from statistical mechanics γsl consists of two parts ~ γ = γ + γ sl s l (78) ~ γ γ where s is solid surface energy and l is that of liquid in the external field created by ~ γ γ the solid. Although s>0 but l can be negative; physically which means that the ~ γ liquid density increases near the solid-liquid interface, it is difficult to see why l > -

γs. In principle, γsl can have any value and γsl=0 deserves no special status. Thus assumption 4, although probably true, becomes irrelevant. On the other hand Johnson and Dettre [53] doubted if arbitrarily small (positive) solid/liquid interfacial energies were not possible when the contact angle approaches zero. They tested the assumption on hexane-water system, where the equivalent

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3. Surface Characterisation: Paper IV Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001 contact angle is zero even when the interfacial tension is about 51 mJ/m2. The test assumes that the equation of state at liquid-liquid interface of the form 70 also exists. γ = f (γ ,γ ) (79) l1l2 l1v l2v The equivalent contact angle is calculated using the following equation and the liquid-liquid interfacial tensions are evaluated using the equation of state. γ − γ cosθ = l1l2 llv (80) Eq γ l2v The second test depends on the corollary, deduced from the equation of state approach, that if one liquid has the same contact angle on two different substrates, Zisman plots for the surfaces should superimpose. Since the PTFE, water and stearic acid, as pointed out by Johnson and Dettre [53] cross but do not superimpose, they conclude that the equation of state approach is neither applicable to liquids and nor to solids. Li [54] refuted Johnson and Dettre´s [53] assumption that the thermodynamics of equation of state does not require the substrate to be solid. Almost the same period when Johnson and Dettre´s comments were published, Li and Neuamnn [47] proposed an alternate proof for the existence of the Equation of state using the phase rule for heterogeneous system containing moderately curved surfaces (Eq. 46). This phase rule (Eq. 46) also predicts that the number of degrees of freedom for two-component liquid-liquid lens-fluid system (Fig. 10a) is generally three; only for the special case where one of the interface in the liquid lens system is planar is the number of degrees of freedom two. These results tell us that generally for a two-component liquid-liquid lens fluid system an equation of state of the form Eq. 79 does not exist. Neumann [55], defend their previous [42] assumption of taking γsl=0 as the least possible value and further discussed the possibility of negative solid-liquid interfacial tension. The first argument is that it is generally accepted interfacial tension between liquid-liquid and liquid-solid under conditions of stable equilibrium cannot be negative. The negative tension if exist would cause mixing of two fluids or dissolution of solid in the liquid phase leading to formation of a homogeneous phase. Pairs of polymer melts which are insoluble to a degree comparable with that of polymer solids and low-molecular-weight liquids are considered [55,56]. In the plots of φ vs γ12 a straight line is achieved with limiting values of φ=1 and γ12=0, even when the free energy of spreading is negative for some and positive for some pairs, and condition for negative interfacial tension is conducive for some pairs. This is in excellent agreement with the assumption that the minimum possible value of γsl is zero. Neumann [55] also considered the interaction of small particles initially suspended in liquid, with an advancing solidification front, i.e., solidification front experiment. When the particle encounters the solidification front, it is engulfed in the solid or is swept along by the solidification front. Particle engulfment or rejection depends upon the free energy of adhesion or engulfment. ∆ Adhesion = γ − γ − γ G ps pl sl (81 a) ∆ Engulfment = γ − γ G ps pl (81 b) Negative free energies mean that particle will be engulfed by the solidification front otherwise not. The surface tension of the melt, γlv, and the contact angles on the solid matrix material and particle material are measured. From these contact angles the solid surface tension, γsv, is evaluated using the equation of state approach and then the 107

3. Surface Characterisation: Paper IV Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001 interfacial tensions required in Eq. 81a and 81b are also evaluated using the equation of state approach. The straight lines in Fig. 12 can be represented by φ = β − αγ sl (82) The solidification front observations are used to test the validity of β=1 which means that γsl=0 is the minimum possible value of γsl. The free energy of adhesion and engulfment (81a, 81b) are evaluated using first β=1 and then by using 5% higher value of β=1.05, which means negative values of γsl. The free energy values are reported in Table I of Neumann et al., 1984 for pairs of polymer particles suspended in polymer melts. Only when β is taken to be 1 the free energies predict the engulfment or rejection correctly. When β=1.05 is taken the free energy values fail to predict the engulfment. Morrison [57] critically examined the thermodynamic proof given by Ward and Neumann [44] for the existence of Equation State (Eq. 41) at the solid-liquid interface. The work draws attention to three errors committed during the derivation. The first error is the error of omission, while writing the Gibbs-Duhem equations for the three interfaces i.e. Eq. 34-36 and then transforming them into Eqs. 37-39. The equations written show that the interfacial energies to depend on the properties of only one of the two components, the chemical potential of the fluid, µ2, and the temperature, T. Since the three equations (34-36) for the interfacial energies are shown as function of T and µ2 only, the interfacial energies appear to be independent of the solid. Ward and Neumann [44] have gone from the relations between differentials dγ, dT and dµ to undefined relations between integral quantities- γ, T and µ2 without adequate consideration of the dependence of the thermodynamic functions on the properties of both the fluid and the solid. The relations between integral quantities must be as follows and not Eq. 34-36 γ sv = sv − sv − Γsv µ u1 TS1 2(1) 2 (34a) γ sl = sl − sl − Γ sl µ u1 TS1 2(1) 2 (35a) γ lv = lv − lv − Γlv µ u TS 2 2 (36a)

Each of the interfacial energies is a function of T and µ2 but they are also functions of specific internal energy, u, specific entropy, S, and specific adsorption, Γ, all depend both upon the liquid and the solid. The significance of being explicit about the dependence of interfacial energy on each of the thermodynamic functions if that it makes clear the dependence of the interfacial energy on chemistry of both of the components, something which is lacking in the equation used by Ward and Neumann [44] saying that γ = γ µ (T, 2 ) (37b) is true but incomplete, as it does not show the material dependence of the internal energy on both the fluid and solid. Showing the explicit dependence of the surface tension on material-dependent quantities point out that the equations for interfacial energy will be different for every pair of materials. Therefore, Eq. 37b is not only incomplete but also incorrectly implies that the equations are same for all materials. The more exact representation is as follows γ sv = γ sv ()µ 2(1) 2(1) T, 2 (37a) γ sl = γ sl ()µ 2(1) 2(1) T, 2 (38a)

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γ lv = γ lv ()µ 2(1) 2(1) T, 2 (39a) Here the extra set of subscript show that each equation will be different for different pairs of fluids and solids. The rearrangement would lead to a different function for every pair of materials. No universal expression for any interfacial energies, γsv, γsl and γsv can be shown to exist. Johnson and Dettre [53] also pointed out this discrepancy and supports that the set of equations 26a-28a will be different for different sets of solids and liquids. Otherwise two liquids having same surface tension will have the same contact angle on the same solid surface, which does not have any scientific justification. The dependence of interfacial energy on the chemical nature of the components can be made even more explicit through statistical mechanics, where the interfacial energy can be derived from an analysis of the grand canonical ensemble for this system γ = ()kT ln Ξ / A (83) where the grand partition function, Ξ , is given by, − E ( N ) N  j   Nµ     2  Ξ = ∑e kT e kT  (84) j

Ej is the total energy of N molecules in the interface of area A. Note that by combining equations 83 and 84 the functional form of equation 37b can be written. The possible energy level of each interface depends on the detail of the intermolecular interactions and are different for every pair of materials. The total energy of the molecules in the interface depends on the chemistry of both fluid and solid. The second error is an error in thermodynamics. Ward and Neumann [44] claim that at equilibrium the interfacial energies are function of only two independent variables, T and µ2 which is not true. The confusion is about the number of independent quantities necessary to analyse the sessile drop, that is; between the number of degree of freedom of the system and the number of independent parameters necessary to calculate a thermodynamic property of the system. Considering the ideal system of a sessile drop of liquid and its vapour contacting a uniform solid substrate in which the liquid and solid are insoluble in each other. The numbers of degree of freedom can be calculated by applying phase rule, Eq. 41, here P=3, C=2 therefore degrees of freedom is one, most conveniently temperature. Which means that the fundamental equations of Ward and Neumann [44] could just be written as function of only temperature. γ=γ(T) (85) If one argues that the curvature of a phase can introduce a new degree of freedom hence making T and µ2 as the factors. But this is only true when the curvature is less than a micron. For observable systems, such as the sessile drop being considered by Ward and Neumann and for systems to which the equation of state approach has been applied, the usual phase rule applies and the number of degrees of freedom is one. The third error is the error in mathematics and is consequence of the first two errors. The statement that the three equations depend on only two variables and therefore can be solved to eliminate one variable is incorrect i.e. γsl ≠ f(γsv,γlv) (86) In fact these three equations are functions of unknown number of material variables as well as temperature. None of the interfacial energies is necessarily a function of just the other two

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Considering that all interfacial energies are fixed at thermal equilibrium, and hence the three interfacial energies for the sessile drop (37a-39a) would be determined when the chemical difference between the fluid and the solid can be characterised, at least approximately, by two material variables, one for the fluid and one for solid. The energy levels, Ej, in the grand partition function, eq. 84, would then be functions of only two material-dependent parameters. Examples of such systems are fluids and solids that interact purely by dispersion forces, where each material is characterised by single London constant and the intermolecular distances of closest approach are similar. According to Gaydos et al. (1990) the above three criticisms by Morrison [57] are unfounded. The error of omission is unfounded because µ2 used in the equations are interfacial chemical potentials:  ∂U (lv)  µ (lv) =   (87) 2  ∂ (lv)  N 2 (lv ) ( lv ) (lv )  S ,A ,N1 (lv) where N1 =0 by definition and not the bulk liquid chemical potential defined by  ∂U (l )  µ (l ) =   (87 a) 2  ∂ (l )  N 2 (l ) (l ) ( l )  S ,V ,N1 (lv) and µ2 is dependent upon both the liquid and vapour phases. It is not surprising that (lv) µ2 has properties which reflect the explicit nature of its interface as similar effects occur with surface tension. For example of oil-water and nitrogen-water interface is ow nw (ow) (nw) considered, then it is not expected that γ =γ and similarly µ2 ≠ µ2 . This (lv) suggests that the value of µ2 is a reflection of the material properties of the interface's adjacent phases. The difference between the "surface of tension" dividing surface and the dividing surface of "zero mass" is not understood by Morrison [57]. For any planar interface it is possible to shift these dividing surfaces to the same location. Consequently in the relations like Eq. 34a - 36a one discovers that the µ2 terms are evaluated at a dividing surface position such that Γ1 ≡ 0 (zero mass of solid). It is also explicitly stated by Ward and Neumann [44] that "there is no dissolution of the solid nor is there any adsorption of any components from the liquid or gaseous phase by the solid". Taken together, it is not surprising that the solids properties do not appear explicitly in the final expression. Lee [58] categorically rejects this claim. He states that all writers on equilibrium thermodynamics point out that, at equilibrium, the chemical potential of each component has a constant value everywhere in the closed system. There can only be one chemical potential whether in bulk or at any interface, whether curved or flat. There is no such thing as a distinct "interfacial chemical potential" that is not identical with that in each of the bulk phases. Of course, there exists a concentration gradient across any interface. But definitely, there is no corresponding gradient of any (lv) l v chemical potential; thus µ2 ≡ µ2 ≡µ2 at equilibrium. The second error is in thermodynamics, where according to Morrison only one thermodynamic variable is necessary to define the system, which most conveniently be the temperature. But Gaydos [59] points out that the normal Gibbs phase rule is not applicable in the present case but the phase rule for heterogeneous system Eq. 38 is applicable according to which the degrees of freedom are two- temperature and interfacial chemical potentials. The third criticism of Morrison [57] is error of mathematics, while proceeding from differential relations Eq. 34-36 to integral relations 37-39. According to Gaydos

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[59] the requirements necessary to proceed from the differential relation to the integral relations are fulfilled in this case. Morrison [60] analysed the second proof given by Li [45] for the existence of the equation of state using the modified phase rule. Morrison agrees that the Gibbs phase rule need to be modified for the systems with curved surfaces. Li [45] correctly maintain that Laplace pressures are created across all curved interfaces no matter how slight is the curvature so that the pressure cannot be constant throughout the system containing curved interfaces. But Morrison is of the opinion that, Laplace pressures are often significant enough to have mechanical effects, such as capillary rise. But Li [45] is at error when they claim that these Laplace pressures influence the thermodynamics. Morrison [60] illustrate the insignificance of Laplace pressures on the thermodynamics of observable systems by two examples A hemispherical liquid drop on a solid substrate, The Laplace pressure is ∆PL=2γ/R (88) where ∆PL is the Laplace pressure, γ is the interfacial tension and R is the drop radius. Apart from the Laplace pressure there is another pressure is the hydrostatic pressure at the bottom of the drop. ∆PH=ρgR (89) where ρ is the density and g is the acceleration due to gravity. The relative magnitude of these pressures can be compared by calculating the radius of the drop when the two pressures are the same R=(2γ/ρg)½ (90) For water this radius is 4 mm. A full analysis shows that the two pressures differ only by a small Laplace pressure across the top of a drop. For usual kind of studies by interfacial tensions and contact angles, Laplace pressures are no more significant that gravitational pressures. Gravitational effects are insignificant for liquids until the depth is greater than several meters, similarly Laplace pressures are insignificant for the liquid drops are 3 orders of magnitude smaller, or on the order of a few micrometers. Laplace pressures exist in all systems containing curved interface; they are just insignificant to the thermodynamics until the curvatures are less than a micrometer. The size of drops necessary to make capillary pressures significant on the thermodynamics can be estimated by Kelvin equation for the effect of an increase in curvature on the vapour pressure of a liquid. A numerical example is given by Defay and Prigogine [61] and presented in Table I of [60]. The pressure of a drop with a radius of one micron is only 0.1% higher than a drop with infinite radius i.e. a flat surface, the pressure is 3 times higher only when the drop is 10-3 microns size. Therefore, the increase in vapour pressure of water drop due to curvature is not significant until the drop is much less than a micrometer in radius. A one-micron drop is barely visible with a light microscope and features like contact are indistinct. Liquids with lower surface tensions or liquid-liquid interface of lower tension must be even smaller. Therefore all liquids for which interfacial tensions and contact angles have been measured directly are too large for capillary pressures to change the thermodynamics of the phase rule. Morrison [60] re-derived the phase rule for capillary systems following the treatment by Defay and Progogine [61]. The capillary system is where the pressure difference cause by the curved interfaces is significant enough to change the thermodynamics properties of the system. F´=1+C (91) 111

3. Surface Characterisation: Paper IV Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001 where C is the number of components in bulk phase. For the Gibbs phase rule (Eq. 41), the number of phases is just the number of bulk phases, surface phases are not counted. For the modified phase rule for capillary systems, the number of phases is not needed as long as all the interfaces are significantly curved. For every interface insignificantly curved, the mean curvature is no longer a variable, and the number of degrees of freedom is reduced by one. Morrison [60] also studied the two systems (as studied by Li [45]) i.e. sessile drop on solid substrate and liquid-liquid lens-fluid system. Considering a sessile drop on a solid substrate as shown in Fig. 3a. If the drop is large (radius greater than a micrometer) then its curvature is insignificant and the classical Gibbs phase rule (41) applies. For two components and 3 phases the number of degrees of freedom is one. Specifying the temperature completely specifies the intensive state of this system. Considering the sessile drop on fluid surface as shown in Fig 10a. If the sessile drop is large and the vapour consists only of molecules of liquid 2 and liquid 3, then the number of phases are 3, the number of components are 2 and by applying the classical Gibbs phase rule (41) we get the degrees of freedom to be 1. Specifying the temperature completely specifies all the intensive variables of the system. If all the interfaces are highly curved then the modified phase rule (91) applies and the number of degrees of freedom are 3. Specifying the curvature of the two phases and the temperature specifies the system. If the substrate liquid is plentiful then its interface with the vapour is flat and hence one can reduce the numbers of degrees of freedom by 1 because one more pressure equality equation is possible. This makes the total degrees of freedom 2 and hence the equation of state like eq. 44 possible. Therefore, Morrison [60] concludes that the classical phase rule does not need modifications simply because a system contains curved surfaces. It need be modified only when the curvatures are sufficient to cause significant pressure gradient, which happens for drops less than one micrometer. The number of degrees of freedom for a sessile drop on a substrate is the same (F=1) whether the substrate is flat solid or extended liquid and hence a equation of state of the type 40 cannot exist. Since the system of sessile drop on both the substrates - flat solid or extended liquid are similar hence, the experimental evidence (used by Johnson and Dettre [53]) taken on liquid- liquid systems to analyse and disprove the equation of state approach is well justified. The equation of state relating the interfacial tensions exist when the curvature of the interface is high (drop size < 1µm) but is different for different materials and is by no means a universal function. Both Li and Neuamnn [62] and Gaydos and Neumann [63] defended the original proof for the equation of state [45] from the criticisms of Morrison [60]. Li and Neumann [62] used the experimental data to justify and defend the existence of equation of state. Fig. 11 shows the variation of the liquid surface tension, γlv, with the γlvcosθ term. Different solids (1 to 8) have different curves with the most hydrophobic solid lying in the topmost regions of the γlv Vs γlvcosθ plots and the least hydrophobic in the lowermost region of the γlv Vs γlvcosθ plots. Changing continuously the liquid surface tension from low surface tension to higher surface tension causes the data points to move along one smooth curve (Fig 11) depending on the solid under consideration. It is quite clear that the contact angle can vary, and because of Youngs equation, γsl can vary by changing the γlv and γsv through simply changing the liquid and solid. If there was only one degree of freedom, according to Morrison [60], then this would not have been possible.

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Other approaches like Fowkes approach [31,64], LW-AB [65] approach divide the surface energy into components. Which means that γsl depends not only the γlv and γsv but also on intermolecular forces or the different components of the surface energies of solid and liquid. In other words the equation like 32 will have more than two variables on the right hand side. This would require more than 2 degrees of freedom, but Morrison [60] does not allow more than 1 degree of freedom. Therefore, Morrison´s claim that there is only one degree of freedom does not contribute to a real debate. Morrison´s [60] most important argument is the insignificance of Laplace pressure on the thermodynamics. He has tried to justify by considering two examples- comparison of Laplace pressure to hydrostatic pressure at the bottom of a sessile drop and the increase in equilibrium vapour pressure of a drop in comparison to flat surface. Though, Li and Neumann [62] agree that the increase in equilibrium vapour pressure of a 1 µm drop is only 0.1% higher in comparison to flat surface, but this is only one aspect of curvature effect. If the effect of curvature on the increase in boiling is considered then the scene is totally different. Taking water as an example at 1 atm it has been shown by Lupis [66], that a bubble of 1 µm in radius will require a boiling point of 1210C. In comparison with a flat surface (1000C) this is a 21% increase. Clearly, a dimension of the order of 1µm or any other dimension should not be used to justify the significance or insignificance of curvature or Laplace pressure effects in general. Li and Neumann [62] also pointed out that it is wrong when Morrison reduces the total degrees of freedom by 1 when the substrate liquid is plentiful and has almost flat interface with the vapour. It is true that far enough from the drop; the liquid- vapour interface is flat. However, a drop interacts with the liquid substrate only in drop's immediate vicinity, where the interface is curved. And it is wrong to consider the liquid substrate-vapour interface to be flat. Gaydos and Neumann [63] claim that classical Gibbs phase rule under predicts the actual degrees of freedom for capillary systems and most importantly the phase rule in general makes no statement regarding the significance of the influence which capillary pressure has on other intensive variables. Kelvin relation is taken as an example because it had been discussed by Morrison [60]. The Kelvin relation gives an expression for the manner by which the bulk saturation pressure must change in order to keep a spherical drop of radius, R, in equilibrium. If a two-phase water-steam system is considered where water is in form of sphere suspended in the vapour phase and the system is in gravitationally free zone. The Gibb's phase rule predicts for C=1 and P=2 then F=1 (Eq. 41). But if the Clausius-Clapeyron equation is written for one- component, two-phase system (water drop suspended in water vapour) − ()υ (α ) −υ (β ) (β ) +υ (α )  2 γ (αβ )  = ()(α ) − (β ) dP d    s s d T (92)  R   α α α where (α) represents water and (β) represents vapour phase, ν( )=V( )/N( ), β β β ν( )=V( )/N( ), V and N are volume and mole number, R is the radius of the drop, T is the equilibrium temperature. Even if the temperature is fixed, so that one degree of β freedom is removed from the system, that the saturation pressure P( ) outside the drop will need to acquire different values for drops of different radii so as to maintain equilibrium. Obviously, this demonstrates in a theoretical sense that the classical phase rule does not correctly predict the 2 degrees of freedom which, exists in this system. Furthermore, it should be understood that the conclusion that there are 2 degrees of freedom in this droplet-vapour system is independent of any decision 113

3. Surface Characterisation: Paper IV Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001 regarding the significance or magnitude of the influence of the droplet's radius upon β the saturation pressure P( ) which is needed to maintain the droplet in equilibrium. Therefore, to claim that capillary effects are significant to thermodynamics until the curvature is less than one micrometer [60] or so is meaningless without specifying what thermodynamic properties are of interest. On the same lines as Li and Neumann [62], Gaydos and Neumann [63] also point out that Morrison [60] took only the intensive property which are hardly influenced by the presence of capillary pressure, it would make far more sense to study those quantities which are. They site an example from Defay and Prigogine [61] which, pertains to the existence of small vapour bubbles in water. Accordingly, they β calculated that at a temperature of 180C the saturation pressure P( ) required to maintain the water-vapour equilibrium across the planar interface is approximately 0.02033 atm, while the corresponding vapour pressure inside a bubble of radius 1µm is just slightly smaller at 0.02032 atm. However, to assume that there are no significant thermodynamic changes in the system [60] is to ignore the fact that there is a significant "difference between the pressures in the bubble and in the liquid which, is approximately 1.441 atm. It follows that "for bubble to exist under these conditions the liquid must be under negative pressure" [61] of 1.412 atm. Both Li and Neumann [62] and Gaydos and Neumann [63] maintain that the case of a sessile drop on the curved liquid surface in form of lens is different from the system of sessile drop resting on flat solid surface. Both maintain that Classical Gibbs phase rule is applicable in this liquid-liquid lens fluid system (Fig. 10a) and accept the Gibbs phase rule's prediction of 3 degrees of freedom in this system. Accordingly they point out that that the two systems, sessile drop on solid substrate and sessile drop on liquid substrate, are different and that the later have one higher degree of freedom than the former. Therefore, in theoretical sense the data taken from liquid- liquid lens system cannot be used to disprove the potential existence or validity of a two-variable equation of state (as done by Johnson and Dettre [53]). Moy and Neumann [67] attempted to use direct force measurement data carried out by Claesson [68], on surface and interfacial tensions for comparison with their data calculated with equation of state approach. Despite the problem of the surface force formula, in terms of 3πγsvR versus 4πγsvR, they do not realise that the force equation was originally derived on the basis of the van der Waals interactions between sphere and flat surface. Thus, the data obtained by equation of state are comparable with those directly derived from apolar, van der Waals interactions. Hence, Lee [58] agrees with the conclusion of Morrison [57] and Fowkes [69], that the equation of state approach is applicable to apolar systems involving solely physisorption. Though physisorption between a liquid and solid is universal and more common than chemisorption, it is generally much weaker in magnitude. Without considering chemisorption the equation of state, as persistantly asserted and vigorously defended by Neumann and his co-workers, is incomplete and definitely not universal for interfacial tensions. Furthermore, the equation of state approach cannot be extended to the system involving a thin liquid film at the interface when the disjoining pressure consists of at least three components: van der Waals, structural,, and electrostatic. On the other hand Lee [58] supports the LW-AB approach as it is applicable to both apolar and polar systems involving physisorption and/or chemisorption at the interface. Drelich and Miller [70] used the advancing contact angles of water, glycerol, diidomethane, and ethylene glycol on Teflon, polyethylene and different bitumen, bitumen asphaltenes and bitumen resins. The surface tensions were evaluated using 114

3. Surface Characterisation: Paper IV Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001 the equation of state approach. They observed a significant scatter in the surface tension values of the solids when contact angle values with varying surface tension and polarity were used. It was found that the surface tensions of Teflon and polyethylene could be successfully estimated with the Equation of state, but only for systems with an apolar liquid (diiodomethane). A lack of agreement, for other systems, in which contact angles of polar liquids were used, points out the limitation. They conclude that the equation of state approach is applicable to polymer-apolar liquid systems (polymers with surface tension from a range of approximately 21 to 42 mJ/m2 were considered in their study) involving physisorption. Gaydos [71] used thermodynamics to prove that the equation of state type of relation is only possible for sessile drop resting on ideal solid and not for liquid-liquid lens system. Equation of state is formulated using the appropriate Gibb's adsorption equation which, does not apply to the liquid-liquid capillary system. For an interface between two immiscible bulk phases, denoted by α and β the equation is as follows, r ρ (α ,β ) µ + (αβ ) + γ (αβ ) = ∑ i d i s dT d 0 (93) i=1 (αβ) where ρi is the surface density of component-i with corresponding chemical (αβ) (αβ) potential µi, s is the specific surface entropy, T is the temperature and γ is the surface tension. In the original derivation Ward and Neumann [44] the position of the (αβ) (αβ) dividing surface choosen in such a way so that one of the quantities e.g., s or ρi becomes zero, this process is convenient and often desirable. The effect of such a choice is that r γ (αβ ) = − (αβ ) − ρ (αβ ) µ d s(1) dT ∑ i(1) d i (94) i=2 where the subscript (1) implies that the dividing surface position is selected such that (αβ) ρ1 =0. Because of this, two results follow ()αβ ()β ()α  ∂γ  () ρ − ρ ()αβ   = −ρ αβ − 2 2 ρ (95)  ∂µ  2 ρ ()α − ρ ()β 1 2 µ µ 1 1  T , 3 ,.... r and () () () () ∆ω αβ = ∆γ αβ = ∆λ()P α − P β (96) αβ where, ω( ) is the specific grand canonical potential for the surface i.e. the surface energy when the surface is in a state of thermal and chemical equilibrium, λ is the α β distance into (β) phase and P( ) and P( ) are the pressures on adjacent sides of the interface. From equation 95 it follows that if the shift is performed to a new location such (αβ) that ρ1 =0 then the distance of the shift is ρ ()αβ λ = 1 (97) ρ ()α − ρ ()β 1 1 (αβ) and the value of ρ2 will change to ()β ()α ()αβ ()αβ ()αβ ρ − ρ ()αβ  ∂γ  ()αβ ρ = ρ + 2 2 ρ so that   = −ρ (98) 2(1) 2 ρ ()α − ρ ()β 1  ∂µ  2(1) 1 1 2 µ µ  T , 3 ,..... r But it is impossible to shift the dividing surface from the surface tension position to (α) (β) any other position if ρ1 =ρ1 . From equation 96 we see that corresponding to the shift (97) one has a change in energy as given by eq. 92. However, for any system (capillary or otherwise) to be in equilibrium, the energy must be stationary. The requirement of a stationary state 115

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α β can only be accomplished in one way for these systems i.e. by choosing P( ) = P( ) so αβ αβ that ∆ω( )=∆γ( )=0. This means that Gibb's moderately curved theory of capillarity only permits one to shift the dividing surface if the surface is planar. Thus, when the surface is planar, the pressures on the adjacent sides of the surface will be equal and any shift in dividing surface position will not change the value of the energy. Therefore, it is possible to arbitrarily move the dividing surface for solid-vapour and solid-liquid surfaces as these are assumed to be planar a priori during the derivation of the equation of state. It is apparent that the equation of state derivation α will not apply to liquid-liquid lens systems since the corresponding conditions P( ) = ε ε β α β ε P( ) and P( ) = P( ) (where P( ), P( ) and P( ) denote pressures in three contacting bulk ε β α phases with densities subjected to the condition ρ( ) >ρ( )>ρ( )) is rarely met. One can say that 2 component liquid-liquid systems with 3 degrees of freedom are not equivalent to 2 component liquid-solid system with 2 degrees of freedom and hence data taken with liquid-liquid lens system cannot be used to disprove the potential existence or validity of a two variable equation of state. Its is also obvious from the form of these equations and the presence of surface densities beyond i=2 that there is nothing which restricts the equation of state to exclusively binary systems with just two components. To this Lee [72] replied stating various points. The interfacial tensions between liquids, for moderately curved surfaces, have never been found to be dependent on curvature. Drops of the third liquid between two immiscible liquids can be of any size, including diameters approaching infinity. Therefore, the argument that such surfaces are curved cannot be used to object to the employment of liquid-liquid interfacial tension to test the equation of state approach. This fact raises strong questions about any thermodynamic derivation that implies otherwise. The consequence of having a curved interface is creation of small Laplace pressure that is thought, by proponents of equation of state approach, to increase the degree of freedom which has been proved untrue by Morrison [57,60]. It is simple to show that the Laplace pressure is less than the gradient caused by gravity, but no one have suggested adjusting contact angle measurements for that. The idea of displacement of the dividing surface, as used by Wrad and Nuemann [44] is unacceptable as shown and proved by Markin and Kozlov [73] on account of energy conservation principle.

(LW-AB) Lifshitz-van der Waals/acid-base approach:

This approach came into existence when the thermodynamic nature of interface was re-examined by van Oss [65] in the light of Lifshitz theory of forces. The role of van der Waals forces and hydrogen bonds was studied in order to explain the strong attachment of biopolymers (Human serum albumin, human immunoglobulin) on low energy solids (Polytetrafluoroethylene, polystyrene) which was previously attributed to the hydrophobic interactions. The apolar interaction between protein and low energy solid is repulsive and hence only the apolar interaction cannot explain the strong attachment of biopolymer on the low energy solids. A new polar term- Lewis Acid/Base interaction, AB (previously referred to as short-range (SR) interactions in van Oss et al. 1986) was introduced in order to explain the attraction between biopolymers and low energy solids. The definition of the Lewis AB term has been ambiguous till van Oss [74,75] defined them properly. The importance of distinguishing apolar and polar contributions to the surface tension of liquids and solids has been stressed already in 116

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1962 [21]. In the beginning only the London dispersion force contribution was considered as apolar and the rest of the following contributions were considered as polar:

1. Dipolar compounds, i.e., substances that have appreciable dipole moment 2. Hydrogen bonding compounds: treated under the Brφnsted (proton donor-proton acceptor) theory of acids and bases 2.1 Substances that are both proton-donors (acids) and proton acceptors (bases), e.g., water, termed as bipolar, the proton donor and acceptor functionalities of a molecule may not be of equal strength. 2.2 Substances that are very much more effective as proton donors then as proton acceptors, e.g., CHCl3. 2.3 Substances that are very much more effective as proton acceptors then as proton donors, e.g., Ketones. 3. Compounds that interact as Lewis acids (electron acceptors) and bases (electron donors) 3.1 Substances that have both kinds of functionalities, electron acceptors and donors, termed as bipolar- the same as the class 2.1, because the Lewis acid- base theory encompasses the Brφnsted acid-base theory 3.2 Substances that are much more effective as electron acceptors then electron donors 3.3 Substances that are much more effective as electron donors then electron acceptors

The three electrodynamic interactions present at the interface are- Keesom (randomly oriented dipole-dipole also called "orientation") interactions, Debye (randomly oriented dipole-induced dipole also called "induction") interactions and London (fluctuating dipole-induced dipole also called "dispersion") interactions. It is known that a possession of a large dipole moment is not necessary condition for hydrogen bond formation [76], e.g., nitriles and nitro compounds both have large dipole moments, yet, nitriles form hydrogen bonds and not nitro compounds. Moreover, in the condensed, macroscopic system using the Lifshitz approach- Chaudhury [77] showed that surface tension components arising from the three electrodynamic interactions must be treated in the same manner and should be grouped together. When grouped together, these electrodynamic interactions are alluded to as Lifshitz-van der Waals interactions (LW). γ LW = γ Keesom + γ Debye + γ London (99) The rest of the interactions i.e. 2 and 3 constitute the Lewis Acid/base contribution. The symbol γ- is used to indicate the parameter of surface tension that is due to proton acceptor or electron donor functionality and γ+ is used to indicate the surface tension parameter due to proton donor and electron acceptor functionality. The joining together of the two kinds of basic behaviour, (Brφnsted bases and Lewis bases, using the symbol γ-) is justified because the proton acceptor group is necessarily and electron donor group. The grouping together of Brφnsted acids and Lewis acids for the purpose of surface tension under the term of γ+ is done for operational reasons and because during the surface chemical measurements i.e. contact angle, adsorption and interfacial tension, Brφnsted and Lewis acid behaviour and not easily distinguished.

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The clear subdivision between apolar (LW) and polar (AB) interactions, also has made it possible to arrive at the quantitative definition of hydrophobic interactions which is also referred to as the total interfacial interaction ∆G Total = ∆G LW + ∆G AB (100) The apolar part γLW, follows the Fowkes treatment (Eq.12) [74,75,78-81] γ LW = γ LW + γ LW − γ LW γ LW sl s l 2 s l (101a) 2 γ LW = ( γ LW − γ LW ) or sl s l (101b) Then the cohesion energy of a liquid is expressed as ∆ LW = − γ LW Gll 2 l (102) Using Vissers notations where 1 and 2 denotes solids and liquids and 3 only liquids and Dupré work of adhesion [82] 12 = γ + γ − γ WA 1 2 12 (103) translated in terms of free energy change ∆ = γ − γ − γ G12 12 1 2 (103a) The apolar (or LW) energy of adhesion between 1 and 2 is expressed as ∆ LW = γ LW − γ LW − γ LW ∆ LW = − γ LW γ LW G12 12 1 2 = G12 2 1 2 (104) and the LW interaction of 1 and 2 in 3 is expressed as ∆ LW = γ LW − γ LW − γ LW G132 12 13 23 = ∆ LW = ( γ LW γ LW + γ LW γ LW − γ LW γ LW − γ LW ) G132 2 2 3 1 3 1 2 3 (105) Unlike the LW interactions, which are mathematically symmetrical, the acid/base interactions are essentially asymmetrical van Oss [74,75] in the sense that for a polar substance i the electron acceptor and the electron donor parameters are quite different. Also, one parameter is not manifested at all, unless the other parameter is either present in another part of the same molecule of the substance i or in another polar molecule j with which molecule i can interact. Thus, at the solid-liquid interface the electron acceptors of solid will interact with the electron donors of liquid, and vice versa. van Oss [74,75,78-81] expressed the acid/base interactions in the following manner ∆ AB = − ( γ +γ − + γ +γ − ) G12 2 1 2 2 1 (106) This was based on Kollman [83] where he analysed the non-covalent interactions in wide variety of intermolecular complexes (van der Waals molecules, H-bonded complexes, charge-transfer complexes, ionic association, radical complexes and three body interactions) and used an expression like Eq. 106 for the asymmetrical interactions. In anology with the Drago´s approach in solution thermodynamics [84] γAB is expressed in a more rigorous manner as γ AB = 2 γ +γ − (107) Dupré equation (Eq. 103 a) is valid for acid/base interactions also hence, ∆ AB = γ AB − γ AB − γ AB γ AB = ∆ AB + γ AB + γ AB Gsl sl l s or sl Gsl l s (108) ∆ AB γ AB γ AB γ+ Putting the value of Gsl from Eq. 106 and expanding s and l in terms of and γ- (Eq. 107) we get, γ AB = ( γ +γ − + γ +γ − − γ +γ − γ −γ + ) sl 2 s s l l s l s l (109)

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γ AB = ( γ + − γ + )( γ − − γ − ) or sl 2 s l s l (109 a) Therefore, for a binary system like the solid-liquid interface the total free energy of interaction is ∆ Total = − ( γ LW γ LW + γ +γ − + γ −γ + ) G 2 s l s l s l (110) and the total interfacial tension is 2 γ Total = ( γ LW − γ LW ) + ( γ + − γ + )( γ − − γ − ) sl s l 2 s l s l (111) Combining this with the Youngs equation (Eq. 1) we get ()+ θ γ = ( γ LW γ LW + γ +γ − + γ −γ + ) 1 cos l 2 s l s l s l (112) For a ternary system the acid/base interaction energy is ∆ AB = [ γ + ( γ − + γ − − γ − )+ γ − ( γ + + γ + − γ + )− γ +γ − − γ −γ + ] G132 2 3 1 2 3 3 1 2 3 1 2 1 2 (113) and the total interaction energy is ∆ Total = ( γ LW γ LW + γ LW γ LW − γ LW γ LW − γ LW ) G132 2 2 3 1 3 1 2 3 + [ γ + ( γ − + γ − − γ − )+ γ − ( γ + + γ + − γ + )− γ +γ − − γ −γ + ] 2 3 1 2 3 3 1 2 3 1 2 1 2 (114) Later it has been clearly demonstrated that, while the apolar-γLW and polar-γAB surface components are additive, the Lewis acid-base electron acceptor-γ+ and donor- γ- surface tension parameter are not additive [85]. LW + - Equation 112 contains 3 unknowns- γs , γs and γs , hence contact angle measurement has to be performed using 3 standard (2 polar and 1 apolar) liquids (well characterised) on the solid surface in order to totally evaluate the surface energy of the solid. Hence, by means of contact angle measurements with the number of different well-characterised, standard, polar and apolar liquids, the apolar (Lifshitz-van der Waals) surface tension component (γLW), the polar (Lewis acid-base) surface tension component (γAB) and the latter's electron-acceptor (γ+) and electron-donor (γ-) parameters, can be determined for a number of polar surfaces. LW + - The standard liquids are pre-characterised with respect to their γl , γl and γl Total values. The absolute value of γl is available from the surface tension LW measurements and the absolute value of γl is obtained by using the Fowkes AB Total LW approach [31]. The γl is obtained from the difference of γl and γl . Relatively few such well-characterised polar, or apolar contact angle liquids are present at our disposal. One requirement that all such liquids must fulfill is to have a rather high surface tension (γl), in order not to spread on most polar surfaces. Usually this means 2 a γl>44 mJ/m . This especially limits the number of available apolar liquids, but in 2 2 practice, α-bromonapthalene (γl=44,4 mJ/m ) and diiodomethane (γl=50,8 mJ/m ) are quite adequate for most purposes. Among polar, hydrogen bonded liquids, water (γl= 2 LW 2 AB 2 72,8 mJ/m , γl =21,8 mJ/m , γl = 51 mJ/m ) is extremely suitable. Other high 2 LW 2 AB 2 tension liquids available are glycerol (γl= 64 mJ/m , γl =34 mJ/m , γl = 30 mJ/m ) 2 LW 2 AB 2 and formamide (γl= 58 mJ/m , γl = 39 mJ/m , γl = 19 mJ/m ) + - No absolute value of γl or γl are known at present for any compound (not even for water). One is reduced to making an arbitrary estimate of the ratio of γ+ and γ- for a reference compound (e.g. for water). However, for obtaining the absolute values of AB AB AB AB γsl , ∆Gsl , ∆G121 and ∆G132 , it is not necessary to know the absolute values of + - + - γi and γi of any substance i. It is sufficient to use the polarity ratio of γi and γi + - + - relative to the γR and γR of the reference compound. The γ /γ ratio for water is 119

3. Surface Characterisation: Paper IV Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001 assumed to be 1 because of its equivalence to the pH convention, and hence γ+ and γ- take the values of 25.5 mJ/m2 each as the of γAB for water is 51 mJ/m2 [74]. Although Gutman [86] has reported the H-donor and acceptor number in the ratio 1/3, which makes water a Lewis base, however, the relation between Gutman´s scale of "electron acceptor numbers" and "electron donor numbers" and the relative values of γ+ and γ- for water has not yet been fully explored. The polarity ratio- γ+/γ- for other standard liquids (e.g. glycerol and formamide) is obtained by measuring contact angle with water, glycerol and formamide on various monopolar (Lewis) basic solid surfaces e.g. poly (methylmethacrylate) (PMMA), poly (ethylene oxide) (PEO), clay films, corona-treated poly (propylene) (CPPL), dried agarose gel, dried zein (a water insoluble corn protein, cellulose acetate and dried film of human serum albumin (HAS) [87]. The Eq. 112 for water contact angle on the solids is of the form γ ()+ θ = ( γ LW γ LW + γ +γ − + γ −γ + ) w 1 cos sw 2 s w s w s w (115) and similarly for glycerol the equation is of the following form γ ()+ θ = ( γ LW γ LW + γ +γ − + γ −γ + ) g 1 cos wg 2 s g s g s g (116) The Lifshitz van der Waals parameter of the solids is determined before hand by measuring contact angle using apolar liquids. Since only monopolar (Lewis bases) + solids are used meaning the term γs =0, hence the above two equation reduce to − + γ γ γ = w ()1 + cosθ − γ LW γ LW (115a) s w 2 sw s w γ γ −γ + = g ()1 + cosθ − γ LW γ LW (116a) s g 2 wg s g + + - - The ratio- γw /γg is evaluated from the above two equations which, yields γw /γg also. Using this method on ten monopolar solids, the averages thus obtained were + 2 - 2 (Glycerol) γg = 3,92 ± 0,7 mJ/m (which yields γg = 57,4 mJ/m ) and (Formamide) + 2 - 2 γf =2,28 ± 0,6 mJ/m (which yields γf = 39,6 mJ/m ) [87].

Objections on Lifshitz-van der Waals Acid/Base approach

The LW-AB approach appears successful to separate the surface polar components and determine negative interfacial tension as an indication of solubility. The LW-AB approach has been successfully applied by many. However, unavoidably there have been some criticisms. Li [45] applied phase rule for capillary systems and showed that only two degrees of freedom are allowed in the system where sessile drop is sitting on a solid substrate (Fig. 3a). They expressed solid-liquid interfacial energy in the form of expression 40 where the solid-vapour and liquid-vapour interfacial energies are the two degrees of freedom. But according to LW-AB approach the solid-liquid interfacial energy is expressed in form of equation 89, which means that the expression like 40 will have 6 variables on the right hand side. This is not allowed according to the modified phase rule. Fowkes [88] pointed out that the use of single parameter for expression of acidity and basicity ignores the hard and soft character of acids and bases, which is accommodated in the Gutmann´s acceptor and donor numbers [86]. He also expressed scepticism about the accuracy of the electron donor and electron acceptor values obtained for the solid surfaces as the degree of acidity and basicity of the standard liquids are still under investigation (assumption: γ+/γ-=1 for water).

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Use of LW-AB approach to evaluate the acidic and basic components of the solid surface free energy shows that most of the solid surfaces are overwhelmingly basic, with a small or negligible acidic component. Berg [89] doubts this to be a general law for the solid surfaces and suggests the checking of the consistency of the method. Van der Mei and Busscher [22] reason that biosurfaces are predominantly electron-donating as a consequence of prevalence of oxygen in the Earth´s lower atmosphere and the hydration of microbial cell surface. Lee [90] reported a correlation between the Lewis acid-base surface interaction components and linear solvation energy relation (LSER) solvatochromic parameters α and β. The solvatochromic approach gives separate subfactors of hydrogen-bond-accepting and hydrogen-bond-donating abilities for many acids and bases. Where α is an empirical quantitative measure of the hydrogen-bond-donating (HBD) ability of a bulk solvent towards a solute and β is an empirical quantitative measure of the hydrogen-bond-accepting (HBA) or electron-pair-donating (EPD) ability of a bulk solvent towards a solute for a hydrogen bond or a Lewis coordination bond. For non-HBD solvents, such as apolar, aliphatic and aromatic hydrocarbons, the α value is zero, for polar aliphatic-alcohols, 0.5<α<1.0, while for fluoro-substituted alcohols and phenols, α>1, reaching a maximum of 1.96 for hexafluoro-2-proapnol. In contrast the β scale is fixed by setting β=0 for cyclohexane and is the same for all apolar aliphatic hydrocarbons. However, for aromatic hydrocarbons the β value is 0.1, for aliphatic ethers, β≈0.7-0.9, for aliphatic amines β= 0.5 to 0.7, β=1 for methylphosphoric triamide and maximum value of β is 1.43 for 1,2-diaminoethane. Lee further comments that "…if the interaction is limited only to hydrogen bonding instead of the broadly defined acid-base interaction, then γ+ resembles HBD parameter α and γ- the HBA parameter β. Marcus [91] have compiled a list of solvatochromic parameters for about 170 liquids. It is interesting to note that there are many more HBA than HBD compounds in the list, coincidentally in terms of Lewis acid-base classification, this means that there are many more Lewis bases than Lewis acids. This observation, in a way, supports the LW-AB approach´s evaluation of most of the solid surfaces as basic. For liquid water at ambient temperature, the HBD ability is stronger than HBA ability. Thus unlike other Lewis acid-base standards like Gutmann donor-acceptor scale, water appears to be acidic with α/β=1.8. Various polymers has been reported as predominantly basic by the LW-AB approach [92], specially the case of polyvinyl chloride (PVC) is doubted by many workers [89,93]. PVC is accepted to be monofunctionally acidic polymer [93]. The acidic behaviour has been reported by measurement of work of adhesion against PVC of monofuncationally acidic and basic liquids [94], by peeling experiment involving a polymeric film of PVC on glass and strengthening of PVC polymer by acidic filler (glass powder) (Fowkes, 1987). But PVC is evaluated to be a basic polymer using LW-AB approach - γ+=0.04 and γ- =3.5 mJ/m2 [92]. Lee [58] used γ+/γ-=1.8 for water and re-evaluated the surface energies of many polymers using the LW-AB approach. With the new γ+/γ- ratio the major improvement was in lowering of the surface Lewis base component for all the polymers, but still PVC came out to be predominantly basic with γ+=0.1 and γ- =2.4 mJ/m2. It is important to stress that the problem is broader, and by no means limited to PVC surface alone. Van Oss [80] finds, by contact angle measurement, that cellulose is basic, while results of Berg [89] on the characterisation of cellulose surface by

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3. Surface Characterisation: Paper IV Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001 inverse gas chromatography (IGC) experiments show that it has primary acidic sites on its surface. Kwok [96,97] tested the internal consistency of the LW-AB approach by evaluating the surface energy of fluorocarbon (FC721), Teflon (FEP) and polyethylenetetrapthalate (PET) using different sets of polar (water, formamide, glycerol, ethylene glycol, (DMSO) dimethyl sulfoxide) and apolar liquids. Equation 104 needs the contact angle data from 3 standard liquids in order to be solved and to evaluate γLW, γ+ and γ- parameters for the solid. Kwok tested the two different strategies to solve the equation. First, to use contact angle data from 3 polar liquids and then solve the 3 simultaneous equations to get the parameters. Second, to LW determine the γs for the solid first by using contact angle with a high-energy apolar liquid and then the γ+ and γ- parameters using contact angle data from 2 polar liquids. LW In the second case the value of γs showed a strong dependence on the apolar liquid surface tensions. In both the cases a wide range of values were obtained for the solid surface parameters when different sets of liquids were used, negative values for (γ+)½ and (γ-)½ were obtained which is anomalous and the negative values of total solid surface energy, γs, is not allowed in the LW-AB approach. For Teflon, which is most hydrophobic, it was expected that the polar component of the surface energy should AB 2 be negligible but the γs component range from -64 to 0.38 mJ/m and the best value is 0.14 for water-formamide pair. It was also shown the anomalies of large variations in the solid surface energy and occurrence of negative solid surface tension and square roots of surface tension components was not due to errors in the contact angle data. Therefore, Kwok et al. concluded that LW-AB approach could not give consistent value of γs. Solid surface tension components are not unique properties of the solid. Contact angle does not contain information about putative surface tension components and contact angle is a function of only the total solid surface tension (as in equation of state approach) and not of its components. Holländer [98] partially attributed the lack of internal consistency of LW-AB approach to the mathematical instability of the model and proposed a selection criterion for the standard liquids for evaluation of the acid-base properties of solid surfaces contact angle goniometry. He agrees with the proponents of the LW-AB approach that the most severe constraint for the use of the approach is the lack of broad spectrum of test liquids with sufficiently high surface tension, which are well characterised. While the choice of basic liquids is adequate, this is not the case for predominantly acidic liquids (monopols). Strong monopoles like, chloroform or nitromethane, have much too low LW interaction capabilities that they spread on most of the surfaces of interest. He observed that the solid surface energy values obtained by a pair of widely different liquids, with respect to their acidic and basic nature, like water-formamide and water-glycerol were similar but the values obtained by the combination of basic liquids i.e., formamide-glycerol, exhibit large differences. This reason becomes obvious from Fig. 14, where the parameters γ+ and γ- are plotted as a function of one contact angle, when the other contact angle in the pair is kept constant. The functional variation for the glycerol-formamide pair are seen to be very steep, and small deviations of the contact angles (within the range of experimental error) results in large changes in the values of γ+ and γ- derived from these contact angles. The data of the pairs water-formamide and water-glycerol are less critical. Therefore, the liquids - + used in the pair must have large difference in their γ /γ (Qr) ratios. In order that the simultaneous equations be least sensitive towards experimental errors then the

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3. Surface Characterisation: Paper IV Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001 difference in their Qr numbers (i.e. ∆Qr) should be more than 3 (∆Qr ≥ 3). In order to obtain reliable results from the contact angle data, ∆Qr ≥ 15 seems to be appropriate But, if the data from Kwok [96,97] is re-examined in the light of Holländer´s selection criterion, then we see that the criterion works but not completely. Table I show the solid surface energy data for two solids arranged according to decreasing ∆Qr of the pair of the polar liquids used for the determination of solid surface energy parameters. It is safe only if ∆Qr ≥ 15 because for both water-formamide and water- glycerol pairs we get positive solid surface energy and square roots of the surface energy components (good parameters). Though, water-ethylene glycol (∆Qr = 9) and water-DMSO (∆Qr = 6,7) gives good parameters but formamide-DMSO, with a higher ∆Qr (=9,6), evaluates bad surface energy parameters for both the solids. By Holländer´s criterion it is expected that DMSO-ethylene glycol with ∆Qr=2,3 should evaluate bad surface energy parameters but, for FC721 the pair of liquids give good parameters and the total surface energy is only 0,04 mJ/m2 off the average which is not bad when the standard deviation is 1,5 mJ/m2. Since the two solids are apolar AB AB hence negligible values for γs is expected. The best value for γs is obtained when water-formamide pair is used (FC721-0,04 and FEP-0,14 mJ/m2) though, it is AB unexpected that fluorocarbon have lower γs than Teflon. Another important point, which becomes obvious, is that all the liquid pairs, which contain water give good parameters even if their ∆Qr is poor. Although, the above analysis is based on only two apolar solids but we can see that Holländer´s selection criterion further narrows down the choice of liquids which can be used to determine the solid surface energy using LW-AB approach. Water- formamide pair (∆Qr=16,4) seems to be best. Water-glycerol pair (∆Qr=14,6) is AB borderline case both according to Holländer´s criterion (∆Qr≥15) and that the γs values jumped from 0,04 to 0,24 and 0,14 to 0,34 for fluorocarbon and Teflon (apolar solids) respectively when glycerol was used with water instead of formamide. The proponents of the LW-AB approach [99] refuted the statement put by Kwok [96]. Firstly, it is pointed out that while determining the solid surface energy of FEP and FC721 by using 3 polar liquids the choice of liquids was not properly done. Good and van Oss [100] pointed out that- "mathematically, it is possible to use three polar liquids and a set of three equations in the form of equation 112. Such tactics work if the values of the parameters (e.g. γ-) for the three liquids are not too close together. If they are close, the calculated values of the three parameters for the solid will be unduly sensitive to small errors in the values of the parameters of the liquids, and in measured contact angles". But neither Wu [99] nor Good and van Oss [100] suggest the selection of those three polar liquids which are most suitable or a selection criterion to select the 3 polar liquids for the purpose of determining the solid surface LW energy parameters accurately. For the second method where γs is pre-determined using an apolar liquid and then 2 polar liquids are used to determine the remaining 2 parameters (γ+ and γ-). Van Oss [99] gives the same reason as Holländer [98] for the failure and suggest that the two polar liquids used must be very different in their acidic and basic behaviour. Differences in the solid surface energy parameters are still obtained when different sets of liquids are used even according to the selection criterion of Holländer [98] and Wu [99]. In absence of any guidelines from the proponents of LW-AB approach and other supporters it is difficult to judge which liquid set gives the most + - accurate solid parameters. Though, van Oss [87] mentions that " γs and γs can be entirely defined with two polar liquids, the availability of a third liquid could yield 123

3. Surface Characterisation: Paper IV Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001 useful set of control values " but this does not help in arriving at the most accurate set of parameters. Wu [99] used least square method to solve the contact angle data for liquid pairs - water-formamide, water-glycerol, water-ethylene glycol and water- DMSO the results of which are presented in Table-I. The least square values are very different from the ones obtained using a single set of liquids. If the situation is seen in totality, both FEP and FC721 are apolar and hence + - AB their γs , γs and γs components must be negligible. This happens only when water- formamide pair is used for the determination of the solid surface energy. This pair Total gives the minimum γs out of all the liquid pairs. Volpe and Siboni [101] investigated the acid-base solid surface free energy theories in detail. They also studied the mathematical instability inherent in the simultaneous equations to be solved for the solid surface energy parameters using LW-AB approach. The simultaneous equations of the form 112a are written in matrix form A.x=B (Eq. 117) with the contact angle data of various liquids on the solid. The system can be over determined with more than 3 liquid contact angles. ()+ θ γ LW LW + − − + 1 cos γ γ + γ γ + γ γ = i li (112a) s li s li s li 2

γ  l  LW + − ()+ θ 1  γ γ  LW 1 cos 1 l l l  γ   2   1 1 1  s  γ LW + − −    γ γ γ  γ  = ()1 + cosθ l2 (117) l2 l2 l2 s  2 2   LW + −  +   γ  γ γ γ γ l  l l l  s ()+ θ 3  3 3 3    1 cos 3   2  While solving the matrix its condition number governs whether meaningful solid surface energy parameters can be obtained from that matrix (or the liquid system). The condition number of the A matrix determines its nearness to singularity (i.e. the norm of matrix is almost equal to a norm of some singular matrix). The condition number is defined as a nonnegative real scalar given by Cond (A) = A . A−1 (118) where A is the norm of a matrix is a scalar that gives some measure of the magnitude of the elements of the matrix. High condition number means that the system is ill-conditioned, i.e., the system is more prone to the errors in the contact angle and measured liquid parameters. In other words the condition number of a matrix measures the sensitivity of the solution of a system of linear equations to errors in the data. It gives an indication of the accuracy of the results from matrix inversion and the linear equation solution [102]. The norm of the matrix is defined as 1 n p  p  A = A Where p≥1 (119) p ∑ i   i=1  Many functions qualify to be called as matrix norms according to the following rules = A max aij (Column norm) (119a) 1 j ∑ i 1  n 2  2 A =  a  (Euclidean norm) (119b) 2 ∑ i   i =1  124

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= A ∞ max aij (Row norm) (119c) i ∑ j Table II lists the liquid systems according to increasing condition numbers. Three different set of liquids are presented, LW I. 2 polar liquid system where γs is pre-determined using contact angle of an apolar liquid. II. 3 liquids out of which 2 are polar and the third one is apolar, all three parameters LW + - γs , γs and γs are determined using the liquid set. LW + - III. 3 polar liquids, all three parameters γs , γs and γs are determined using the liquid set. IV. More then 3 liquids- a combination of polar and apolar liquids, Least square method is used to solve the system of equations. The first two groups i.e. II and I are essentially the same as they give the same LW + − values for the parameters γs , γs and γs , but their condition numbers are different. Kiely [103] reported contact angle data with 3 polar and 2 apolar liquids, for two strains of Brevibacterium linens used as taste enhancer in the Danish Cheddar cheese (Table III). The cell surface energy parameters are analysed from liquid systems using Matlab 5.3 and presented in Table IV. LW Case-I is when the γs is predetermined using contact angle data with some apolar liquid and then polar liquids are used in pairs or in overdetermined system + − when all three liquids are used for the determination of γs and γs . Out of the 4 possible liquid groups only water-formamide (cond. no. 3,5) group gives positive solid surface energy and square roots of the surface energy components. Even the over-determined system with water-formamide-Glycerol (cond. no. 3,9) does not give all positive parameters. The condition numbers for the formamide-glycerol pair is very high, i.e., 106 and the system is ill-conditioned and should never be used. Case-II is when 2 polar and 1 apolar liquid is used. This case is the same as first case since the solid surface energy parameters obtained are essentially the same. But the condition numbers are higher than the respective pairs in case I. In the 3 liquid system also only water-formamide-bromonapthalene (cond. No. 5.14) and water- formamide-methyleneiodide (Cond. No. 5.17) give positive solid surface energy and square roots of the surface energy components. Similar to the 2 liquid system the formamide-glycerol-bromonapthalene and formamide-glycerol-methyleneiodide have very high condition numbers of 148 and 151 respectively and hence are highly ill- conditioned systems and should never be used. Case-III is when 3 polar liquids are used to determine all the solid surface energy parameters i.e. without any apolar liquid in the group then the condition + number is high (18.5). Highly negative √γs values are obtained. In the fourth case overdetermined system of equation is used with polar and apolar liquids. Even though the condition numbers are 5 to 6, negative parameters are obtained. Therefore, we can see that when biological surfaces are involved then the choice of liquid systems, which can be used for the evaluation of solid surface energy from contact angle data, is very restricted. Only water-formamide pair along with bromonapthalene or methyleneiodide gives positive surface energy and square root of surface energy parameters. Errors in the contact angle measurements can be one of the reasons for this. The bacterial layers, deposited on cellulose nitrate membranes, used for contact angle determination are not smooth, an inherent surface roughness of the order of bacterial cell size is involved. The spreading pressure, moisture content of the bacterial layer, layer thickness and liquid purity are the other factors, which influence 125

3. Surface Characterisation: Paper IV Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001 the accuracy of contact angle measurements. Volpe and Siboni, 1997 are of the opinion that the large inconsistencies of the experimental data in current literature are not acceptable without coherent explanation. Published contact angle data frequently show kinetic effects interpreted as thermodynamic ones (receding angles greater than advancing ones), lack of any statistical analysis, of whether the angles are advancing or receding, of the temperature values at which they were taken etc. Many years ago Padday [104] defined the situation of contact angles data as "the comedy of errors".

Volpe and Siboni [101] compared the LW-AB approach with other scales available to quantify the acid-base properties. They concluded that from mathematical point of view the LW-AB theory can be classified in the same realm as Drago [105], Taft [106,107] and Abraham [108] scales. They commented that the use of LW-AB approach is easy but results must be considered with attention because 1. The base component of the analysed surface has systematically higher base components 2. The results strongly depend on the choice of three solvents used for measurements (as seen in Table IV) 3. The unknowns of the calculation are the square roots of the surface tension components, and in some cases they assume negative values (as seen in Table IV) 4. The values of the coefficients used for acid-base properties of test liquids are completely dependent on the choice made for water: the acid-base ratio at 20oC is set at 1, i.e., each components contributes 25.5 mJ/m2 4.1. This does not mean that water is equally strong as a Lewis acid as a Lewis base. Actually no definition of strength of acids and bases is given in the LW- + − 2 AB approach, γw = γw = 25.5 mJ/m is just an assumption. 4.2. One cannot compare the acid and base components of the same solvent, but, eventually, the acid (or base) components of different solvents can be compared. This is similar to Drago´s theory [105]. But different to Taft scale [106,107] where the electron acceptor parameters of many solvents are put in such a form that one can compare the two parameters of the same solvent; in that scale, water is considered an electron acceptor or Lewis acid about 6,5 times stronger. 4.3. An ambiguity exists with regards to the monopolarity, when a component (acid-base) is very small as compared to the other. According to LW-AB approach a substance is monopole if its component ratio with respect to water is less than 25,5, corresponding to the value of 1 mJ/m2. But this argument is not sufficient because of 4.1 5. In the set of solvents usually employed, water, glycerol, ethylene glycol, formamide, dimethyl sulfoxide, diiodomethane and bromonapthalene, and on the scale proposed by LW-AB, none is prevalently acid and all the polar once have base components stronger than water, so the situation is strongly asymmetric, with a pronounced role for base functions. These choices are probably at the origin of the systematic base predominance in tabulated LW-AB coefficients 6. The equation system, whose solution provides the surface tension components can be ill-conditioned for an improper choice of solvents, unfortunately LW-AB approach does not examine this subject closely. Different liquid systems are studied before and presented in Table IV. A consequence is that no evaluation of the goodness of liquid and solid parameters has been done in the LW-AB approach, so no standard deviation or error estimate of reckoned values is known.

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Keeping all the above considerations Volpe and Siboni [101] suggested another method in order to arrive at the surface energy parameters of solids and solvents. A wider set of equation containing Eq. 112a for a large set of solid-liquid couples and the following equations for every liquid i and solid j. γ Total = γ LW + γ + γ − l,i l,i 2 l,i l,i (120a) γ Total = γ LW + γ + γ − s, j s, j 2 s, j s, j (120b) The symbols have the same meaning as in Eq. 112a with an additional index j to distinguish the various solids. Similarly the contact angle of ith liquid on jth solid is denoted by θij. For L liquids and S solids we have a set of SL+S+L equation and 4S+3L unknowns; the four unknowns for each solid include the total surface free energy because for liquids the total surface energy is experimentally measured but there is no commonly accepted technique for solids. The equation system (Eq. 112a, 120a, 120b) is nonlinear and overdetermined (except for very low values of S and L). The procedure of solution is similar to Drago [105] for his set of equations leading to the well-known acid-base scale. The system has no exact solution but can be solved by best-fit criterion (least squares in our case) searching for the minimum of the objective function (113). This system has infinite number of best-fit solutions L S L S 2 T Total 2 T Total 2  T 1 Total  U = [][]X RX − γ + Y RY − γ + X RY − ()1 + cosθ γ ∑ i i l,i ∑∑∑i i s,i  i j ij l,i  i=1 j===111i j  2  (121) where R is a 3x3 symmetric orthogonal matrix, and  γ LW   γ LW   l,i   s, j  X =  γ +  and Y =  γ +  (121a) i  l,i  j  s, j   γ −   γ −   l,i   s, j 

Among the infinitely many solutions, one can eventually choose that which is based on some conventionally assigned components of reference solvents. Infinitely many equivalent scales are possible, but a scale based on realistic ratio between acid- base components of a reference solvent (as for water acid-base ratio of 1 in LW-AB or 6.5 in Taft scale or 5.5 in Abrahams scale). This procedure is difficult to realise, due to the disagreement in the values of contact angles of common nonpolar and basic solvents on common polymers and to the lack of data about acid solvents, however in their opinion this proposed method represents the most correct way to apply the LW- AB approach. A less restrictive, but very useful, constraint would be simply to impose acidic and basic components of a solvent to be equal, without assigning a particular value to them. In this case the scale is not completely determined. But in the present form this choice is incorrect due to reliable values of contact angles and solvent surface energy parameters. The reference solvents and components have to be judiciously chosen because a wrong choice could be inconsistent with the experimental data. The possibility of estimating the errors of the coefficients, we can confirm that the location of minima for the nonlinear best fit function is obviously influenced by the knowledge of the constant parameters, which comes from experiments. Errors in 127

3. Surface Characterisation: Paper IV Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001 the estimate of parameters result in a certain amount of uncertainty about the minima positions: owing to nonlinear coupling of variables. Even a small change in even a single parameter will yield a global rearrangement of minima, all of whose location will generally be modified, as will, therefore, the estimated value of the acid-base components for all the chemical species involved. Volpe and Siboni [101] claim that the necessity of a wide, accurate and homogeneous file of experimental data, collect according to standard procedures for methods and materials employed, specimen treatment, working temperature etc. The number of available data for each solid-solvent pair should be sufficiently large to clearly provide significant statistics, to ensure good reproducibility, and to allow a reasonable estimate of the experimental error. A round robin test is proposed where wide group of laboratories should work together, realising a round robin with solvents and polymer surfaces chosen by mutual consent; the mean results of all laboratories, both advancing and receding, could be used as a base set of proper contact angles to calculate the most satisfactory acid-base components of liquids and solid surface free energies. Kwok [50,97] once again put the LW-AB approach to test using another method, i.e., by comparing the liquid-liquid interfacial tension from calculations using LW-AB approach and by measurement using the ADSA-P (asymmetric drop shape analysis-profile) technique. Though, Van Oss [75] have reported that by the LW-AB approach some interfacial tensions for the solute/solvent systems with good solubility are negative. Lee [90] supports this, where he reported the solubilities and interfacial tensions of many polymers, carbohydrates and proteins, and found that indeed the interfacial tensions were negative for those with good solubility. Kwok [109] reported the interfacial tensions of liquids-Glycerol, formamide, diiodomethane, bromonapthalene, ethylene glycol and DMSO against pentane, decane, dodecane, tetradecane and hexadecane. The percentage error between the measured and calculated (by LW-AB approach) interfacial tensions for the liquid pairs which are immiscible range from 34% lower to 112% higher but the scatter diagram between the measured and calculated show a definite correlation. Kwok [50,97] also reported a previous work where they studied the miscible liquid systems [110]. LW-AB approach reported negative interfacial tensions for the miscible system of water- glycerol, water-formamide, water-ethyleneglycol and water-DMSO. But did not give negative interfacial tension values for many other miscible liquid systems like glycerol-formamide, glycerol-ethylene glycol, glycerol-DMSO, formamide-ethylene glycol, formamide-DMSO, diiodomethane-bromonapthalene, diiodomethane-DMSO, ethylene glycol-DMSO and bromonapthalene-DMSO. They concluded that LW-AB approach does not predict the correct interfacial tensions of a large number of arbitrarily chosen miscible and immiscible liquid-liquid system hence, caution should be exercised when this approach is used to determine solid surface tension components from contact angles.

Determination of spreading pressure

Spreading pressure is the reduction of the solid surface energy due to the vapour adsorption. In general the approaches assume this term to be negligible, which is true for low energy solids in contact with high energy liquids [31,64]. The spreading pressure term is significant for high-energy surfaces when the low energy liquid spontaneously spreads and forms a very thin layer in order to reduce the total energy of the system. It has been made possible to quantify the spreading pressure on

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3. Surface Characterisation: Paper IV Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001 polymer surfaces but spreading pressure quantification due to water-bacterial surface has not yet been reported. Busscher [111] used the Geometric mean approach in order to obtain the d spreading pressure. First the γs is determined by using contact angle with apolar liquid, then contact angle with a series of water+propanol mixtures is obtained. A plot γ ()θ + − γ dγ d γ p γ p of lv cos 1 2 l s Vs l gives straight line the slope of 2 s and intercept on Y-axis of −πe according to Eq. 122. γ ()θ + − γ dγ d = γ p γ p − π lv cos 1 2 l s 2 l s e (122) The water+propanol mixture surface tension parameters are obtained by contact angle d 2 p measurement on Paraffin wax surface (γs =25.5 mJ/m and γs =0) Bellon-Fontain and Cerf [112] evaluated the spreading pressure using the fact that the work of adhesion, WA, is a maximum value when there is no vapour adsorption on the solid surface. A plateau region is observed in the plot between work = γ ()θ + γ of adhesion WA lv cos 1 and lv for different liquids on the solid surface. The spreading pressure is determined from the difference between the plateau work of adhesion and the work of adhesion of the liquid. Erbil [113] combined one-liquid and two-liquid (Fig. 9)(where the contact angle with one liquid is measured in presence of another immiscible liquid and not air) contact angle data and Geometric mean approach to obtain the spreading pressure of water-polymer interactions. Later Erbil [114,115] used the Lifshitz-van der Waals acid/base approach to determine the spreading pressure by combining the one-liquid and two-liquid contact angle methods. Most of the methods use either Geometric mean approach or Lifshitz-van der Waals acid/base approach. Equation of state approach applies to ideal solids, which does not absorb vapours (essentially true for low energy solids e.g. Teflon etc.). But deviation of some of the points from the γlv vs γlvcosθ (Fig. 11) curves gives a possibility to determine the spreading pressure [116,117]

Comparison of the Thermodynamic approaches

Studies has been performed in different fields to find out the best approach for evaluation of solid surface energy from contact angle data. Bellon-Fontaine [118] studied the adhesion behaviour of dairy microorganisms (L. mesenteroides and S. thermophilus) on FEP, polypropylene, PMMA and glass, and used the different approaches to predict the possibility and extent of adhesion. They concluded that all the approaches yield similar surface energies, but equation of state approach with different liquids did not give consistent values for high surface energy surfaces. Geometric mean approach accounting for spreading pressure was the best approach, which could predict the adhesion phenomenon. Gindl [119] compared the different approaches to evaluate the surface energy of wood samples. Surface energy of 10 Norway spruce were evaluated using different approaches and was shown that LW-AB approach delivered results very similar to geometric mean and, to a lesser degree, to the equation of state approach. The results achieved with harmonic mean and Zisman approach deviated clearly from the group. It was concluded that if the right group of liquid contact angles are used then LW-AB approach delivers a maximum information about the chemical composition, in giving

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3. Surface Characterisation: Paper IV Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001 the values for electron-donor and electron-acceptor components of surface, which is especially valuable with a chemically heterogeneous material like natural polymer wood, containing cellulose, lignin and variety of hemicellulose. LW-AB approach is most suitable to explain the coating properties (adhesion) of wood surfaces. Schneider [120] studied the adhesion of Gram-negative bacterium SW8 on various substratum- Germanium, stainless steel, polypropylene, perspex coated with conditioning films. The surface energy of the bacterium and the solid substrates were evaluated using the different approaches. The bacterium-substrate interfacial energy and free energy of adhesion were calculated from the solid surface energies. He concludes that the LW-AB approach provides the most consistent treatment of acid- base and apolar interfacial interactions. Results obtained with LW-AB were not in conflict with its theoretical framework but the fact that negative square roots were obtained with some regularity for the determination of γ+ and hence suggested to be cautious while interpreting the results obtained from this theory.

Experimental

Measurement of contact angle on bacterial cells

The contact angle of bacterial cells is measured by producing a uniform layer of cells on agar or the bacterial lawn is deposited on membrane filters as [121-123]. The membrane technique is preferred over the agar techniques as the cells are less apt to detachment in the liquid phase, secondly by depositing the bacterial lawns on the membrane they can be dried of that only bound water is present on the bacterial surface. The membrane method is described in detail by Busscher [122] and van der Mei [23,124]. The bacterial substrate for measuring contact angles are prepared by depositing bacterial cells, suspended in water, on cellulose triacetate filter (preferable pore diameter of 0.45 µm) by applying negative pressure. The bacterial lawns are deposited to a density of 108 cells/mm2 [122] or approximately 50 layers of bacteria [124], 800-900 layers [16]. To establish constant moisture content the filters with bacteria are placed in a petri dish on the surface of a layer of 1% (wt./vol.) agar in water containing 10% (vol./vol.) glycerol. The filters are left in the petri dish till they can be used for contact angle measurement. This serves two purposes- Agar acts as a moisture buffer for about 3-5 hours and it does not allow the filter to dry, secondly the moisture content in all the filters with bacterial lawns are brought to the same level before they are dried under controlled conditions (the moisture content left after the filtration process may not be the same). The filter with bacterial lawns is cut in strips of appropriate width (about 1 cm) and fixed on the sample holder with the help of double-sided adhesive tape. The bacterial lawns are allowed to air-dry till a physiologically relevant state [121] is achieved, where only bound water is present on the bacterial surface. This physiologically relevant state is characterised by attainment of a plateau region in the water contact vs drying time curves, this state lasts for 30- 60 mins [23]. The contact angle behaviour after the drop has been positioned on the bacterial lawn is different for polar and apolar liquids. For the apolar liquids contact angle along with the drop volume, height and base diameter stays constant with time under the period of monitoring the drop i.e. 30 seconds. But the contact angle changes with time for polar liquids (shown in Fig. 15). There are three different regions in the 130

3. Surface Characterisation: Paper IV Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001 contact angle vs time curve. In the beginning the contact angle drops till about 0.1 to 0.3 seconds this happened because the drop spreads on the surface as can be inferred from the fact that the drop volume stays constant, the drop height decreases and the base diameter increases. After this stage the drop comes in equilibrium where there is no change in drop contact angle, volume, height or base diameter. The equilibrium stage lasts till about 1-3 seconds. Again after some time the contact angle starts decreasing this is because the polar liquid wets the bacterial lawns and absorption of the liquid takes place. This stage is characterised by steep decrease of volume and height, the drop keeps on spreading as seen from the increase in the drop diameter. The relative spans of the three stages depend on the bacterial lawn thickness and probably on the bacterial strain hydrophobicity. Sharma [16] reported that 800- 900 bacterial layers were required to get a reasonably long equilibrium stage for wild and sulfide mineral adapted Paenibacillus polymyxa. If too less bacterial layers are taken then the spreading can directly merge with the absorption stage and hence no equilibrium stage is available to measure the contact angle value. Though, other authors have also observed this decrease in contact angle after positioning of the drop [125] but they have considered it be an anomaly and have proposed extrapolation of the curve to t=0 in order to account for the stability problems. In our view the initial drop in contact angle takes place for the drop to attain equilibrium and van der Mei [124] are also of the same opinion that it takes about 5-7 seconds for the contact angle equilibrium.

Axisymmetric Drop Shape Analysis-Contact Diameter (ADSA-CD)

Neumann and co-workers has been using the ADSA-CD technique to measure the contact angles on bacterial lawns - Lin [126]; Drumm [127] and Duncan-Hewitt [128]. ADSA-CD is a modified version of ADSA-P, which was developed by Rotenberg and implemented by Skinner [129]. This technique was initially developed to measure low contact angles. The technique requires the contact diameter, the drop volume and the liquid surface tension, the density difference across the liquid-liquid interface, and the gravitational constant as inputs to calculate the contact angle by means of numerical integration of the Laplace equation of capillarity. As the contact angle decreases, the profile of sessile drop becomes increasingly flat about the apex, and the accuracy of directly methods, such as goniometry, is adversely affected. The lack of curvature in the profile also presents a problem for all methods that rely on the profile of a drop to determine the contact angle. The success of drop profile methods is dependent on the existence of comparable surface tension and gravitational effects. For very flat drops, the effect of gravity dominates, and the surface tension has negligible effect on the shape of the interface. Thus, using the shape of the interface to determine the surface tension is not effective method for such experimental situations. Therefore, the ADSA-P technique and goniometry are not suitable. ADSA-CD circumvents this problem by utilising top view of the drop instead of the side view. Essentially, the contact angle is computed by numerically minimising the difference between the volume of the drop, as predicted by Laplace equation of capillarity and the experimentally measured volume. Although, ADSA-CD was originally developed to measure the contact angles of very flat drops but since it uses the top view of the drop, it is found to be very useful for measurement of drops on non-ideal surfaces, which are relatively rough and heterogeneous. It is practically impossible to form axisymmetric drop on such

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3. Surface Characterisation: Paper IV Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001 surfaces. The irregularities in the three-phase contact line are averaged to an average diameter of the drop by a least-square fit of a circle to the experimentally measured points along the three-phase boundary and then an average contact angle is determined. All these facts have made ADSA-CD methodology particularly useful for biological materials e.g. layers of bacterial cells which are necessarily rough and absorb water and other liquids so that the drops sink into the layer of cells, in addition the hydrophilic, bacterial layers produce small time dependent contact angles.

The Contact angle data on bacterial lawns

Ven der Mei and Busscher [23] has compiled a list of contact angles measured on bacterial lawns with Water, Formamide, Methyleneiodide and α- Bromonapthalene. This review provides a reference guide to microbial cell surface hydrophobicity based on contact angles with the above diagnostic liquids and involves Acinebacter, Actinobacillus actinomycetemcomitans, actinomyces, Brevibacterium linens, various Candida species, Capnocytophaga gingivalis, Enterococci, Escherichia coli, lactobacilli, Leuconostoc mesenteroides, peptostreptococci, Porphyromonas gingivalis, Prevotella intermedia, pseudomonads, Serratia marcescens, staphylococci, and streptococci adding up to 142 isolates. Only those strains or species are included in the reference guide where the plateau contact angles with the four diagnostic liquids were available in the literature. This compiled list of contact angles is used along with the contact angles of 5 wild and sulfide mineral adapted Paenibacillus polymyxa [16] cells.

Analysis of the aforementioned approaches

In the following discussion two issues have been addressed independently without mixing them wherever possible: (1) the surface energy and its components for bacterial cell surface and (2) the similarities, dissimilarities and internal consistency of the three approaches namely- Equation of state, Geometric mean and Lifshitz-van der Waals acid/base (LWAB). The contact angles of 147 different isolates with 4 liquids have very different distribution, contact angles with polar and apolar liquids have totally different distribution (Fig. 16). Except the Equation of state approach, the other approaches (Fowkes, Geometric mean and LW-AB) allow that the total surface energy can be divided in two separate parts- the apolar, so called dispersion or Lifshitz van der Waals and polar or acid-base part. The surface energy using the Equation of state approach is evaluated using the conversion tables from contact angle to surface energy by Neumann [130]. Since the conversion is available only at fixed liquid surface tensions therefore, γl=73 is used for Water (72,8), γl=58 is used for Formamide (58), γl=44 is used for α- Bromonapthalene (44,4) and γl=51 is used for Methyleneiodide (50,8). The detailed parameters for the standard liquids are presented in Table VI. Recently Balkenende [131] measured liquid contact angles on FC722 and have reported different surface LW 2 energy parameters for methyleneiodide (γl=50,8 & γl =33,5 mJ/m ) and α- LW 2 Bromonapthalene (γl=43,9 & γl =37,6 mJ/m ), but since they are not universally accepted yet, the widely accepted parameters (Table VI) has been used in the analysis. The contact angle values for the 147 microbial isolates along with the surface energies evaluated by the different approaches have been presented in Table V. The 132

3. Surface Characterisation: Paper IV Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001 total surface energy by Equation of state approach and Geometric mean approach (along with the acid-base component) can be evaluated by water or formamide contact angle and least square method. Similarly either Methyleneiodide or α- Bromonapthalene can be used to evaluate the Lifshitz-van der Waals component of surface energy. But due to lack of space, the surface energies evaluated by using water and α-Bromonapthalene is only presented in Table V. Data evaluated using Formamide and Methyleneiodide is only presented in the figures. For the evaluation of surface energy Excel 97 spreadsheet is used and for the over-determined system of equations, Matlab 5.3 is used.

Contact angle of the bacterial cells with apolar liquids

Majority of the contact angles with the apolar liquids for the bacterial isolates fall in a very narrow region. The contact angle values with Methyleneiodide have a mean of 53o and standard deviation of 9.3; 95% of the contact angles fall between 36- 72o. The contact angle values with α-Bromonapthalene have a mean of 37o and standard deviations of 11.17; 95% values fall between 20-60o. This is evident from Fig. 16 and the cluster formation in Fig. 17a. This means that majority of the bacterial cells have very similar interaction with the apolar liquids.

Lifshitz van der Waals (dispersion) component of surface energy using Fowkes and Equation of state approach:

When the contact angles of bacterial surfaces with apolar liquids are used in the Fowkes equation then we arrive at the dispersion component of the surface energy of the bacterial lawns. These apolar liquids namely Methyleneiodide and α- Bromonapthalene interact with the bacterial cell surfaces through apolar interactions only. Because of the fact that the contact angle values with apolar liquids group together the dispersion component of the surface energy also forms cluster around an average value (Fig. 17a). There is no straight-line correlation between the dispersion energies evaluated from methyleneiodide and α-Bromonapthalene. 95% of the bacterial cells have γLW values in the range of 20 - 40 mJ/m2 with a mean of 32,7 and standard deviations 5,1 when Methyleneiodide contact angle is used. Similarly when α-Bromonapthalene contact angle is used then, 95% of bacterial cell γLW lies between 25 - 45 mJ/m2 with a mean of 35,6 and standard deviation of 4,88 mJ/m2 (Fig. 18). When the contact angles of apolar liquids are used to evaluate the bacterial surface energy by Equation of state approach then it follows the same behaviour as LW the dispersion energy, γs , evaluated by the Fowkes approach (Fig. 17a). The evaluated surface energies form clusters with slightly higher average value than the dispersion energies evaluated by Fowkes approach. 95% of the bacterial cells have γ values in the range of 20 - 40 mJ/m2 with a mean of 34,5 and standard deviations 4,48 when Methyleneiodide contact angle is used. Similarly when α-Bromonapthalene contact angle is used then 95% of bacterial cell γ lies between 30 - 50 mJ/m2 with a mean of 41,5 and standard deviation of 4,98 mJ/m2 (Fig. 18). The average values are in accordance with the observation made by van Oss [80] that the Lifshitz van der Waals component of surface energy of biopolymers is close to 40 mJ/m2 ± 10%. Since the bacterial cells surfaces are composed of biopolymers like Peptidoglycan, Phospholipids (bilayer), Lipopolysaccharides,

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Lipoproteins, Teichoic acid, other proteins etc, bacterial cells also have Lifshitz van der Waals component of surface energy in this range. For the approaches to have internal consistency, it is expected that the apolar component of surface energy of bacterial cell surface evaluated by using two different liquids must be the same, i.e., instead of forming a cluster in Fig. 16a the points are expected to lie on the 45o line. Which does not happen, because the contact angle for different bacterial cells with the two liquids just forms a cluster and does not follow the expected trend by either equation of state or Fowkes approach. The lack of internal consistency is also evident from Fig. 17. The expected correlation between the contact angle with Methylene iodide and α-Bromonapthalene by Fowkes approach (Eq. 14 without the reading pressure term) is evaluated using the following relations and presented in Table VII. First o LW θMethyleneiodide is assumed to be 35, 40, 45…75 , then γs is evaluated and, then θα- Bromonapthalene is calculated. 50,8 γ LW = ()cosθ + 1 (123) s 2 Methyleneiodide

LW 44,4 γ = ()cosθα − +1 (124) s 2 Bromonapthalene Similar values are presented in Table V for Equation of state approach, which is evaluated using the conversion tables [130]. The difference in θα-Bromonapthalene values for the two approaches is in the range of 5.46o to 3.32o. Fig.17 b correlates the surface energies evaluated using the two approaches for different bacterial strains. The curves are plotted for Methyleneiodide and α- Bromonapthalene contact angles. Equation of state approach consistently gives higher surface energy than Fowkes approach by 5.24 mJ/m2 for the whole range of α- Bromonapthalene contact angles. The energies evaluated are well correlated by a straight line fit with a slope of 1.02 (45.56o) and an intercept of 5.42 mJ/m2 on the Equation of state axis. Surface energies evaluated by the two approaches using Methyleneiodide contact angle also correlate well with each other by a straight line fit. The Intercept of the straight line fit is 5.76 mJ/m2, i.e., similar to the case of α-Bromonapthalene but now the dispersion energy evaluated from Fowkes approach increase at a slower rate than the surface energy evaluated by Equation of state approach (slope =0.87 i.e. 41o). The reason for this difference from the case of using α-Bromonapthalene contact angle becomes clearer from Fig. 19. Plots of surface energy vs contact angle for two different approaches and the two contact angles are shown in Fig. 19a. The plots fit very well with polynomials of 3rd degree w.r.t θ. We can see that when Equation of state approach is used then the surface energies evaluated from contact angles with two different apolar liquids follow exactly the same curve, though the individual points do not overlap. But this is not true for γLW, as evaluated from Fowkes approach, plot with respect to contact angles of two different apolar liquids. The γLW vs α-Bromonapthalene contact angle plot is almost parallel to the γ vs contact angle plots for Equation of state approach, with an offset of 5.24 mJ/m2 (Same as Fig. 17b). The γLW Vs Methyleneiodide contact angle plot has totally different slope. At low contact angles (< 40O) when Fowkes approach is used the Methyleneiodide contact angles give similar γLW as the Equation of state surface energies and at high contact angles the evaluated γLW comes close to the γLW Vs α-Bromonapthalene contact angle plot.

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Plots of γBacterial cell surface Vs cosθ for the two approaches and the two different LW apolar liquid contact angles are shown in Fig. 19b. As expected the γ Fowkes vs cosθ plots follow the quadratic relation of type Eq. 14 without the spreading pressure term, which can also be written as γ γ γ γ LW = l + l cosθ + l cos2 θ (125) s 4 2 4 LW Since γl =γl for apolar liquids, the actual second degree polynomial fits of LW γFowkes vs cosθ for two different liquids are: γ LW = + θ + 2 θ Fo 12,7 25,4cos M 12,7cos M for Methyleneiodide (125a) γ LW = + θ + 2 θ α Fo 11,1 22,2cos Br 11,1cos Br for -Bromonapthalene (125b)

Surprisingly the plots for γEquation of state vs cosθ also follow the quadratic relation of the type 125, the quadratic fit for the plots are- γ LW = + θ + 2 θ Eq.st 16,6 23,8cos M 9,53cos M for Methyleneiodide (125c) γ LW = + θ + 2 θ α Eq.st 16,7 21,95cos Br 11,9cos Br for -Bromonapthalene (125d) Surprisingly the coefficients of cosθ and cos2θ in both the equations- 125c and LW 125d are similar to the equation 125b (γFowkes vs cosθα-Bromonapthalene in Fig. 19b). But these equations give the value of γ, higher by 5,5 mJ/m2 than γLW evaluated by Eq. 125b. LW The γFowkes vs cosθMethyleneiodide plot follows the quadratic fit (Eq. 125a), as it should, but the coefficients of cosθ and cos2θ are very different from Eqs. 125b, 125c and 125d, hence the slope of the curve is very different from the others. The plots of Fig. 19b can also be interpreted as reverse Zisman plots. Instead LW of plotting γl vs cosθ for different liquids on a solid surface, here γs vs cosθ is plotted for one liquid on many solids. The quadratic fits cross cosθ=1 i.e. θ = 0 line at LW a critical γs . The liquid will give zero contact angle for the bacterial cell surface (or LW LW solid surface) which have the critical γs . These γs values are not measured but calculated ones using the two approaches therefore, the two approaches are compared LW based on this critical γs . According to the Fowkes approach the apolar liquids will spread on that LW LW bacterial cell surface which have the same γs as the liquid surface tension (γl=γl ). LW Therefore, Methyleneiodide will spread on the bacterial cell surface with γs =~51 2 2 mJ/m (γl=50,8 mJ/m ) and α-Bromonapthalene will spread on the bacterial cell LW 2 2 surface with γs =~44 mJ/m (γl=44,4 mJ/m ). When Equation of state approach is used then irrespective of the liquid used LW 2 the critical γs is the same, i.e., ~50 mJ/m . If the analysis with two liquids can be generalised then we can say that all the apolar liquids will spread on the bacterial cell LW 2 surface with γs =~50 mJ/m . Since equation of state approach does not believe on the division of surface energy into different components Although the γ vs θ (Fig. 19a) and cosθ vs γ (Fig. 19b) curves for equation of state approach overlap each other, the surface energy evaluated by the two liquids are different. This is evident from Fig. 18 where the distribution of γ is different for the two liquids. There are few points, which become very clear by the above analysis of the bacterial cell surface energy evaluated by the two approaches and when contact angle with apolar liquids are used.

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1. 147 microbial cell surfaces have very similar contact angles to each other with an apolar liquid. This is same for the two-tested apolar liquids. This indicates that the LW Lifshitz van der Waals component of their surface energy (γs ) is not very different from each other and lies in the vicinity of 35-40 mJ/m2 (Fig.s 16 & 18). 2. Although, the proponents of equation of state approach reject the division of solid surface energy into components, but when apolar liquid contact angles are used then the equation of state approach evaluates the surface energy very close to the LW Lifshitz van der Waals component of surface energy (γs ) evaluated by the Fowkes approach. The difference is only 0 to 6 mJ/m2 depending on the liquids used (Fig. 17b). 3. When α-Bromonapthalene is used than the surface energy obtained from equation LW of state approach is consistently higher then the γs by Fowkes approach by 5-6 mJ/m2 (Fig. 17b). 4. The proponents of equation of state approach have totally refuted the use of the geometric mean for combining the components of solid-liquid surface energies. But the γ vs cosθ plots follow a quadratic relation of the type Eq. 125 which originates from geometric mean and is very different from the proposed Eq. 60 or 71 or 76 (Fig. 19b).

Contact angle with polar liquids-Water and Fomamide: Fig. 16 shows the distribution of water and formamide contact angles on the bacterial cell surface. As can be seen the contact angle values are spread over a large range from close to zero to about 120o. Since water and formamide interact with the bacterial cells both by polar and apolar interactions we can say that the polar characteristics of different bacterial cells is very different, as we have already seen that the apolar characteristics of bacterial cells are not very different from each other.

Total and acid-base (polar) component of surface energy evaluated by Geometric mean and Equation of state approach:

Geometric mean approach needs contact angle values with one polar and one apolar liquid in order to give the polar and apolar component of the solid surface energy. The data available is with 2 apolar liquids (water and Formamide) and 2 apolar liquids. As seen before, the apolar-Lifshitz van der Waals component of the surface energy is found using Fowkes approach and we get different values when the contact angle values with two different apolar liquids are used. The polar or acid-base component of the surface energy can be evaluated using either water or formamide LW contact angle. For further analysis the apolar component γs of the bacterial cell surface evaluated by using α-Bromonapthalene contact angle is used, which have an average value of 35.6 mJ/m2. Apart from using the liquid contact angles pair-wise, the acid-base and Lifshitz-van der Waals component of solid surface energy are evaluated by solving the over-determined system of four equations by Least square method. Fig. 20a shows the total and acid-base component of the surface energy for the 147 bacterial isolates decreases as a function of water contact angle. The acid-base component of the surface energy for the bacterial cell surface varies between 0 to 50 mJ/m2 (can also be seen from Fig. 34b). γAB vs θ plot follows a straight relation till the contact angle of 90o after which γAB approaches zero value and becomes plateau, i.e., the straight-line behaviour does not continue into negative values of γAB. The γLW values are distributed between 30-40 mJ/m2 and are independent of the water contact

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3. Surface Characterisation: Paper IV Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001 angle. Total surface energy, γTotal, which is the sum of acid-base component and the Lifshitz-van der Waals component, follows the same behaviour as γAB vs θ and range between 75 to 35 mJ/m2. For the bacterial cells having water contact angle above 90o the acid-base component is zero and the total surface energy is entirely due to apolar contribution. Fig. 21a shows that the total and acid-base component of the surface energies for the 147 bacterial isolates decrease as a function of formamide contact angle. The acid-base component of surface energy for bacterial cells range from 0 to 30 mJ/m2. For the bacterial cells having formamide contact angle above 60o, the acid-base component of surface energy approaches zero. The Lifshitz-van der Waals component of surface energy is independent of the formamide contact angle. The total surface energy also follows the same trend as the acid-base component and ranges between 35 to 60 mJ/m2 and after a formamide contact angle of 60o, it is entirely composed of the Lifshitz-van der Waals component of surface energy. As expected the γTotal and γAB evaluated by Geometric mean approach follows a quadratic relation with cosθ like Eq. 28. The acid/base (polar) component of surface energy for the bacterial cell surface follow a relation of the type 126 γ 2  γ 2 γ γ LWγ LW  γ AB = l cos2 θ +  l − l s l cosθ + s 4γ AB  2γ AB γ AB  l  l l   γ 2 γ LWγ LW γ γ LWγ LW   l + s l − l s l  (126)  4γ AB γ AB γ AB   l l l  LW where γs is previously know from the Fowkes approach. For further calculations the LW 2 γs value is taken to be an average of 35.6 mJ/m for α-Bromonapthalene contact angle. LW Using Eq. 126, average γs and the liquid parameters the coefficients for cos2θ, cosθ and the constant can be evaluated. For water the coefficients are 25.97, 12.19 and 1.42; for Formamide the coefficients are 44.26, -25.21 and 3.58 respectively. The total surface energy is evaluated using two contact angles one with polar liquid (water or formamide) and one apolar liquid therefore the relation for total surface energy is the same as Eq. 126 but higher by the Lifshitz van der Waals part of the solid surface energy (Eq. 127) γ 2  γ 2 γ γ LWγ LW  γ Total = l cos2 θ +  l − l s l cosθ + s 4γ AB  2γ AB γ AB  l  l l   γ 2 γ LWγ LW γ γ LWγ LW   l + s l − l s l  + γ LW (127)  4γ AB γ AB γ AB  s  l l l  2 AB Therefore, the expected coefficients for cos θ, cosθ remain the same as for γs but LW 2 the constant will higher by the γs value (on an average by 35.6 mJ/m ). AB The real quadratic fit for γs Vs cosθW is as follows γ AB = + θ + 2 θ GM 1,4 13cos W 25,4cos W (126a) with R2 of 0.937 and S.D. of 3.25. The R2 statistics indicates that the model (second- AB degree polynomial fit) explains 93.7% of the variability in γs and S.D. gives the standard deviation of the experimental points from the model. AB The quadratic fit for γs vs cosθF is as follows

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γ AB = − θ + 2 θ GM 5,9 29cos F 46cos F (126b) with R2 of 0.514 and S.D. of 5.17 Total The quadratic fit for γs vs cosθW are as follows γ Total = + θ + 2 θ GM 37,5 12,8cos W 24,8cos W (127a) with R2 of 0.973 and S.D. of 2.01 Total The quadratic fit for γs vs cosθF are as follows γ Total = − θ + 2 θ GM 37,9 22,5cos F 43,8cos F (127b) with R2 of 0.914 and S.D. of 1.82 The coefficients of the real fit are close to the expected coefficients, difference LW is due to the fact that the expected coefficients are calculated by using an average γs value of 35.6 mJ/m2. Total Figure 24a shows the plots of γs vs θ evaluated by the Geometric mean approach using Water and Formamide contact angles. The figure also contains the surface energy evaluated by Equation of state approach and by using water and formamide contact angle. A clear conclusion which can be drawn from the plots is that when contact angle of polar liquids are used for evaluation of surface energy of Total bacterial cell surface than the values are similar to the γs evaluated by that particular polar liquid contact angle by the Geometric mean approach. This conclusion is valid for water contact angles below 70o and for formamide contact angles of 55o. Figures 22a and 23a show the bacterial surface energy evaluated by using equation of state approach and contact angle values with polar liquids. They are Total Total designated as γ because the surface energies are similar to the γs evaluated by the geometric mean approach. Since Lifshitz-van der Waals component of the surface energy is already known from apolar liquid contact angles, acid-base component of surface energy can be evaluated from the difference γTotal-γLW, which is also plotted in the figures. Fig. 22a shows that the total surface energy evaluated decreases with the water contact angle and ranges in between 20 to 70 mJ/m2 when equation of state approach was followed. The marked difference from geometric mean approach is that the total o surface energy evaluated, for θw > 66 , is less than the Lifshitz-van der Waals component of the surface energy and hence gives rise to negative acid-base component. The acid-base component of surface energy ranges in between -25 to 40 mJ/m2. Fig. 23a shows the total surface energy evaluated by Equation of state approach decreases with the formamide contact angle and ranges in between 20 to 57 mJ/m2. Above the formamide contact angle of 50o, the total surface energy evaluated by formamide contact angle is less than the lifshitz-van der Waals component of surface energy evaluated using α-Bromonapthalene contact angle. Hence, the acid- base component is negative after 50o, ranging between -20 to 25 mJ/m2. Surprisingly the total surface energy evaluated by Equation of state approach also follows a quadratic relation when γ is plotted with cosθ (Fig. 22b; 23b) Total The second degree polynomial fit for the γs vs cosθW is as follows γ Total = + θ + 2 θ Eq.st 28,7 33,3cos W 9,4cos W (127c) with R2 of 0.998 and S.D. of 0.597 Total The second degree polynomial fit for the γs vs cosθF is as follows γ Total = + θ + 2 θ Eq.st 20,6 22,9cos F 14cos F (127d)

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3. Surface Characterisation: Paper IV Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001 with R2 of 0.999 and S.D. of 0.13 AB Total LW The γs , which is equal to γ -γ evaluated from equation of state approach using polar and apolar liquid contact angles, also follows a quadratic fit with respect to cosθ. AB The second degree polynomial fit for the γs vs cosθW is as follows γ AB = − + θ + 2 θ Eq.st 13,3 33,5cos W 10,2cos W (126c) with R2 of 0.895 and S.D. of 5.03 AB The second degree polynomial fit for the γs vs cosθF is as follows γ AB = − + θ + 2 θ Eq.st 17,8 18,7cos F 14cos F (126d) with R2 of 0.621 and S.D. of 4.85 Total The second-degree polynomial fits for the γs vs cosθ evaluated by equation of state approach are very good which is clear from the R2 values above 0,99. Whereas when Geometric mean approach is used then the second-degree polynomial fits have R2 values in the range of 0,91 to 0,97. This clearly shows that the quadratic Total fit explains 99% of the variability of γs with respect to cosθ even when the equation of state approach is used. As mentioned earlier Fig. 24a shows that the total surface energy evaluated by Geometric mean approach is very similar to the surface energy evaluated by the equation of state approach using polar liquid contact angles. This is evident from Fig. 38a. When formamide contact angle is used, then, the total surface energy evaluated Total Total by the two approaches is same as the γs vs θ and γs vs cosθ overlap shown in Fig. 24a and 24b respectively. This similarity is also evident from Fig. 25a where the correlation between the total surface energy evaluated by the two approaches follow 45o line. This similarity continues till the formamide contact angle of 55o, where the Total 2 o Total γs has reached about 40 mJ/m . Above 55 formamide contact angle the γs evaluated by the two approaches start to become different. For equation of state Total approach γs continues to drop but for Geometric mean approach it almost becomes constant at 35.6mJ/m2 because after this point total surface energy is composed entirely by the Lifshitz-van der Waals component and acid-base component approaches zero. When water contact angle is used than the Geometric mean approach gives the total surface energy similar to the one given by Equation of state approach but about 2,5 mJ/m2 higher. For water contact angle higher than 70o the difference between the surface energies evaluated by the two approaches increases. The surface energy 2 o evaluated by Geometric mean approach is 45 mJ/m at θW ≈ 70 and after that it gradually decrease to a value of 35.6 mJ/m2 and stays constant. At higher water contact angle the surface energy is entirely composed by Lifshitz-van der Waals component and acid-base component contribution approaching zero. On the other hand the total surface energy evaluated by Equation of state approach decreases o continuously after θW ≈ 70 hence, giving rise to negative acid-base component. This is also evident from Fig. 25a where, the plot for surface energy evaluated by the two approaches is parallel to the 45o line and follow a relation of Eq. 128, untill the surface energy value of 45 mJ/m2 and then it curves and becomes horizontal. γ Total = + γ Total GM ;W 2,5 Eq.st;W (128) In the whole range of surface energy the total surface energies evaluated by the two approaches follow a third degree polynomial relation-Eq. 129. γ Total = − γ Total + ()γ Total 2 − −4 ()γ Total 3 GM ;W 49,7 1,41 Eq.st;W 0,04 Eq.st;W 2,25X10 Eq.st;W (129)

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Fig. 25b correlates the acid-base component of surface energy evaluated by the two approaches. For formamide contact angle, the acid-base component evaluated by the two approaches follow Eq. 130. This means that the acid-base component evaluated by the two approaches is similar but the one evaluated by Equation of state approach is about 6 mJ/m2 lower than the one evaluated by Geometric mean approach. Because of this, Equation of state approach gives rise to negative acid-base component of surface energy. γ AB = + γ AB GM ;Fo 6 Eq.st;Fo (130) The acid-base component of surface energy from water contact angle and evaluated by the two approaches is also correlated in Fig. 24b. In the whole range, the points follow a third degree polynomial (Eq. 131). But a straight line relation of Eq. 132 can correlate most of the points in the range of 10-37 mJ/m2, which means that the acid-base component evaluated by Equation of state is about 9 mJ/m2 lower to the one evaluated by Geometric mean approach. γ AB = − γ AB + ()γ AB 2 − −4 ()γ AB 3 GM ;W 9,57 0,84 Eq.st;W 0,012 Eq.st;W 2,99X10 Eq.st;W (131) γ AB = + γ AB GM ;W 9 Eq.st;W (132) Total Fig. 24b shows the plot of γs Vs cosθ for water and formamide contact Total angles. If we consider these plots as reverse Zismann plots and since the γs is evaluated by using Equation of state and Geometric mean approach, we can compare the two approaches based on this. When water is used for measuring contact angle on the bacterial surfaces, then according to Equation of state approach water will spread or give zero contact angle for a bacterial cell surface having the surface energy of 71.4 mJ/m2 (and higher), which is very close to the water surface tension. According to the geometric mean approach water will spread on the bacterial cell surface with surface energy of 75 mJ/m2 (and higher) which, is close to the water surface tension but higher by 3 mJ/m2. The difference can be due to the fact that Equation of state approaches needs only one (water) contact angle to evaluate the surface energy but Geometric mean approach needs two contact angle values. Geometric mean approach evaluates the total surface energy of the bacterial cell surface by adding the Lifshitz- van der Waals component (evaluated from apolar liquid contact angle) and acid-base component (evaluated from polar liquid contact angle) hence, the errors in the contact angle measurement propagating to the final surface energy value is much higher. Total When formamide contact angle is used, the γs vs cosθ plots by the two approaches overlap each other. According to Equation of state approach formamide will spread on the bacterial cell surface having a surface energy of 57.5 mJ/m2 or more, whereas according to Geometric mean approach formamide will spread on bacterial cells with surface energy of 59.2 mJ/m2 and more. Both the values are quite close to each other and are very close to the formamide surface tension of 58 mJ/m2. Figure 26 shows the correlation between the surface energy evaluated by different liquids but by using the same approach. The total surface energy evaluated by water contact angle does not correlate to the one evaluated by formamide contact angle, this is true for both the approaches. If the approaches were internally consistent then the surface energies evaluated by them should be the same irrespective to the liquid contact angle used. The acid-base component of the surface energy also follows the same trend. The acid-base component evaluated by Geometric mean approach confines itself in the first quadrant with all positive values but acid-base component o o evaluated by Equation of state approach give negative values for θW > 70 or θF > 50 . This elucidates another shortcoming of the Equation of state approach.

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Polar and dispersion component of surface energy using Least square method to fit the contact angle data with 4 liquids to Geometric mean approach:

The matrix in Eq. 33 is solved for all the 147 microbial surfaces. This method gives both the polar and dispersion component of the surface energy. Fig. 18 shows the distribution of the dispersion component of surface energy for the 147 microbial isolates. It can be seen clearly the distribution of is similar to the distribution of dispersion energy evaluated by the Fowkes approach with the use of Methyleneiodide liquid contact angle. This become more obvious in Fig. 27a where the dispersion energy evaluated by the least square method correlates well with the dispersion energy evaluated by Fowkes approach using Methyleneiodide and the data points fall on the 45o line. The dispersion energy evaluated by Fowkes approach using α-Bromonapthalene contact angles are higher than the ones obtained by the least square method (also seen from Fig. 18) and hence the points do not fall on the 45o line, but correlate well. Fig. 27b correlates the acid-base component obtained by the least square method to the ones obtained by solving pair-wise equation from water/α- Bromonapthalene and formamide/α-Bromonapthalene. The least square method evaluates the acid-base component similar to the ones obtained from pair-wise solution using water and α-Bromonapthalene as the points lie on the 45o line. There is no correlation between the acid-base component obtained from least square method and pair-wise solution using formamide and α-Bromonapthalene. This is also evident from the distribution diagram in Fig. 34b where the distribution of acid-base component of surface energy evaluated by least square method is very similar to the one obtained by pair-wise solution using water and α-Bromonapthalene, but very different from the one obtained by pair-wise solution using formamide and α- Bromonapthalene. Fig 27c correlates the total surface energy obtained by the least square method to the ones obtained by solving pair-wise equation from Water/α-Bromonapthalene and Formamide/α-Bromonapthalene. Total surface energy evaluated by least square method is similar to the ones obtained by pair-wise solution using water and α- Bromonapthalene, although the data points follow the straight line fit for Eq. 128, which crosses the 45o line at 40 mJ/m2 but we can see the all the data points lie very close to the 45o line. There is no correlation between the total surface energy obtained from least square method and pair-wise solution using formamide and α- Bromonapthalene γ Total = γ Total − GM ;W ,α −Br 1,185 GM ;LeastSq. 5,63 (133) The above analysis brings out few important points, which needs to be highlighted.

1. Equation of state approach and Geometric mean approaches are not internally consistent. They evaluate different surface energies when contact angle with different liquids are used (Fig. 26). 2. When water contact angle is used, the Equation of state approach gives similar surface energy values as Geometric mean approach with a difference of 2-3 mJ/m2 in the contact angle range of 16-70o and only at higher contact angles the difference becomes larger (Fig. 24a; 25a).

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3. When formamide contact angle is used then both the approaches evaluate the same surface energy values in the contact angle range of 7-55o and only above this the difference in surface energy values evaluated by the two approaches increase (Fig. 24a; 25a). 4. When water contact angle is used then the 147 bacterial cell isolates have surface energy values of about 75-35 mJ/m2 when Geometric mean approach is followed. But when Equation of state approach is used, then the value ranges from about 70- 20 mJ/m2. 5. When formamide contact angle is used then the bacterial surface energy ranges from about 60-35 mJ/m2 in Geometric mean approach. But when Equation of state approach is used, it ranges between about 60-20 mJ/m2. 6. The proponents of equation of state approach have totally refuted the use of the geometric mean for combining the components of solid-liquid surface energies. But the γ Vs cosθ plots follow a quadratic relation of the type Eq. 127 which originates from geometric mean and is very different from the proposed Eq. 60 or 71 or 76 (Fig. 19b, 24b). Although the coefficients for cos2θ, cosθ do not correspond to the expected ones (From Geometric mean approach) but the second degree fit of γ Vs cosθ for Equation of state approach is better than that for Geometric mean approach itself. 7. When the least square method is used to fit the geometric mean to the contact angles of Water, Formamide, Methyleneiodide and α-Bromonapthalene, then the total and acid-base component of the 147 microbial isolates is similar to the ones obtained from Water/α-Bromonapthalene pair and the Lifshitz.van der Waals component is similar to the one obtained by using Methyleneiodide (Figs. 27a, b, c).

Polar liquid contact angles analyses by LW-AB approach:

The Lifshitz-van der Waals/acid-base (LW-AB) approach uses equation 112 to evaluate the surface energy of the bacterial cell surface. This equation has three unknowns and hence needs three simultaneous equations to be solved. As discussed before it is preferable that contact angle with one apolar liquid and two polar liquids are to be used. The Lifshitz-van der Waals component of surface energy is evaluated by using the Fowkes approach (Eq. 14 without the spreading pressure term) and contact angle with apolar liquid. The acid-base component and the electron-donor/electron-acceptor characteristics are evaluated by using the two polar liquid contact angles. The data available is with water and formamide liquids, which is good because this pair gives stable results as analysed in Table IV. Figure 28 plots the experimentally measured water contact angles Vs the formamide contact angle for 147 bacterial isolates. As can be seen that the contact angles are scattered and do not follow a well defined behaviour. The figure also contains the curves showing expected correlation between θW and θF by Equation of state and Geometric mean approach along with the θW vs θF scatter expected by LW- AB approach. The expected contact angle of water and formamide are presented in Table VIII. An average value of 35.57 mJ/m2 is assumed for γLW and then the γAB is assumed from 1 to 26 mJ/m2. Equation 23 is solved using the solid and liquid surface energy parameters and, cosθ and then θ are determined for Geometric mean approach.

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For Equation of state approach the total surface energy is assumed in the region 20 to 57 mJ/m2 and the water and formamide contact angle are read from the conversion table [130] LW The θW vs θF scatter for LW-AB approach is generated by first assuming γ to be 35.57 mJ/m2. The electron accepting and electron-donating parameters are then alloted values between 1.8x10-5 to 11 and 0.01 to 80 respectively. By combining γ+ − and γ the acid-base component is evaluated (Eq. 107) and then equation 112 is solved using the liquid surface energy parameter for water and formamide to obtain Water and Formamide contact angles. The values obtained are presented in Table IX. Similar approach has been used by de Meijer [132] to correlate the water and formamide contact angles. It is clear from Fig. 28 that the contact angle of the two liquids does not follow the expected curve that is expected by Equation of state approach and the Geometric mean approach. But the scatter predicted by the LW-AB approach almost covers the scatter of the two contact angles. The incomplete coverage may be due to the fact that the scatter of contact angle values using LW-AB approach are generated using the γLW of only 35,57 mJ/m2. This shows that the LW-AB approach can explain the natural behaviour of the water and formamide contact angles. The LW-AB approach evaluates the electron-donor, electron-acceptor and acid-base component of the surface energy of the bacterial cells using water and formamide contact angle. Fig. 29 correlates the aforementioned parameters to water contact angle. There is no straightforward correlation between the parameters and water contact angle but the random scatter of the different parameters vs θW is overlapped by the theoretically estimated behaviour for the parameters vs θW (Table IX). Similarly Fig. 30 correlates the formamide contact angle to the evaluated parameters, in this case also the theoretically generated scatter at γLW = 35.57 mJ/m2 (Table IX) overlaps the scatter in the plots. The LW-AB approach needs contact angles with two polar liquids and one with apolar liquid. For the 147 bacterial isolates, contact angle data is available with two polar liquids and two apolar liquids therefore the internal consistency of LW-AB approach can be checked only by changing the apolar liquid contact angle. Fig. 30 shows the correlation of surface energy parameters - γ-, γ+, γAB and γTotal when they are evaluated using Water, Formamide and either Methyleneiodide or α- Bromonapthalene contact angles. Fig. 31 shows the different surface energy parameters evaluated by LW-AB approach using two different apolar liquids contact angle. Fig. 31a shows the electron-acceptor (γ+) characteristics of microbial cells. The value of γ+ ranges from 1,8x10-5 to 15 mJ/m2, more than 90% of microbial cells have the γ+ value within 1,8x10-5 to 5 mJ/m2 (Inset figure). The correlation of the γ+ evaluated by using Methyleneiodide or α-Bromonapthalene contact angles along with water and formamide does not follow the 45o line but is a scatter with a straight line fit with a slope of 0.74 − Fig. 31b shows the electron-donor (γ ) characteristics of the microbial cells. − Values of γ range from 0.03 to 105 mJ/m2. For a microbial cell surface the value of − γ does not depend on the apolar liquid used in the calculation, which is evident from − the fact that all the data points lie on the 45o line when γ values evaluated using Methyleneiodide are plotted against the ones evaluated using α-Bromonapthalene. − For the 147 microbial isolates under study, the γ parameter lie in two different − groups. For about 38% of the microbial cells the γ parameter lie between 0.03-30 143

3. Surface Characterisation: Paper IV Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001 mJ/m2 and for the other 57% it lies between 40-70 mJ/m2 with a very less number of − microbial isolates having the γ parameter between 30-40 mJ/m2 (inset figure). This clearly shows that the 147 isolated have two groups with different types of surfaces. Fig. 31c shows the polar component i.e. acid-base component of the surface energy of the microbial cells. The values of γAB range from 0.06-70 mJ/m2 but more than 97% of the microbial cells have the γAB between 0.06-30 mJ/m2 (Inset figure). The correlation of γAB values evaluated using Methyleneiodide and α- Bromonapthalene show that the data points are scattered and follow a straight line fit with a slope of 0,82. Fig. 31d shows the total surface energy of the microbial isolates evaluated by the LW-AB approach. The total surface energy for the 147 microbial isolates range between 30 to 100 mJ/m2 but more than 98% of the microbial cells have the surface energy between 30-60 mJ/m2 (Inset figure). The correlation of γTotal evaluated by using Methyleneiodide and α-Bromonapthalene is a scatter, which follows a straight- line fit of Eq. 134. γ Total = + γ Total LW − AB;Br 7,43 0,85 LW − AB;M (134) − The presence of two peaks in the distribution of γ parameter for 140 bacterial isolates (Inset of Fig. 31b) is further investigated in Fig. 32, where the distribution of − γ parameter is plotted against frequency in form of numbers. Out of the 140 bacterial isolates 29 are gram-negative and the other 111 are gram-positive. The distribution of − γ parameter for gram-positive, gram-negative and overall 140 bacterial isolates is plotted separately in Fig. 32. If we consider the overall distribution then 35.7% of the − − bacterial isolates have the γ parameter between 0-25 mJ/m2 and 57.9% have γ parameter between 35-65 mJ/m2. Gram-positive bacterial cells have the same behaviour as the overall distribution, i.e., having two separate groups, with 38.7% in the range 0-25 mJ/m2 and 54.9% in between 35-65 mJ/m2. But for the gram-negative − bacterial cells 69% have the γ parameter between 35-65 mJ/m2 and only 24.1% in between 0-25 mJ/m2. This shows that most of the gram-negative bacterial isolates − have higher γ characteristics. The X-ray photoelectron spectroscopic (XPS) surface composition for 116 out of the 140 bacterial isolates is available from van der Mei [23]. Fig. 32 shows the distribution diagrams for N/C, O/C and P/C ratios for 116 bacterial isolates. Out of these 116, 26 are gram-negative and 90 are gram-positive bacterial strains. Fig. 33a shows the distribution of nitrogen to carbon ratio (N/C) for 90 gram- positive, 26 gram-negative and overall 116 bacterial isolates. The N/C ratio for the 116 bacterial isolates lie in between 0.026-0.199 and the distribution is similar to the − distribution of their γ parameter in Fig. 32 with two groups of bacterial isolates having different nitrogen to carbon ratio on their surface. 67,3% of the bacterial cells have 0.026 < N/C ≤ 0.105 and 32,7% have 0,105 < N/C < 0.199. About 59% of gram- positive bacterial cells have 0.026 < N/C ≤ 0.105 and 41% have 0.105 < N/C < 0.199. But 96% of gram-negative bacterial isolates have the 0.026 < N/C ≤ 0.105 and only 4% have 0.105 < N/C < 0.199. This shows that most of the gram-negative bacteria have a lower nitrogen content on their surface, whereas equal proportion of gram- positive bacterial cells which have high and low nitrogen concentration on their surface. This delineates a clear difference in the surface composition of gram-positive and gram-negative bacterial isolates under investigation. Out of all the surface polymers nitrogen is present in the peptodiglycan and proteins [23,133]. In general gram-negative cell surface lacks peptidoglycan which could have got reflected for the gram-negative cells under investigation. But the other reason can be that they have 144

3. Surface Characterisation: Paper IV Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001 lower protein content on their surface whereas gram-positive cells have both high and low protein or peptidiglycan content on its surface. Fig. 33b shows the distribution of oxygen to carbon ratio (O/C) for gram- positive, gram-negative and overall 116 bacterial isolates. The O/C ratio for the 116 bacterial isolates lie in between 0.203-0.655 and the distribution of the oxygen to carbon ratio shows the presence of two different groups but the demarcation is not so clear. For the 116 bacteria along with the 90 gram-positive bacteria have the O/C ratio distributed in between 0.203-0.705, but most of the gram-negative bacteria (> 96%) have the O/C between 0.203-0.55. Here also we can see that most of the gram- negative bacteria have lower oxygen content on their surface as compared to gram- positive bacteria. Fig. 33c shows the distribution of phosphorus to carbon ratio (P/C) for the gram-positive, gram-negative and overall bacterial isolates. The P/C ratio for the 116 bacterial isolate lie in between 0-0.05 and the distribution shows a presence of 3 groups with different phosphorus contents on their surfaces. Gram-positive bacterial isolates also have the P/C ratio in the range 0-0.05, but 26 gram-negative bacterial cells have the P/C ratio in the range 0.002-0.027. Out of all the surface polymers phosphorus is present in Teichoic acid (Gram +) and Lipopolysaccharides (Gram −) [23,133]. − The plots inset in Fig. 33 a to 33 c show the correlation between the γ parameter of gram-negative bacterial cells to N/C, O/C and P/C ratios respectively as obtained by XPS and taken from van der Mei [23]. From the inset plots we can see that there is no direct correlation between the surface composition of gram-negative − bacterial cells (in form of N/C, O/C and P/C ratios) to the γ parameter of the surface energy.

From the above analysis of LW-AB approach we can conclude that:

1. The 98% of the 147 microbial isolates under investigation have total surface in between 30-60 mJ/m2. The acid-base component varying between 0.06 to 30 mJ/m2 (Fig. 31 c, d). 2. Most of the microbial cells (99%) have low electron-accepting characteristics between 1,85x10-5 to 10 mJ/m2. But the microbial cells have from very low (0.03) to very high (105) electron-donating characteristics (Fig. 31 a, b). 3. Unlike Equation of state and Geometric mean approach, the LW-AB approach with Water and Formamide contact angles on bacterial cell surface are expected to form a scatter and not to follow a well-defined polynomial fit. This is actually observed in the experimentally measured water and formamide contact angle on 147 microbial cell surfaces (Fig. 28). 4. When the electron-accepting, electron-donating, acid-base component and total surface energy of bacterial cells are correlated to either water or formamide contact angle, then they give rise to a scatters which are overlapped by the theoretically generated scatters for LW-AB approach (Fig. 29, 30). 5. The electron-donating parameter of bacterial cells remains unchanged when the apolar liquid for contact angle is changed for evaluation of surface energy by LW- AB approach (Fig. 31b). 6. Electron-accepting parameter, acid-base component and the total surface energy are different when the apolar liquid for contact angle is changed (Fig. 31 a, c, d).

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7. The 140 bacterial isolates under investigation can be divided in two groups having different surface properties, in form of their electron-donating characteristics (inset Fig. 31b). 8. The gram-negative bacterial cells have predominantly higher electron-donating parameter between 35-65 mJ/m2, whereas some gram-positive bacterial cells have high (35-65 mJ/m2) and some low (0-25 mJ/m2) electron-donating parameter of surface energy (Fig. 32). 9. Although there is no direct correlation of the electron-donating parameter of gram-negative bacterial cells surface composition (XPS), the gram-negative bacterial cells have lower nitrogen, oxygen and phosphorous content on their surface (Fig. 33).

Comparison of LW-AB approach to Equation of state and Geometric mean approach: Figures 34a and b show the distribution of the total surface energy and acid- base component respectively, evaluated using LW-AB approach with water, formamide and α-Bromonapthalene contact angles, while using equation of state and geometric mean approaches with water and formamide contact angles. For the 147 microbial isolates when water contact angle is used, the total surface energy evaluated by both equation of state and geometric mean approach is distributed over a wide range (~10-80 mJ/m2), but when formamide contact angle is used then the surface energy is distributed only between ~30-60 mJ/m2 (34a). Total surface energy evaluated using LW-AB approach varies in between ~30-60 mJ/m2 and the distribution overlaps to the distribution for equation of state and geometric mean approach using formamide contact angle. The distributions again highlight the internal inconsistency of equation of state and geometric mean approaches but shows the similarity between the two approaches when the same polar liquid contact angle is used. The distribution diagrams of acid-base component (Fig. 34b) show that when LW-AB and geometric mean approaches are used then minimum value obtained is zero and there are no negative values, whereas with equation of state approach negative values are obtained. When water contact angle is used with equation of state and geometric mean approaches, the γAB is spread over a wide range (0-48.9 for geometric mean and -24.2 to 44.2 for equation of state approach). But when formamide contact angle is used for the two approaches then the γAB is spread in narrow range (0-25 for geometric mean and -20 to 22 for equation of state approach). The distribution of γAB for the 147 microbial isolates obtained by LW-AB approach is similar to the distribution of γAB obtained by using formamide contact angle and other two approaches. Figure 35 correlates the total surface energies for the 147 microbial isolates obtained by LW-AB approach to the ones obtained by geometric mean and equation of state approaches when water contact angle is used for the later (Fig. 35a) and when formamide contact angle is used for the later (Fig. 35b). We can see that the surface energies obtained by LW-AB approach does not correlate with the surface energies obtained by the other two approaches upon using water contact angle. But there is a definite correlation of surface energy by LW-AB to the surface energy by other two approaches upon the use of formamide contact angle, and the data points fall on the 45o line (135) for γTotal > 50 mJ/m2. For γTotal < 50 mJ/m2 the points fall near the 45o line. Similarly Fig. 35 correlates the γAB for the two cases. There is no correlation

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3. Surface Characterisation: Paper IV Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001 between the γAB obtained by LW-AB approach and γAB obtained by the other two approaches upon the use of water contact angle (Fig. 36a). But there is a definite correlation between the γAB obtained by LW-AB and other two approaches upon the use of formamide contact angle (Fig. 36b). The γAB values obtained from geometric mean approach using formamide contact angle is same as the ones obtained by LW- AB (136) approach but equation of state approach gives the γAB values lower to the ones given by LW-AB approach by ~7 mJ/m2 as the data points follow the straight line (Eq. 137) γ Total = γ Total GM ,Eq.st;Fo LW − AB (135) γ AB = γ AB GM ;Fo LW − AB (136) γ AB = γ AB − Eq.st;Fo LW − AB 7 (137)

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Table I Polar liquid sets arranged according to decreasing ∆Qr, data from Kwok et al. 1994. Rows in Bold letters are for liquid pairs, which give positive solid surface energy and square roots of the surface energy components for FEP and FC721 All values in mJ/m2.

FC721 - Fluorocarbon

LW * + ½ - ½ + - AB Total Liquids ∆Qr γ (γ ) (γ ) γ γ γ γ Wa-Fo 16,4 9,07 0,02 0,9 0,0004 0,81 0,04 9,11 Wa-Gl 13,6 9,07 0,16 0,76 0,0256 0,5776 0,24 9,31 Fo-DM 9,6 9,07 0,53 -1,19 0,2809 1,4161 0.04 7,81 Wa-Eg 9 9,07 0,5 0,42 0,25 0,1764 0,42 9,49 Fo-Eg 7,4 9,07 2,44 -9,16 5,9536 83,9056 -44,7 -35,63 Gl-DM 6,9 9,07 0,39 -0.13 0,1521 0,0169 -0,1 8,97 Wa-DM 6,7 9,07 0,3 0,63 0,09 0,3969 0,38 9,45 Gl-Eg 4,6 9,07 1,36 -3,82 1,8496 14,5924 -10,39 -1,32 Fo-Gl 2,8 9,07 -1,1 5,58 1,21 31,1364 -12,28 -3.21 DM-Eg 2,3 9,07 0,04 2,7 0,0016 7,29 0,22 9,29

Avg. 9,07 0,0735 1,8501 0,26 9,33 S.D. 0,1051 3,0498 0,150 0,150

Least Sq.# 9,15 0,07 0,44 0,34 9,40

FEP - Teflon

LW * + ½ - ½ + - AB Total Liquids ∆Qr γ (γ ) (γ ) γ γ γ γ Wa-Fo 16,4 15,29 0,08 0,86 0,0064 0,7396 0,14 15,43 Wa-Gl 13,6 15,29 0,24 0,7 0,0576 0,49 0,34 15,63 Fo-DM 9,6 15,29 0,47 -0,79 0,2209 0,6241 -0,74 14,55 Wa-Eg 9 15,29 0,64 0,3 0,4096 0,09 0,38 15,67 Fo-Eg 7,4 15,29 2,92 -10,9 8,5264 118,81 -64,18 -48,89 Gl-DM 6,9 15,29 0,33 0,38 0,1089 0,1444 0,25 15,54 Wa-DM 6,7 15,29 0,29 0,65 0,0841 0,4225 0,38 15,67 Gl-Eg 4,6 15,29 1,63 -4,6 2,6569 21,16 -15 0,29 Fo-Gl 2,8 15,29 -1,31 6,63 1,7161 43,9569 -17,37 -2,08 DM-Eg 2,3 15,29 -0,15 4,2 0,0225 17,64 -1,26 14,03

Avg. 15,29 0,1333 0,3773 0,298 15,58 S.D. 0,1590 0,2658 0,103 0,103

Least Sq.# 15,25 0,13 0,34 0,41 15,66 - + - + ∆Qr= (γ /γ )Liquid 1 - (γ /γ )Liquid 2 (Holländers selection criterion) * Predetermined using α-Bromonapthalene Wa-Water, Fo- Formamide, Gl-Glycerol, DM- DMSO, Eg-Ethylene glycol Average and SD only for the pairs which give positive surface energy and square root of the surface energy components

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# Wa-Gl, Wa-Fo, Wa-Eg and Wa-DM contact angle data solved using least square method (From Wu et al., 1995) Table II Condition numbers of the system of simultaneous equation required to be solved in evaluation of solid surface energy parameters using LW-AB approach. Rows in Bold letters are for liquid pairs, which give positive solid surface energy and square roots of the surface energy components both for FEP and FC-721 (In Table I) Two Liquid systems Condition numbers Using norms Liquid ∆ A A A ∞ Qr System 1 2 Column Euclidean Row Two liquid system where γLW is known before hand Wa-Gl 4,5 3,6 4,5 13,6 Wa-Fo 4,7 3,5 4,7 16,4 Wa-Eg 5,6 4,1 5,6 9 Wa-DM 6,3 4,7 6,3 6,7 Fo-DM 23,4 18,5 23,4 9,6 Gl-DM 31,9 23,7 31,9 6,9 Fo-Eg 35,1 28,6 35,1 7,4 Gl-Eg 54,1 40,9 54,1 4,6 Eg-DM 62,2 49,9 62,2 2,3 Fo-Gl 136,5 106,4 136,5 2,8 Three liquid system with one apolar and two polar liquids Wa-Gl-Br 6,12 4,85 7,35 13,6 Wa-Fo-Br 7,35 5,14 7,12 16,4 Wa-Eg-Br 8,91 5,91 8,38 9 Wa-DM-Br 10,95 7,06 10,19 6,7 Fo-DM-Br 39,3 29 45,7 9,6 Gl-DM-Br 47 34,6 61,2 6,9 Fo-Eg-Br 54,6 42,8 63,6 7,4 Gl-Eg-Br 74,1 56,7 95,9 4,6 Eg-DM-Br 107,2 79,3 117,4 2,3 Fo-Gl-Br 184,7 148,9 245,3 2,8 Three liquid system with all polar liquids Wa-Gl-DM 17,2 11,6 13,3 Wa-Fo-Gl 25,5 18,7 20,3 Wa-Gl-Eg 31,8 21 24,6 Wa-Fo-DM 34,5 25,5 28,7 Wa-Eg-DM 35,2 26,5 30,6 Fo-Gl-DM 53,4 43,1 68,9 Fo-Gl-Eg 67,1 50,1 69,9 Fo-Eg-DM 76,4 47,8 61,6 Gl-Eg-DM 872,5 674 1,09x103 Wa-Fo-Eg 1,55x103 1,14x103 1,24x103 - + - + ∆Qr= (γ /γ )Liquid 1 - (γ /γ )Liquid 2 (For Holländers Selection Criterion) = −1 = Condition Number Cond (A) A A , A max aij 1 j ∑ i 1  n 2  2 =   = A ai , A ∞ max aij , 2 ∑  i ∑  i =1  j Wa-Water, Fo- Formamide, Gl-Glycerol, DM- DMSO, Eg-Ethylene glycol 153

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Br- Bromonapthalene

Table III Mean contact angles (degrees) for two strains of Bervibacterium linens (BL) strains. Data taken from Table I of Kiely, 1997.

BL-MGE BL-9174 Mean SD Mean SD θWater 30 2 18 1 θFormamide 13 1 11 1 θGlycerol 65 1 65 1 θα-bromonapthalene 43 3 35 3 θMethyleneiodide 48 1 49 1

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Table IV. Surface energy parameter of two strains of Bervibacterium linens (BL) using LW-AB approach. The determination of surface energy parameters using 2,3 or over-determined set of equations. Condition number is reported along with the liquid systems.

Condition BL-MGE BL-9174 + - LW + - + - LW + - Liquid System Number √γs √γs γs γs γs √γs √γs γs γs γs LW + - I-γs Pre-determined using θBr, only γs and γs are determined using the polar liquid systems Wa-Fo 3,5 1,87 6,27 33,2 3,5 39,32 1,42 7,05 36.7 2,02 49,71 Wa-Gl 3,6 -0,74 8,88 33,2 0,55 78,86 -1,15 9,64 36.7 1,33 92,93 Fo-Gl 106 24,9 -89,6 33,2 620,01 8028,16 24,19 -87,8 36.7 585,16 7708,84 Wa-Fo-Gl* 3,9 0,37 7,71 33,2 0,14 59,45 -0,05 8,48 36.7 0,01 71,92

LW + - II- 2 polar and 1 apolar liquid, evaluation of γs , γs and γs Wa-Fo-Br 5,14 1,87 6,27 33,3 3,5 39,32 1,42 7,05 36,7 2,02 49,71 Wa-Gl-Br 4,85 -0,74 8,88 33,3 0,55 78,86 -1,15 9,64 36,7 1,33 92,93 Fo-Gl-Br 148 24,9 -89,6 33,3 620,01 8028,16 24,19 -87,8 36,7 585,16 7708,84 Wa-Fo-Mi 5,17 1,6 6,3 36,4 2,56 39,69 1,59 7,04 34,7 2,53 49,57 Wa-Gl-Mi 4,88 -0,93 8,83 36,4 0,87 77,97 -1,04 9,67 34,7 1,09 93,51 Fo-Gl-Mi 151 23,9 -86,7 36,4 571,21 7516,89 24,7 -89,6 34,7 610,09 8028,16 III- 3 polar liquids Wa-Fo-Gl 18,7 -7,03 7,04 212 49,43 49,57 -7,38 7,82 217 54,47 61,16 IV-Overdetermined system of polar and apolar liquids Wa-Fo-Gl-Br* 5,25 0,18 7,69 35,8 0,04 59,14 -0,24 8,47 39,4 0,06 71,75 Wa-Fo-Gl-Mi* 5,27 -0,01 7,68 38,6 0,01 58,99 -0,083 8,48 37,1 0,01 71,92 Wa-Fo-Gl-Br-Mi* 5,48 0,17 7,69 36,26 0,03 59,14 -0,07 8,48 36,9 0,01 71,92 Mean values reported by Kiely et al., 1997 The Liquid systems used are not 34 2 59 36 2 71 clear from the paper 1 n 2 2 −   Condition Number Cond (A) = A A 1 , The norm used is A =  a  2 ∑ i   i =1  * Are overdeterimned system of equations, and least square method is used for solid parameter determination Wa-Water, Fo- Formamide, Gl-Glycerol, DM- DMSO, Eg-Ethylene glycol, Br- Bromonapthalene, Mi- Methyleneiodide

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Table V Summary of microbial cell surface properties-Type of Gram staining, contact angle with Water, Formamide, Methyleneiodide and α- Bromonapthalene (van der Mei, 1998) and surface energy parameters evaluated using different thermodynamic approaches. Equation of state Geometric mean approach LW-AB approach using θW, θF approach and θBr − Bacterial isolate Gram θW θF θM θBr γTotal γLW γTotal- γLW γAB by γTotal γ+ γ γAB γTotal by θW by θBr γLW by θBr θW , θBr Acenobactor calcoacenticus MR-481 − 32 25 46 37 63,44 41,83 21,61 35,91 30,28 66,19 1,63 42,51 16,64 52,55 Acenobactor calcoacenticus RAG-1 − 48 49 43 38 54,57 41,39 13,18 35,49 21,28 56,77 0,04 40,15 2,54 38,03 Actinobacillus actinomycetemcomitans − 93 36 76 61 27,24 30,40 -3,16 24,48 2,55 27,02 14,48 2,39 11,75 36,22 HG1098 Actinobacillus actinomycetemcomitans − 87 47 41 54 31,07 33,87 -2,80 27,99 3,63 31,62 5,98 0,07 1,25 29,23 HG1099 Actinomyces israelii PK16WT + 60 35 44 30 47,51 44,68 2,83 38,66 12,83 51,48 1,73 14,14 9,88 48,54 Actinomyces naeslundii 147 + 53 45 45 31 51,64 44,3 7,34 38,29 16,97 55,26 0,19 29,24 4,66 42,94 Actinomyces naeslundii 5519 + 62 51 48 32 46,34 43,9 2,44 37,91 12,01 49,92 0,09 21,33 2,69 40,60 Actinomyces naeslundii 5951 + 62 45 45 31 46,34 44,3 2,04 38,29 11,87 50,15 0,54 17,21 6,08 44,37 Actinomyces naeslundii PK29 + 90 41 43 31 29,16 44,3 -15,14 38,29 1,11 39,39 4,39 0,37 2,54 40,82 Actinomyces naeslundii T14V-J1 + 64 37 43 29 45,17 45,06 0,11 39,01 10,55 49,56 1,70 10,90 8,59 47,60 Brevibacterium linens BL-9174 + 18 11 49 35 69,62 42,68 26,94 36,74 35,79 72,53 2,05 49,67 20,15 56,88 Brevibacterium linens BL-MGE + 30 13 48 43 64,45 39,14 25,31 33,28 32,96 66,23 3,53 39,15 23,48 56,76 Candida albicans ATCC10261(30C), DYM 59 29 33 46 48,10 37,74 10,36 31,88 16,25 48,13 4,83 12,36 15,45 47,32 Candida albicans ATCC10261(30C), GSB 78 40 44 30 36,74 44,68 -7,94 38,66 4,38 43,03 2,76 1,71 4,34 42,99 Candida albicans ATCC10261(37C), DTM 21 - 49 24 68,49 46,82 21,67 40,65 32,35 73,00 - - - - Candida albicans ATCC10261(37C), GSB 36 34 49 27 61,34 45,79 15,55 39,70 26,03 65,73 0,34 44,17 7,75 47,45 Capnocytophaga gingivalis PC1000 − 32 59 68 57 63,44 32,39 31,05 26,49 36,67 63,16 0,06 77,83 4,28 30,77 Capnocytophaga gingivalis PC1000/1-7 − 37 74 67 60 60,80 30,90 29,90 24,98 34,82 59,80 2,06 95,93 28,08 53,06 E. coli 917 − 23 22 52 38 67,67 41,39 26,28 35,49 34,75 70,24 1,63 50,11 18,03 53,52 E. coli C1212 − 25 23 50 34 66,80 43,09 23,71 37,14 32,87 70,00 1,27 48,95 15,76 52,90 E. coli C1214 − 25 23 50 34 66,80 43,09 23,71 37,14 32,87 70,00 1,27 48,95 15,76 52,90 E. coli O111K38 − 18 23 60 50 69,62 35,82 33,80 29,96 40,54 70,50 2,74 54,27 24,38 54,33 E. coli O157 K- − 19 17 56 46 69,26 37,74 31,52 31,88 38,76 70,64 2,93 50,58 24,31 56,19 E. coli O161K- − 26 37 57 51 66,35 35,36 30,99 29,47 37,58 67,04 1,17 57,86 16,41 45,88 E. coli O2K2 − 57 30 59 35 49,28 42,68 6,60 36,74 15,30 52,03 2,74 15,10 12,87 49,60

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Table V Continued E. coli O2K7 − 29 28 60 40 64,94 40,51 24,43 34,62 32,59 67,21 1,41 47,68 16,35 50,97 E. coli O39K1 − 21 25 54 40 68,49 40,51 27,98 34,62 36,11 70,73 1,42 53,63 17,45 52,07 E. coli O8K(A)28 − 46 29 57 36 55,73 42,26 13,47 36,33 22,07 58,40 1,96 27,64 14,71 51,04 E.coli Col − 21 25 52 38 68,49 41,39 27,10 35,49 35,54 71,03 1,25 53,73 16,37 51,86 E.coli Hu734 − 17 18 48 33 69,96 43,50 26,46 37,53 35,61 73,13 1,36 53,13 16,99 54,51 E.coli O83K − 54 36 57 39 51,05 40,95 10,10 35,06 17,83 52,89 1,83 21,44 12,53 47,58 Enterococci faecalis 1131 + 23 30 42 27 67,67 45,79 21,88 39,70 32,15 71,85 0,31 55,97 8,28 47,97 Enterococci faecalis 29212 + 30 68 46 21 64,45 47,75 16,70 41,50 28,09 69,59 6,02 99,25 48,88 90,38 Enterococci faecalis C1030 + 100 91 46 39 22,82 40,95 -18,13 35,06 0,12 35,18 3,73 5,83 9,32 44,38 Enterococci faecalis IC14 + 35 75 23 26 61,88 46,14 15,74 40,03 26,39 66,41 8,60 104,25 59,88 99,90 Lactobacillus acidophilus 68 + 74 39 52 35 39,22 42,68 -3,46 36,74 6,45 43,19 3,00 3,50 6,47 43,20 Lactobacillus acidophilus 75 + 66 56 50 39 44,00 40,95 3,05 35,06 10,89 45,94 0,07 19,60 2,20 37,25 Lactobacillus acidophilus RC14 + 102 47 55 38 21,57 41,39 -19,82 35,49 0,03 35,51 5,58 4,67 10,21 45,70 Lactobacillus acidophilus T13 + 80 39 46 27 35,49 45,79 -10,30 39,70 3,47 43,17 2,96 0,85 3,17 42,86 Lactobacillus casei 36 + 19 29 44 33 69,26 43,50 25,76 37,53 34,95 72,48 0,53 58,54 11,10 48,63 Lactobacillus casei 43 + 46 29 55 38 55,73 41,39 14,34 35,49 22,50 57,99 2,17 27,57 15,44 50,93 Lactobacillus casei 55 + 36 27 44 33 61,34 43,50 17,84 37,53 27,21 64,74 1,30 39,11 14,22 51,75 Lactobacillus casei 62 + 19 30 50 32 69,26 43,90 25,36 37,91 34,71 72,62 0,42 59,38 9,89 47,80 Lactobacillus casei 65 + 58 32 46 33 48,69 43,50 5,19 37,53 14,39 51,92 2,30 14,91 11,69 49,22 Lactobacillus casei 70 + 43 26 44 23 57,46 47,14 10,32 40,95 21,55 62,49 1,18 30,17 11,92 52,86 Lactobacillus casei 8 + 30 33 48 31 64,45 44,3 20,15 38,29 29,88 68,17 0,40 50,68 8,92 47,20 Lactobacillus casei subsp. rhamnosus 81 + 86 37 52 37 31,70 41,83 -10,13 35,91 2,36 38,27 5,77 0,06 1,09 37,00 Lactobacillus casei subsp. rhamnosus + 34 28 51 31 62,4 44,30 18,10 38,29 27,86 66,14 0,96 42,23 12,73 51,02 ATCC7469 Lactobacillus casei subsp. rhamnosus GR1 + 33 38 44 26 62,93 46,14 16,79 40,03 27,42 67,44 0,06 51,55 3,45 43,46 Lactobacillus casei subsp. rhamnosus RC15 + 52 29 48 27 52,23 45,79 6,44 39,70 16,95 56,64 1,70 20,53 11,80 51,49 Lactobacillus casei subsp. rhamnosus RC17 + 54 39 47 30 51,05 44,68 6,37 38,66 16,23 54,88 0,74 23,53 8,33 46,98 Lactobacillus fermentum A60 + 29 27 54 33 64,94 43,50 21,44 37,53 30,81 68,34 0,98 47,30 13,58 51,10 Lactobacillus fermentum B54 + 105 46 55 38 19,71 41,39 -21,68 35,49 0,02 35,51 6,54 7,41 13,91 49,40 Lactobacillus gasseri 56 + 90 46 47 29 29,16 45,06 -15,90 39,01 1,03 40,04 2,80 0,06 0,79 39,80 Lactobacillus gasseri 60 + 67 43 47 24 43,42 46,82 -3,40 40,65 8,53 49,18 0,76 10,69 5,69 46,33

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Table V Continued Lactobacillus jensenii RC28 + 87 40 47 30 31,07 44,68 -13,61 38,66 1,69 40,34 4,06 0,04 0,70 39,35 Lactobacillus plantarum RC20 + 79 43 45 30 36,12 44,68 -8,56 38,66 4,02 42,67 2,22 1,84 4,03 42,68 Lactobacillus plantarum RC6 + 25 31 49 24 66,80 46,82 19,98 40,65 30,79 71,44 0,21 54,86 6,75 47,39 Leuconostoc mesenteroides NCDO 523 + 17 39 52 36 69,96 42,26 27,70 36,33 36,38 72,71 0,05 69,51 3,48 39,81 Paenibacillus polymyxa NCIM 2539 + 40,81 56 66,68 51,84 58,59 34,85 23,74 29,06 29,50 58,56 0,01 59,43 0,21 29,27 Chalcopyrite adapted Paenibacillus + 15,85 21,05 70,6 55,6 70,29 32,88 37,41 27,19 43,46 70,64 3,89 54,31 29,04 56,23 polymyxa NCIM 2539 Galena adapted Paenibacillus polymyxa + 17,55 19,37 51 48,3 69,79 36,79 33,00 30,79 40,07 70,85 2,94 52,67 24,85 55,63 NCIM 2539 Pyrite adapted Paenibacillus polymyxa + 19,85 18,42 71,8 56,66 68,88 32,39 36,49 26,66 42,47 69,12 4,69 49,95 30,61 57,26 NCIM 2539 Sphalerite adapted Paenibacillus polymyxa + 29,97 27,63 69,94 57 64,45 32,39 32,06 26,49 37,81 64,29 3,89 45,45 26,57 53,05 NCIM 2539 Peptostreptococci micros ATCC 33270 + 39 49 72 51 59,70 35,36 24,34 29,47 30,36 59,83 0,25 53,53 7,18 36,64 Peptostreptococci micros HG1108 + 66 69 51 34 44,00 43,09 0,91 37,14 10,16 47,29 1,32 32,07 13,01 50,14 Peptostreptococci micros HG1109 + 33 49 58 43 62,93 39,14 23,79 33,28 31,37 64,65 0,01 62,91 0,07 33,34 Peptostreptococci micros HG1111 + 58 70 55 34 48,69 43,09 5,60 37,14 14,55 51,68 2,34 48,08 21,21 58,34 Pervotella intermedia HG1103 − 38 - 75 71 60,25 25,46 34,79 19,51 38,78 58,28 - - - - Pervotella intermedia HG1104 − 42 55 65 46 58,02 37,74 20,28 31,88 26,98 58,86 0,03 56,46 2,33 34,21 Pervotella intermedia HG1105 − 36 39 67 58 61,34 31,89 29,45 25,99 34,68 60,66 2,22 46,98 20,42 46,40 Porphyromonas gingivalis HG1101 − 30 - 60 30 64,45 44,68 19,77 38,66 29,67 68,32 - - - - Porphyromonas gingivalis HG1102 − 37 51 71 46 60,80 37,74 23,06 31,88 30,00 61,88 0,01 59,33 0,89 32,77 Porphyromonas gingivalis W83 − 61 57 59 50 46,92 35,82 11,10 29,96 15,92 45,88 0,19 27,23 4,55 34,50 Pseudomonas aeruginosa AK1 − 106 52 52 39 19,09 40,95 -21,86 35,06 0,04 35,09 4,63 5,78 10,34 45,40 Pseudomonas fluorescens − 38 38 50 32 60,25 43,90 16,35 37,91 25,9 63,80 0,28 44,66 7,05 44,96 Serratia marcescens 3162 − 50 55 47 39 53,40 40,95 12,45 35,06 20,28 55,33 0,05 43,35 2,70 37,75 Serratia marcescens 3164 − 21 18 58 49 68,49 36,31 32,18 30,45 39,05 69,49 3,37 49,26 25,76 56,20 Serratia marcescens RZ30 − 54 55 88 91 51,05 16,18 34,87 10,72 35,45 46,17 6,82 33,93 30,41 41,13 Serratia marcescens RZ37 − 37 41 97 90 60,80 16,63 44,17 11,10 48,87 59,97 10,05 45,35 42,70 53,80 Staphylococci epidermidis 169 + 37 31 58 45 60,80 38,21 22,59 32,35 29,70 62,05 1,97 39,96 17,70 50,05 Staphylococci epidermidis 236 + 32 31 52 35 63,44 42,68 20,76 36,74 29,79 66,52 0,83 46,58 12,40 49,13 Staphylococci epidermidis 242 + 30 29 54 29 64,45 45,06 19,39 39,01 29,47 68,48 0,62 47,69 10,85 49,86 Staphylococci epidermidis 252 + 29 21 47 19 64,94 48,31 16,63 42,02 28,28 70,29 0,79 44,23 11,76 53,78 Staphylococci epidermidis 26512 + 77 55 53 37 37,37 41,83 -4,46 35,91 5,41 41,32 0,46 6,86 3,55 39,46

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Table V Continued

Staphylococci epidermidis 26585 + 41 57 49 49 58,59 36,31 22,28 30,45 28,49 58,93 0,06 60,66 3,53 33,97 Staphylococci epidermidis 26741 + 77 47 45 33 37,37 43,50 -6,13 37,53 5,01 42,54 1,45 3,89 4,74 42,27 Staphylococci epidermidis 298 + 68 49 51 22 42,83 47,75 -4,92 41,23 7,89 49,12 0,17 12,80 2,88 44,11 Staphylococci epidermidis 3059 + 31 53 46 37 63,95 41,83 22,12 35,91 30,79 66,70 0,36 71,52 10,08 45,98 Staphylococci epidermidis 3081 + 87 50 49 32 31,07 43,90 -12,83 37,91 1,79 39,70 1,76 0,33 1,52 39,43 Staphylococci epidermidis 3294 + 107 44 53 35 18,48 42,68 -24,2 36,74 0,13 36,87 7,17 10,12 17,03 53,77 Staphylococci epidermidis 3399 + 29 56 56 39 64,94 40,95 23,99 35,06 32,32 67,37 0,68 78,78 14,59 49,65 Staphylococci epidermidis ATCC35893 + 66 45 50 34 44,00 43,09 0,91 37,14 10,16 47,29 0,95 12,56 6,89 44,02 Staphylococci epidermidis ATCC35984 + 39 25 77 38 59,70 41,39 18,31 35,49 26,67 62,16 2,18 34,14 17,23 52,71 Staphylococci epidermidis HBH171 + 33 26 53 27 62,93 45,79 17,14 39,70 27,60 67,29 0,89 42,30 12,22 51,91 Staphylococci epidermidis HBH2 + 28 17 49 34 65,42 43,09 22,33 37,14 31,52 68,65 1,96 42,99 18,33 55,46 Staphylococci epidermidis HBH2102 + 69 51 50 29 42,24 45,06 -2,82 39,01 8,07 47,08 0,21 12,75 3,25 42,26 Staphylococci epidermidis HBH2169 + 28 34 46 32 65,42 43,90 21,52 37,91 31,05 68,96 0,32 53,82 8,21 46,12 Staphylococci epidermidis HBH2277 + 42 40 64 36 58,02 42,26 15,76 36,33 24,45 60,78 0,40 40,68 8,02 44,34 Staphylococci epidermidis HBH23 + 36 32 48 22 61,34 47,75 13,59 41,23 25,23 66,46 0,34 42,79 7,54 48,77 Staphylococci epidermidis HBH276 + 29 23 51 34 64,94 43,09 21,85 37,14 31,04 68,18 1,44 44,83 16,06 53,19 Staphylococci epidermidis HBH45 + 28 31 48 36 65,42 42,26 23,16 36,33 32,01 68,34 0,75 51,17 12,34 48,67 Staphylococci epidermidis NCTC 100835 + 27 54 50 37 65,89 41,83 24,06 35,91 32,73 68,64 0,58 78,57 13,44 49,35 Staphylococci epidermidis NCTC 100892 + 19 10 73 50 69,26 35,82 33,44 29,96 40,18 70,14 4,24 47,93 28,50 58,45 Staphylococci epidermidis NCTC 100894 + 27 43 46 13 65,89 49,71 16,18 43,27 28,50 71,77 0,24 64,70 7,73 51,00 Staphylococci epidermidis SL58 + 22 45 49 35 68,09 42,68 25,41 36,74 34,35 71,09 0,04 72,06 3,14 39,88 Staphylococci hominus SL33 + 37 28 57 32 60,80 43,90 16,9 37,91 26,45 64,36 1,17 38,53 13,41 51,32 Staphylococci saprophyticus SAP1 + 20 19 49 12 68,88 49,90 18,98 43,44 31,12 74,55 0,49 51,93 10,06 53,49 Streptococci cricetus AHT + 28 32 51 35 65,42 42,68 22,74 36,74 31,76 68,49 0,60 52,01 11,15 47,88 Streptococci cricetus E49 + 33 31 45 35 62,93 42,68 20,25 36,74 29,27 66,00 0,87 45,37 12,52 49,26 Streptococci cricetus HS6 + 33 51 51 36 62,93 42,26 20,67 36,33 29,51 65,83 0,18 65,89 6,84 43,17 Streptococci gordonii NCTC 7869 + 93 46 51 32 27,24 43,9 -16,66 37,91 0,65 38,56 3,54 0,55 2,79 40,70 Streptococci mitis 244 + 60 38 47 30 47,51 44,68 2,83 38,66 12,83 51,48 1,27 15,60 8,89 47,54 Streptococci mitis 272 + 54 39 51 30 51,05 44,68 6,37 38,66 16,23 54,88 0,74 23,53 8,33 46,98 Streptococci mitis 357 + 53 31 42 33 51,64 43,50 8,14 37,53 17,31 54,83 1,98 20,16 12,62 50,14 Streptococci mitis 398 + 59 33 48 26 48,10 46,14 1,96 40,03 12,86 52,88 1,66 14,40 9,77 49,79

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Table V Continued

Streptococci mitis ATCC33399 + 56 40 45 31 49,87 44,30 5,57 38,29 15,23 53,51 0,79 21,56 8,23 46,51 Streptococci mitis ATCC9811 + 68 53 48 31 42,83 44,30 -1,47 38,29 8,77 47,06 0,1 15,11 2,41 40,69 Streptococci mitis BA + 103 55 49 35 20,94 42,68 -21,74 36,74 0,01 36,74 2,68 2,74 5,42 42,15 Streptococci mitis BMS + 100 43 50 34 22,82 43,09 -20,27 37,14 0,06 37,19 5,98 4,51 10,38 47,51 Streptococci mitis T9 + 91 47 55 36 28,52 42,26 -13,74 36,33 1,14 37,47 3,45 0,12 1,28 37,6 Streptococci mutans HG1003 + 22 27 49 38 68,09 41,39 26,70 35,49 35,15 70,64 1,08 54,20 15,29 50,78 Streptococci mutans HG979 + 26 48 54 38 66,35 41,39 24,96 35,49 33,46 68,94 0,07 71,25 4,15 39,63 Streptococci mutans HG982 + 33 34 51 35 62,93 42,68 20,25 36,74 29,27 66,00 0,58 47,70 10,48 47,21 Streptococci mutans HG983 + 27 28 48 35 65,89 42,68 23,21 36,74 32,23 68,96 0,94 50,08 13,68 50,42 Streptococci mutans HG985 + 23 30 61 43 67,67 39,14 28,53 33,28 36,22 69,49 1,20 55,26 16,26 49,53 Streptococci oralis 34 + 24 7 54 33 67,24 43,50 23,74 37,53 33,05 70,58 2,30 43,96 20,09 57,62 Streptococci oralis H1 + 90 37 43 28 29,16 45,43 -16,27 39,36 0,99 40,35 5,18 0,75 3,94 43,29 Streptococci oralis J22 + 24 31 49 33 67,24 43,50 23,74 37,53 33,05 70,58 0,48 55,55 10,32 47,85 Streptococci oralis PK1317 + 92 37 45 34 27,88 43,09 -15,21 37,14 0,88 38,01 6,44 1,47 6,15 43,29 Streptococci rattus ATCC19645 + 20 22 48 38 68,88 41,39 27,49 35,49 35,91 71,40 1,52 52,73 17,86 53,34 Streptococci rattus BHT + 23 31 50 37 67,67 41,83 25,84 35,91 34,48 70,39 0,66 56,36 12,19 48,10 Streptococci rattus BTHsm + 20 25 56 36 68,88 42,26 26,62 36,33 35,37 71,69 1,07 54,68 15,26 51,58 Streptococci rattus FA1 + 19 29 50 38 69,26 41,39 27,87 35,49 36,27 71,76 0,81 58,31 13,70 49,18 Streptococci salivarius HB + 42 48 56 33 58,02 43,50 14,52 37,53 23,82 61,34 0,01 48,66 0,86 38,38 Streptococci salivarius HBC12 + 21 28 55 32 68,49 43,90 24,59 37,91 34,00 71,91 0,60 56,10 11,56 49,47 Streptococci sanguis ATCC10556 + 22 17 54 27 68,09 45,79 22,30 39,70 32,54 72,23 1,19 48,94 15,21 54,90 Streptococci sanguis CR311 + 74 67 55 47 42,83 37,27 5,56 31,41 8,06 39,46 0,03 17,44 1,30 32,70 Streptococci sanguis CR311VAR1 + 75 59 58 46 38,60 37,74 0,86 31,88 7,43 39,31 0,36 10,66 3,90 35,78

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Table V Continued

Streptococci sanguis CR311VAR2 + 75 63 55 51 38,6 35,36 3,24 29,47 8,22 37,69 0,20 13,08 3,23 32,70 Streptococci sanguis CR311VAR3 + 31 39 55 46 63,95 37,74 26,21 31,88 33,36 65,23 0,68 54,18 12,11 43,98 Streptococci sanguis PK 1889 + 28 - 50 33 65,42 43,50 21,92 37,53 31,28 68,81 - - - - Streptococci sanguis PSH1b + 60 60 53 36 47,51 42,26 5,25 36,33 13,73 50,06 0,19 32,3 4,93 41,25 Streptococci sobrinus HG1025 + 29 33 47 34 64,94 43,09 21,85 37,14 31,04 68,18 0,50 51,73 10,09 47,23 Streptococci sobrinus HG970 + 26 45 54 38 66,35 41,39 24,96 35,49 33,46 68,94 0,01 67,40 0,25 35,73 Streptococci sobrinus HG977 + 27 43 53 33 65,89 43,50 22,39 37,53 31,74 69,27 0,01 64,06 0,34 37,87 Streptococci thermophilus B + 23 34 50 35 67,67 42,68 24,99 36,74 33,96 70,69 0,33 59,05 8,79 45,52 Equation of state Geometric mean approach LW-AB Using θW and θBr approach − Bacterial isolate θW θF θM θBr γTotal γLW γTotal- γLW γAB by γTotal γ+ γ γAB γTotal by θW by θBr γLW by θBr θW , θBr Average 45,81 38,4152,63 37,06 55,1 41,54 13,55 35,58 23,28 58,85 1,93 37,89 11,8 47,37 Standard deviation 24,92 14,43 9,33 11,17 14,29 4,99 15,38 4,89 12,83 12,30 2,37 23,31 9,38 8,86 Minimum 15,85 7,0023,00 12,00 18,48 16,18 -24,20 10,72 0,00017 27,02 1,8x105 0,03 0,07 29,23 Maximum 107,00 91,00 97,00 91,00 70,29 49,9 44,17 43,44 48,87 74,55 14,48 104,3 59,88 99,90

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Table VI Surface energy parameters (mJ/m2) of the standard liquids used for contact angle measurements (van der Mei et al., 1998; Chapter XIII van Oss, 1994; Bellon- Fountaine et al., 1990; Bellon-fontaine, 1996)

Liquid γTotal γd/γLW γh/γAB γ+ γ- Water 72.8 21.8 51 25.5 25.5 Formamide 58 39 19 2.3 39.6 α-Bromonapthalene 44.4 44.4 ≈0 <0.1 <0.1 Methyleneiodide 50.8 50.8 ≈0 <0.1 <0.1

Table VII. Expected correlation between θMethyleneiodide and θBromonapthalene according to Fowkes and Equation of state approach

F Fowkes approach Equation of state approach θ Bromonapthalene − LW LW E θMethyleneiodid γs θBromonapthalene θMethyleneiodide γs θBromonapthalene θ Bromonapthalene 35 41,95 19,46 35 42,68 14 5,46 40 39,53 27,61 40 40,51 23,5 4,11 45 36,94 34,59 45 38,21 31 3,59 50 34,21 41,02 50 35,82 37 4,02 55 31,39 47,11 55 33,38 43 4,11 60 28,52 52,99 60 30,9 49 3,99 65 25,65 58,71 65 28,42 55 3,71 70 22,83 64,32 70 25,95 61 3,32 75 20,09 69,85 75 23,53 66 3,85

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Table VIII. Expected correlation between θWater and θFormamide according to Geometric mean and Equation of state approach Geometric mean appraoch γLW γAB γTotal cosθW cosθF θW θF 35,57 1 36,57 -0,04 0,43 92,32 64,37 35,57 2 37,57 0,04 0,49 87,67 60,34 35,57 3 38,57 0,10 0,54 84,08 57,14 35,57 4 39,57 0,16 0,58 81,05 54,35 35,57 5 40,57 0,20 0,62 78,35 51,80 35,57 6 41,57 0,24 0,65 75,89 49,43 35,57 7 42,57 0,28 0,68 73,61 47,16 35,57 8 43,57 0,32 0,71 71,45 44,98 35,57 9 44,57 0,35 0,73 69,41 42,85 35,57 10 45,57 0,38 0,76 67,45 40,75 35,57 12 47,57 0,44 0,80 63,72 36,59 35,57 13 48,57 0,47 0,82 61,94 34,49 35,57 14 49,57 0,50 0,84 60,19 32,37 35,57 15 50,57 0,52 0,86 58,47 30,19 35,57 16 51,57 0,55 0,88 56,78 27,94 35,57 17 52,57 0,57 0,90 55,11 25,58 35,57 18 53,57 0,60 0,92 53,46 23,08 35,57 19 54,57 0,62 0,94 51,82 20,38 35,57 20 55,57 0,64 0,95 50,18 17,36 35,57 22 57,57 0,68 0,99 46,91 9,16 35,57 24 59,57 0,72 1,02 43,61 NA 35,57 26 61,57 0,76 1,05 40,24 NA Equation of state approach γTotal θW θF 57,00 44 11 56,00 46 15,5 55,00 47 19 54,00 49 22 50,00 56 32 45,00 64 43 40,00 73 52 35,00 81 61,5 30,00 89 71 25,00 96,5 80 20,00 104,5 90

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Table IX. Expected correlation between θWater and θFormamide according to LW-AB approach. Surface energies in (mJ/m2) and angles in (o)

− − γLW γ+ γ γAB γTotal θW θF γLW γ+ γ γAB γTotal θW θF 35,57 1,8x10−5 0,01 0,001 35,58 102,74 73,12 35,57 1,5 1 2,45 38,02 85,78 52,96 35,57 1,8x10−5 0,1 0,003 35,58 100,99 72,44 35,57 1,5 10 7,75 43,32 68,07 44,32 35,57 1,8x10−5 1 0,009 35,58 95,49 70,28 35,57 1,5 20 10,96 46,53 56,27 38,38 35,57 1,8x10−5 10 0,027 35,6 78,22 63,22 35,57 1,5 30 13,42 48,99 46 33,23 35,57 1,8x10−5 20 0,038 35,61 67,3 58,73 35,57 1,5 40 15,5 51,07 35,68 28,26 35,57 1,8x10−5 30 0,047 35,62 58,31 55,14 35,57 1,5 50 17,33 52,9 23,67 23,1 35,57 1,8x10−5 40 0,054 35,63 49,99 51,98 35,57 1,5 53 17,84 53,41 19,12 21,44 35,57 1,8x10−5 50 0,060 35,63 41,71 49,08 35,57 1,5 55 18,17 53,74 15,48 20,3 35,57 1,8x10−5 60 0,066 35,64 32,84 46,35 35,57 1,5 57 18,5 54,07 10,8 19,11 35,57 1,8x10−5 70 0,071 35,65 22,14 43,72 35,57 1,5 58,9 18,8 54,37 1,59 17,93 35,57 1,8x10−5 72 0,072 35,65 19,48 43,2 35,57 2 0,1 0,9 36,47 89,71 52,58 35,57 1,8x10−5 79,1 0,076 35,65 1,07 41,37 35,57 2 1 2,83 38,4 84,27 49,95 35,57 0,1 0,01 0,064 35,64 100,21 69,02 35,57 2 10 8,95 44,52 66,43 40,84 35,57 0,1 0,1 0,2 35,77 98,47 68,32 35,57 2 20 12,65 48,22 54,44 34,41 35,57 0,1 1 0,64 36,21 93,01 66,1 35,57 2 30 15,5 51,07 43,86 28,65 35,57 0,1 10 2 37,57 75,67 58,79 35,57 2 40 17,89 53,46 33,01 22,79 35,57 0,1 20 2,83 38,4 64,58 54,07 35,57 2 50 20 55,57 19,59 16,06 35,57 0,1 30 3,47 39,04 55,34 50,26 35,57 2 53 20,6 56,17 13,8 13,61 35,57 0,1 40 4 39,57 46,67 46,87 35,57 2 55 20,98 56,55 8,09 11,76 35,57 0,1 50 4,48 40,05 37,83 43,72 35,57 2 56 21,17 56,74 2,05 10,72 35,57 0,1 60 4,9 40,47 27,94 40,71 35,57 3 0,1 1,1 36,67 87,19 47,42 35,57 0,1 70 5,3 40,87 14,17 37,77 35,57 3 1 3,47 39,04 81,72 44,57 35,57 0,1 72 5,37 40,94 9,59 37,19 35,57 3 10 10,96 46,53 63,65 34,36 35,57 0,1 73 5,41 40,98 6,19 36,9 35,57 3 20 15,5 51,07 51,27 26,62 35,57 0,1 73,5 5,43 41 3,4 36,75 35,57 3 30 18,98 54,55 40,08 18,81 35,57 0,5 0,01 0,15 35,72 97,07 63,71 35,57 3 40 21,91 57,48 28,04 7,73 35,57 0,5 0,1 0,45 36,02 95,34 62,98 35,57 3 42,2 22,51 58,08 24,98 0,83 35,57 0,5 1 1,42 36,99 89,9 60,66 35,57 3 0,1 1,1 36,67 87,19 47,42 35,57 0,5 10 4,48 40,05 72,44 52,91 35,57 4 0,1 1,27 36,84 85,05 42,71 35,57 0,5 20 6,33 41,9 61,09 47,81 35,57 5 0,1 1,42 36,99 83,17 38,18 35,57 0,5 30 7,75 43,32 51,47 43,6 35,57 6 0,1 1,55 37,12 81,45 33,66 35,57 0,5 40 8,95 44,52 42,23 39,78 35,57 7 0,1 1,68 37,25 79,87 28,96 35,57 0,5 50 10 45,57 32,43 36,15 35,57 8 0,1 1,79 37,36 78,39 23,85 35,57 0,5 60 10,96 46,53 20,34 32,56 35,57 9 0,1 1,9 37,47 77 17,86 35,57 0,5 65 11,41 46,98 11,02 30,76 35,57 10 0,1 2 37,57 75,67 9,23 35,57 0,5 66 11,49 47,06 8,05 30,39 35,57 10,1 0,1 2,01 37,58 75,54 7,91 35,57 0,5 67 11,58 47,15 2,97 30,03 35,57 10,2 0,1 2,02 37,59 75,41 6,34 35,57 1 0,1 0,64 36,21 93,01 58,82 35,57 10,3 0,1 2,04 37,61 75,22 2,61 35,57 1 1 2 37,57 87,57 56,39 35,57 3,5 0,01 0,38 35,95 87,8 45,94 35,57 1 10 6,33 41,9 69,98 48,19 35,57 4 0,01 0,4 35,97 86,77 43,66 35,57 1 20 8,95 44,52 58,39 42,68 35,57 5 0,01 0,45 36,02 84,89 39,22 35,57 1 30 10,96 46,53 48,43 38,03 35,57 6 0,01 0,49 36,06 83,19 34,81 35,57 1 40 12,65 48,22 38,64 33,69 35,57 7 0,01 0,53 36,1 81,61 30,27 35,57 1 50 14,15 49,72 27,79 29,42 35,57 8 0,01 0,57 36,14 80,14 25,41 35,57 1 55 14,84 50,41 21,16 27,24 35,57 9 0,01 0,6 36,17 78,76 19,86 35,57 1 60 15,5 51,07 11,95 24,99 35,57 10 0,01 0,64 36,21 77,44 12,65 35,57 1 61 15,63 51,2 9,16 24,53 35,57 10,5 0,01 0,65 36,22 76,8 6,93 35,57 1 62 15,75 51,32 5,07 24,07 35,57 10,6 0,01 0,66 36,23 76,68 5,1 35,57 1,5 0,1 0,78 36,35 91,22 55,49 35,57 10,7 0,01 0,66 36,23 76,55 2,04

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Table IX Continued

− − γLW γ+ γ γAB γTotal θW θF γLW γ+ γ γAB γTotal θW θF 35,57 10,7 0,01 0,66 36,23 76,54 1,41 35,57 6 4 9,8 45,37 67,53 23,01 35,57 3 1 3,47 39,04 81,72 44,57 35,57 7 4 10,59 46,16 65,83 15,63 35,57 4 1 4 39,57 79,57 39,6 35,57 7,9 4 11,25 46,82 64,39 2,78 35,57 5 1 4,48 40,05 77,65 34,74 35,57 4 10 12,6548,22 61,24 27,91 35,57 6 1 4,9 40,47 75,91 29,76 35,57 5 10 14,1549,72 59,08 20,79 35,57 6,5 1 5,1 40,67 75,09 27,15 35,57 6 10 15,5 51,0757,08 11,12 35,57 6,7 1 5,18 40,75 74,77 26,07 35,57 6,1 10 15,63 51,2 56,89 9,73 35,57 7 1 5,3 40,87 74,29 24,4 35,57 6,2 10 15,75 51,32 56,7 8,11 35,57 8 1 5,66 41,23 72,78 18,13 35,57 6,3 10 15,88 51,45 56,51 6,1 35,57 9 1 6 41,57 71,35 9,03 35,57 6,4 10 16 51,57 56,32 2,97 35,57 9,1 1 6,04 41,61 71,21 7,6 35,57 4 20 17,8953,46 48,48 17,79 35,57 9,3 1 6,1 41,67 70,93 3,28 35,57 4,5 20 18,98 54,55 47,18 11,9 35,57 9,32 1 6,11 41,68 70,9 2,46 35,57 4,6 20 19,19 54,76 46,93 10,39 35,57 4 0,5 2,83 38,4 81,92 40,95 35,57 4,7 20 19,4 54,97 46,68 8,65 35,57 5 0,5 3,17 38,74 80,02 36,25 35,57 4,8 20 19,6 55,17 46,42 6,48 35,57 6 0,5 3,47 39,04 78,3 31,48 35,57 4,9 20 19,8 55,37 46,18 3,1 35,57 7 0,5 3,75 39,32 76,7 26,44 35,57 3,5 30 20,5 56,07 38,34 12,39 35,57 8 0,5 4 39,57 75,2 20,77 35,57 3,6 20 16,98 52,55 49,57 21,58 35,57 9 0,5 4,25 39,82 73,79 13,52 35,57 3,7 20 17,21 52,78 49,29 20,68 35,57 9,5 0,5 4,36 39,93 73,1 8,06 35,57 3,8 20 17,44 53,01 49,02 19,75 35,57 9,6 0,5 4,39 39,96 72,97 6,47 35,57 3,9 20 17,67 53,24 48,75 18,79 35,57 9,7 0,5 4,41 39,98 72,84 4,34 35,57 4 20 17,89 53,46 48,48 17,79 35,57 9,75 0,5 4,42 39,99 72,77 2,72 35,57 4,5 20 18,98 54,55 47,18 11,9 35,57 9,75 0,5 4,42 39,99 72,77 2,72 35,57 4,8 20 19,6 55,17 46,42 6,48 35,57 9,75 0,5 4,42 39,99 72,77 2,72 35,57 4,9 20 19,8 55,37 46,18 3,1 35,57 9,75 0,5 4,42 39,99 72,77 2,72 35,57 4,92 20 19,84 55,41 46,13 1,78 35,57 9,75 0,5 4,42 39,99 72,77 2,72 35,57 3,5 30 20,5 56,07 38,34 12,39 35,57 9,75 0,5 4,42 39,99 72,77 2,72 35,57 3,6 30 20,79 56,36 38 10,74 35,57 9,75 0,5 4,42 39,99 72,77 2,72 35,57 3,7 30 21,08 56,65 37,66 8,83 35,57 1 4 4 39,57 79,57 52,71 35,57 3,9 30 21,64 57,21 36,99 2,16 35,57 2 4 5,66 41,23 76,2 45,91 35,57 3,1 40 22,28 57,85 27,55 4,34 35,57 3 4 6,93 42,5 73,58 40,12 35,57 3,11 40 22,31 57,88 27,5 3,85 35,57 4 4 8 43,57 71,35 34,63 35,57 3,12 40 22,35 57,92 27,45 3,28 35,57 5 4 8,95 44,52 69,35 29,06 35,57 3,13 40 22,38 57,95 27,4 2,59

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Bacterial adhesion important in Mineral Bioprocessing 1) Biocorrosion

2) Biofouling & Biodeterioration Bio-beneficiation 3) Odontology (Oral cavity)

4) Thrombosis of biomaterial implants Bio-flotation Bio-flocculation 5) Blood platelet and Leukocyte adhesion (Coagulation) 6) Phagocytosis of bacteria Most important factors imparting selectivity (Immune-system) is selective adhesion of bacterial cells 7) Ophthalmology (Contact lens) on the mineral surface

Adhesion of microbes

selectively on solid surface Estimation of possibility and extent of adhesion

Thermodynamic approach Extended DLVO approach

The interfacial free energy of the Balance of Lifshitz van der Waals attractive interacting surfaces i.e. microbe and force, Electrostatic forces and acid/base mineral is compared before and after interaction forces with separation distance adhesion Gtotal=GLW+GEL+GAB ∆ γ γ γ Gadh= sb- sl- bl

GTotal Calculations Calculations vs ∆ Gadh Separation distance ∆ LW ∆ AB ( Gadh , Gadh )

γ LW γ AB γ LW γ + γ - γ sv , sv sv , sv , sv Zeta-potential sv γ LW γ AB γ LW γ + γ - γ bv , bv bv , bv , bv bv γ LW, γ AB γ LW, γ +, γ - lv lv lv lv lv

Equation Geometric mean Lifshitz van der Waals of state approach Acid/base approach

1 polar and 2 polar and 1 Liquid 1 non polar liquid 1 non polar liquid e.g. e.g. Water, e.g. Water, Formamide Water Methyleneiodide Methyleneiodide Contact Angles measurement

and surface tensions

Figure 1. Theoretical estimation of the possibility and extent of microbial adhesion on solid surfaces. Approaches to convert contact angle data into solid surface energy

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1805 Thomas Young proposed the equation for a drop placed on a solid surface, as force balance at the 3 phase boundary in horizontal direction γ = γ + γ θ SV SL LVCOS γ γ In order to obtain the solid surface free energy ( SV) an estimate of SL have to be obtained. Zisman and co-workers tried direct γ estimation of SV by using concept of critical contact angle but otherwise two different approaches have came into being. Surface tension component approach. Equation of state approach γ γ γ γ not only depends on γ and γ but also on the SL depends only upon LV and SV and a thermodynamic SL LV SV γ γ γ specific intermolecular interactions i.e. components of relation of the type SL=f( LV, SV) exists 1898 the surface energy Berthelot´s geometric mean combining rule γ =γ +γ − γ γ SL LV SV 2 LV SV 1907 Antonow proposed the following relation without any theoretical background γ = γ − γ SL LV SV 1937 Bangham and Razouk pointed out the importance of vapour adsorption on the solid surface, introduced a new γ γ π term in the Youngs equation called the spreading pressure where, s= sv+ e γ = γ + γ θ + π S SL LV COS e 1 γ C 1952 Zisman and his associates approximated the solid surface energy by using the concept of cosθ γ Critical contact angle C, which is the surface tension that divides the liquids forming zero contact angles on the solid surface from those forming contact angle greater than zero -1 0 γ 60 L 1957 Good and Garifalco proposed almost the same relationship as Berthelot but with a constant φ which was specific to the liquid- solid system γ =γ +γ − φ γ γ SL LV SV 2 LV SV 1962, Fowkes pioneered the surface component approach. He φ = 1 for “Regular” system i.e. where the adhesive forces 1964 divided the total surface energy in 2 parts. Dispersive part and across the interface are of the same type non-dispersive part, first resulted from the molecular φ < 1 when the predominant forces within the separate interactions due to London forces and the second due to all phases are of unlike types e.g. London-van der the non-London forces. γ=γd+γh Waals forces and metallic bonding φ Proposed the following relation for solid-liquid interacting by > 1 when there is specific interactions between the only dispersion force interaction molecules ofthe two phases γ = γ + γ − γ dγ d SL S L 2 S L 1969 Zettlemoyer explored the Owens and Wendt extended the Fowkes used of arithmetic mean relation and included the hydrogen bonding instead of geometric mean to term also. Used geometric mean to combine combine the dispersion the dispersion force and hydrogen bonding component of solid and liquid components. Called it Geometric mean used by Fowkes. approach γ = γ + γ − (γ d + γ d ) γ = γ + γ − γ dγ d − γ hγ h SL S L S L SL S L 2 S L 2 S L 1971 Wu used harmonic mean to combine the polar and dispersion components of the solid and liquid surface energies to obtain the following relation  γ dγ d γ pγ p  γ = γ + γ − 4 L S + L S SL S L γ d + γ d γ p + γ p  1974  L S L S  Ward and Neuamnn gave thermodynamic proof for existence of equation of state. Neumann derived the first formulation γ = ()γ − γ − γ γ SL SV LV 1 0,015 SV LV 1986 Van Oss et al. further divided the polar component of surface energy into electron-accepting(γ+) and electron-donating parameters (γ-) γ = γ LW + γ AB γ AB = 2 γ −γ + Proposed a new relation. This is called the Lifshitz-van der Waals/acid-base approach (LW-AB) 2 γ = ()γ LW − γ LW + 2()γ + − γ + ()γ − − γ − SL S L S L S L 1990, 1992 Li and Neuamnn derived the second formulation − ()γ −γ 2 γ = γ + γ − γ γ 0,0001247 SV LV SL S L 2 SV LV e

2000 Kwok and Neuamnn derived the third formulation γ = γ + γ − γ γ ()− ()γ − γ 2 SL S L 2 SV LV 1 0,0001057 LV SV

Figure 2. Chronological development of different approaches for evaluation of surface energy of solids from Contact angle goniometry.

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(a) γ lv

θ γ sl γsv

γ = γ + γ θ sv sl lv cos

(b) γ lv γ s

θ γ γ π sl sv e

γ −π = γ + γ cosθ s e sl lv

Figure 3. Contact angle of a sessile drop (a) neglecting the spreading pressure, (b) accounting for spreading pressure

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(a) (b) (c )

Figure 4. (a) Wettability of polytetrafluoroethylene (Teflon) by n-alkanes, (b) Wettability by various liquids on the surface of Polyvenylchloride and

Figure 5. Cosθ Vs γl curve for Poly (ethylene terephthalate): (A) homologous series of hydrocarbon liquids, (B) Calculated for the ethanol/water series, (C) observed for

ethanol/water series (From Dann, 1970 b).

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) 2

mJ/m (

c γ

Critical Surface Tension Tension Surface Critical

γ d l = 1 γ d l γ s Figure 6. Critical surface tension conversion curves (From Dann, 1970a)

Figure 7. Diagram of the two neighbouring monolayers at the an interface in which tension resides (From Fowkes, 1964b)

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θ γ Figure 8. Relation of contact angle to surface tension of liquid l. (From Fowkes, 1962)

γ wh Hydrocarbon

Water θ

γ γ sw sh

Solid

Figure 9. Contact angle of a sessile drop in the two liquid system of water and a hydrocarbon

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(a)

Fluid 1

Liquid 3

Liquid 2

(b) Fluid 1

Liquid 3

Liquid 2

Figure 10. General liquid-liquid lens-fluid system with (a) Three curved interfaces and (b) two curved interfaces

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Figure 11 Plot of γlvcosθ vs γlv of various liquids on 1. Methacrylic polymer A with fluorinated side chains; 2. Methylacrylic polymer S with fluorinated side chains; 3. 17-(perfluoropropylene)-heptadecanoic acid; 4. 17-(perfluoroethyl)-heptadecanoic acid; 5. Polytetrafluoroethylene; 6. 80-20 copolymer of tetrafluoroethylene and chlorotrifluoroethylene; 7. 60-40 copolymer of tetrafluoroethylene and chlorotrifluoroethylene; and 8. Copolymer of tetrafluoroethylene and polyethylene

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Figure 12. Good and Garifalco interaction parameter, φ, as a

function of γsl for the 8 solids in Figure 11.

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3. Surface Characterisation: Paper IV Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001

Figure 13. Goods parameter (φ) Vs solid-liquid interfacial tension for different γ γ Ο γ ∆ γ sv values such that ( ) sl<0 ( ) sl=0 and ( ) sl>0

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γ+ γ- Figure 14. Surface energy components and as a function of the contact angles of (a) water, (b) formamide and (c) glycerol (From Holländer, 1995)

90 6 Stage III Stage I Stage II Spreading Equilibrium Absorption Contact angle µ 80 Drop Volume ( l) Drop Height (mm) Base diameter (mm) 70 4 ) o 60

40 2

Contact Angle(

20 0 0,1 1 10 100 Time (Seconds)

Figure 15. A typical curve for polar liquids-contact angle Vs time lapsed after

positioning of drop over the bacterial lawns. Measured by the FIBRO DAT 1100 dynamic absorption tester.

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60 θ 55 Water θ 50 Formamide θ 45 α-Bromonapthalene

θ 40 Methyleneiodide

35 30

20 15

Relative Frequency (%) Relative Frequency 0

020406080100120 o Contact angle ( )

Figure 16. Distribution of water, fomamide, Methyleneiodide and α-Bromonapthalene contact angles on 147 different bacterial isolates

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2 (a) γ Using θ (mJ/m ) Methyleneiodide 10 20 30 40 50 100 γ =34,51 Contact Angles γ LW =32,74 Eq F σ=4,48 σ=5,1 50 γ

γLW by Fowkes approach U 80 γ =41,54 s γLW Eq i by Equation of state approach ng σ=4,98

40 θ γ LW α =35,6 -Bromonapthalene 60 Fo σ=4,9 30

40 37,05O σ=11,17 -Bromonapthalene

α 20 θ

(

20 m Expected correlation J/ Equation of state 10 m O σ 53 , =9,3 Fowkes 2 0 ) 20 30 40 50 60 70 80 90 100 θ Methyleneiodide (b) 50 Using θα-Bromonapthalene

Using θMethyleneiodide

24 5, 40 + es 76 wk 5, LW Fo + γ s 02 ke 1, LW ow = 7γ F te ,8 ta 0 S = of e n tat 30 tio f S γ ua n o Eq tio γ ua Eq

by Equation of Equation state by approach γ ine o L 45 10 10 20 30 40 50 γLW by Fowkes approach

Figure 17. For the 147 microbial isolates (a) Correlation of Methyleneiodide and α-

Bromonapthalene contact angle and the correlation of the dispersion component of surface energy evaluated by using Methyleneiodide and α-Bromonapthalene contact angles. Correlation shown when either Fowkes or Equation of state approach is used (b) Correlation of dispersion component of surface energy evaluated using Fowkes and α Equation of state approach, correlations shown when either Methyleneiodide and - Bromonapthalene contact angles are used.

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80 Fowkes approach Using θ α-Bromonapthalene Using θ Methyleneiodide 60 Geometric mean Least Square Fit Equation of state approach Using θ α-Bromonapthalene Using θ 40 Methyleneiodide

Relative Frequency (%) Relative Frequency 0

0 102030405060 γLW 2 (mJ/m )

Figure 18. Distribution of Lifshitz-van der Waals component of surface energy

evaluated by using apolar liquid contact angles on 147 different bacterial isolates,

with Equation of state, pair wise and least squared Geometric mean approach

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(a) γLW Using θα-Bromonapthalene Fowkes 50 γLW Using θMethyleneiodide Fowkes γ Using θα-Bromonapthalene Equation of state γ θMethyleneiodide 40 Using Equation of state

) 2 30

(mJ/m

γ 20

20 40θ o 60 80 100 ( )

(b) γLW Bacterial cell Surface 10 15 20 25 30 35 40 45 50 55 ~44

1,0

0,8 ~51

θ 0,6

cos 0,4

γLW Using θα-Bromonapthalene 0,2 Fowkes γLW Using θMethyleneiodide Fowkes 0,0 γ Using θα-Bromonapthalene Equation of state

γ Using θMethyleneiodide Equation of state

Figure 19. Correlation of the Lifshitz-van der Waals component of surface energy as evaluated by Fowkes and Equation of state approaches versus θ (a) and cosθ where the contact angles used are with Methyleneiodide and α-

Bromonapthalene (b)

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(a)

80 γLW Using θ α-Bromonapthalene 70 γAB

γAB γLW γTotal 60 + =

2 40

30 mJ/m γ 20

-10

20 40 60 8090 100 θ Water (b)

γAB 80 AB LW Total 75 γ +γ =γ γ T=37,5+12,8cosθ +24,8cos2θ 70 b w w

2 39,8

mJ/m 30 γ γ AB=1,4+13cosθ +25,4cos2θ b w w 20

-0,4 -0,2 0,0 0,2 0,4 0,6 0,8 1,0 cosθ Water Figure 20. Polar, apolar and total surfa ce energy of bacterial cell surfaces as a function of water contact angle (a) and cosines water contact angle (b), while using Geometric mean approach

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(a) γLW Using θ 70 α-Bromonapthalene γAB 60 γAB+ γLW= γTotal

mJ/m

γ 20

0 20406080 90 θ Formamide

(b) γAB 70 γAB+ γLW=γTotal 60 59,2 γ T=37,9-22,5cos θ +43,8cos2θ b F F 50

2 40

mJ/m

γ γ AB=5,9-29cosθ +46cos2θ 22,9 20 b F F

0,0 0,2 0,4 0,6 0,8 1,0 cosθ Formamide Figure 21. The polar, apolar and total surface energy of bacterial cell

surfaces as a function of formamide contact angle (a) and cosines formamide contact angle (b), while using Geometric mean approach

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3. Surface Characterisation: Paper IV Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001

(a)

2 20 10

mJ/m 0 γ γTotal -10 γLW Using θ α -20 -Bromonapthalene γAB=γTotal-γLW -30 20 40 60 8090 100 θ Wate r (b)

80 γTotal

γ T=28,7+33,3cosθ +9,4cos2θ 71,4 70 γTotal-γLW=γAB b w w

50 40

30 30,4

20 10 mJ/m

γ 0

-10 γ AB=-13,3+33,5cosθ +10,2cos2θ -20 b w w

-30

-0,4 -0,2 0,0 0,2 0,4 0,6 0,8 1,0 cosθ Water

Figure 22. The polar, apolar and total surface energy of bacterial cell surfaces as a function of water contact angle (a) and cosines water contact angle (b), using Equation of state approach

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3. Surface Characterisation: Paper IV Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001

(a)

2 20

10 mJ/m γ 0 γTotal -10 γLW Using θ α-Bromonapthalene

γTotal-γLW=γAB -20

20 40 60 8090 100 θ (b) Formamide 70 γTotal 60 γTotal-γLW=γAB 57,5 γ T=20,6+22,9cosθ +14cos2θ b F F 50

2 20 14,9 γ AB=-17,8+18,7cos θ +14cos2θ 10 b F F

mJ/m γ 0

-10

-20

-30 0,0 0,2 0,4 0,6 0,8 1,0 cosθ Formamide

Figure 23. The polar, apolar and total surface energy of bacterial cell surfaces as a function of formamid e contact angle (a) and cosines

formamide (b) contact angle, using Equation of state approach

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3. Surface Characterisation: Paper IV Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001

(a)

Geometric mean approach

γTotal Vs θ 70 Water γTotal Vs θ Formamide 60 Equation of state approach γTotal Vs θ Water γTotal Vs θ ) Formamide

2 50

(mJ/m γ γ LW=35,6 mJ/m2 30 Average

20 ~55 o ~70o

20 40 60 80 100 θ (o ) (b)

57,5 1,0 71,4 59,2 75

0,8

0,6

θ 0,4

cos Geometric mean approach 0,2 γTotal Vs cosθ Water 0,0 γTotal Vs cosθ Formamide Equation of state approach -0,2 γ Vs cosθ Water γ Vs cosθ Formamide -0,4 10 20 30 40 50 60 70 80 γ (mJ/m 2)

Figure 24. Total surface energy of bacterial cell surfaces evaluated by using Geometric mean and Equation of state approaches vs water and formamide contact angles (a), cosines of water and formamide contact angles (b)

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3. Surface Characterisation: Paper IV Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001

(a) 80 γ Total=49,7-1,41γ Total+0,04(γ Total)2-2,25x10-4(γ Total)3 GM;W Eq.st;W Eq.st;W Eq.st;W γ Total=2,5+γ Total GM;W Eq.st;W 45o Line 70 Using θ Water Using θ Formamide

by Geometric mean approach by 30

Total γ 20 30 40 50 60 70 γTotal by Equation of state approach (b)

γ AB=9,57+0,84γ AB+0,012(γ AB)2 -2,99x10-4(γ AB)3 GM;W Eq.st;W Eq.st;W Eq.st;W 50 γ AB=6+γ AB GM;Fo Eq.st;Fo γ AB=9+γ AB GM;W Eq.st;W 40 Using θ Water Using θ Formamide 30

by Geometric mean approach by i 0 o L 45

AB γ -20-100 10203040 Equation of state approach

γAB=γTotal-γLW α-Bromona pthalene

Figure 25. Comparison of the (a) total surface energy and (b) polar component of surface energy when evaluated using either Geometric mean or Equation of state approaches. The comparison is done when water or formamide contact angles are used

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3. Surface Characterisation: Paper IV Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001

60 50

30 20

Formamide

θ Geometric mean Approach 0 γAB -10 γAB+γLW=γTotal ine o L Using -20 45 Equation of state approach

γ γTotal -30 γTotal- γLW=γAB -40 * γLW Using θ α-Bromonapthalene -50 -20-100 10203040506070 γ Using θ Water

Figure 26. Correlation of total and polar surface energy evaluated using either water or formamide contact angles, the correlation is shown for both Geometric mean and Equation of state approach

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3. Surface Characterisation: Paper IV Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001

γLW Using θ (a) α-Br

40 γLW Using θ M

LW γ 20

10 15 20 25 30 35 40 (b) γLW by Least Square fit

γAB Using θ and θ 50 W α-Br γAB Using θ and θ Fo α-Br 40

AB γ 20

0 1020304050 (c) γAB by Least square fit 80 γTotal =-5,633+1,185 γTotal GM;W,α-Br GM;Least Sq. γTotal Using θ and θ α 70 W -Br γTotal Using θ and θ Fo α-Br ne o Li 45 60

50 Total

30 40 50 60 70 γTotal by Least square fit Figure 27. Correlation of (a) dispersion component, (b) polar component and (c) total surface energy when either least square method is used or

pair-wise solution is used for Geometric mean approach

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3. Surface Characterisation: Paper IV Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001

Formamide θ

0 0 2040608010012 0 θWater

Figure 28. Correlation of water and formamide contact angles ♦Experimental data + Theoretical fit of Equation of state appr oach the relation is irrespective to the LW F -4 W 3 W 2 W value of γ , θ =2,27x10 (θ ) -0,054(θ ) +5,35θ -138,11 * Theoretical fit of Geometric mean approach using γLW=35.5 mJ/m2, θF=3,72x10-4(θW)3-0,089(θW)2+8θW-207 O γLW 2 Theoretical fit of LW-AB approach using =35.5 mJ/m

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3. Surface Characterisation: Paper IV Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001

(a) 80 From Experimental Data Theoretical Fit using γLW=35,5

γTotal γTotal

γAB γAB

) 2 40

(mJ/m γ 20

0 20406080100θ (b) Water 14 14 Experimental

Theoretical 12

) 8

(mJ/m + 4 γ

0 20406080100 (c) θWater 100 Experimental Theoretical 80

60 )

40 (mJ/m

γ 20

0 20406080100 θWater

Figure 29. Correlation of experimentally obtained and the theoretically evaluated- (a) total, acid-base, (b) electron acceptor and (c) electron donor surface energy parameters of bacterial cells with respect to water contact angle using LW-AB approach

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3. Surface Characterisation: Paper IV Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001

(a) 80 From Experimental Data Theoretical Fit using γLW=35,5 γTotal γTotal γAB γAB 60

) 2 40

(mJ/m γ 20

020406080100 (b) θFormamide 14 Experimental 12 Theoretical

) 8

(mJ/m

+ 4 γ

0 2040608010 0 (c) θFormamide 100 Experimental Theoretical 80

60 ) 2

40 (mJ/m -

γ 20

020406080100 θFormamide

Figure 30. Correlation of experimentally obtained and the theoretically evaluated- (a) Total, Acid-Base, (b) electron acceptor and (c) electron donor surface energy parameters

of bacterial cells with respect to Formamide contact angle using LW-AB approach 191

3. Surface Characterisation: Paper IV Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001

) 15 (a) ne

2 i o l 45

+ γ M ,74 (mJ/m +0 10 ,04 =0 γ+ r B

Using θ α 60 -Bromonapthalene Using θ 50 Methyleneiodide 5 40 30 -Bromonapthalene 20 α

θ 10

0 0 0 5 10 15 Relative Frequency (%) γ+ (mJ/m2)

using +

γ 0 5 10 15 20 γ+ using θ (mJ/m2) Methyleneiodide

) 120 2 ne (b) o li 5 100 4

(mJ/m 80

60 Using θ α-Bromonapthalene 30 Using θ 40 Methyleneiodide

20 -Bromonapthalene

α 20

θ 10

0 0

0 20406080100 Relative Frequency (%) γ- 2 using (mJ/m ) -

γ -20 020406080100 γ- using θ (mJ/m2) Meth yleneiodide

Figure 31. Correlation of (a) electron-donor, (b) electron-acceptor, (c) acid-base component and (d) total surface energy parame ters for 147 different bacterial isolates evaluated using LW-AB approach, when the liquid for apolar contact angle is changed keeping the two polar liquids same - water and formamide

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3. Surface Characterisation: Paper IV Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001

) 2 60 e (c) in o l 5 B 50 4 A M γ 82 (mJ/m 0, 3+ 01 40 ,0 -0 = B r γA B 30 Using θ α-Bromonapthalene Using θ 30 Methyleneiodide 20 -Bromonapthalene 20

θ 10 10 0

0204060 Relative Frequency (%) Frequency Relative using γAB 2 0 (mJ/m ) AB

γ 0 10203040506070 γAB using θ (mJ/m2) Meth yleneiodide

) 60 2 (d)

(mJ/m 50

tal γTo M ,85 +0 40 Using θ 43 α-Bromonapthalene 7, Using θ 40 l = Methyleneiodide ota r 30 γT B

-Bromonapthalene 20 α θ 10 30 e lin 0 Relative Frequency (%) o 0 20406080100120140 5 γTotal 2 using 4 (mJ/m ) 20 30 40 50 60

Total γ γTotal using θ (mJ/m2) Meth yleneiodide Figure 31. Continued

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3. Surface Characterisation: Paper IV Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001

Gram negative bacteria Gram positive bacteria

All the bacteria 30

Frequency (Numbers)

020406080100 γ- mJ/m2 Using θ α-Bromonapthalene

Figure 32. Distribution of the electron-donating characteristics for 140 bacterial species, 111 Gram-positive and 29 Gram-n egative bacterial cell surfaces.

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3. Surface Characterisation: Paper IV Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001

(a) 60 50 40 40 30 20 10 For Gram (-) bacteria -

0 30 γ 0,02 0,04 0,06 0,08 0,10 0,12 0,14 N/C

Frequency (Numbers)Frequency 0 0,00 0,05 0,10 0,15 0,20 N/C

(b) 60 50 25 40 30 20 20 10 bacteria (-) Gram For -

0 γ 0,2 0,3 0,4 0,5 0,6 15 O/C

Frequency (Numbers) Frequency 0

0,20,30,40,50,60,70,80,9 O/C

60 30 (c) 50 25 40 30 20 20

10 For Gram (-) bacteria

- 15 0 γ 0,00 0,01 0,02 0,03 10 P/C

Frequency (Numbers) Frequency 0 0,00 0,01 0,02 0,03 0,04 0,05 0,06 P/C

Figure 33. Distribution of the chemical composition of bacterial cells surface in the form of N/C, O/C and P/C obtained by XPS spectroscopy (van der Mei et al. 2000). The distribution is presented for Gram-positive (+ with dashed line) and Gram- negative (− with dotted line) bacterial cells along with the distribution for them − together (Solid square with whole line). Inset figures show correlation between the γ parameter and the surface composition by XPS

195

3. Surface Characterisation: Paper IV Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001

40 Geometric mean approach (a) Using θ and θ Water α-Bromonapthalene Using θ and θ Formamide α-Bromonapthalene Least square fit 30 Equation of state approach Using θ Water Using θ Formamide LW-AB approach 20 Using θ α-Bromonapthalene

Relative Frequency (%) Relative Frequency

0 20406080 γTotal mJ/m2

70 Geometric mean approach

Using θ and θ (b) Water α-Bromonapthalene Using θ and θ 60 Formamide α-Bromonapthalene Least square fit Equation of state approach 50 Using θ and θ Water α-Bromonapthalene Using θ and θ Formamide α-Bromonapthalene 40 LW-AB approach Using θ α-Bromonapthalene 30

Relative Frequency (%) Relative Frequency 0

-60 -40 -20 0 20 40 60 γAB (mJ/m2)

Figure 34. Distribution of total and acid-base component of surface

energy when evaluated using different approaches and contact angles

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3. Surface Characterisation: Paper IV Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001

(a)

50 )

2 40

(mJ/m 30 ine 45o L Total

γ 20

Equation of state approach using θ Water 10 Geometric mean approach using θ and θ Water α-Bromonapthalene 0 30 40 50 60 γTotal by LWAB approach (b)

Equation of state approach using θ Fromamide 60 Geometric mean approach using θ and θ Formamide α-Bromonapthalene

)

(mJ/m 30 Total

γ ine o L 5 4 20

20 30 40 50 60 γTotal by LW-AB approach

Figure 35. Correlation of the total surface energy evaluated by using either Geometric mean approach or Equation of state approach with the one evaluated by LW-AB approach (a) By using water contact angle (b) Using Formamide contact angle along with α-Bromonapthalene contact angle for Geometric mean approach

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3. Surface Characterisation: Paper IV Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001

(a) 60 Equation of state approach γAB=γ -γ θ θ Water α-Bromonapthalene 50 ne Geometric mean approach using o Li θ and θ 45 Water α-Bromonapthalene 40

30 )

2 20

(mJ/m

AB 0 γ -10

-20

0 102030405060 (b) γAB by LW-AB approach 70 Equation of state approach; γAB=γ -γ θ θ α Formamide -Bromonapthalene e 60 Geometric mean approach using Lin 5o θ and θ 4 50 Formamide α-Bromonapthalene

) 2 30 7 X- Y= 20

(mJ/m 10

AB γ 0

-10

-20

0 102030405060 γAB by LW-AB approach Figure 36. Correlation of the Acid- base component of surface energy

evaluated by using either Geometric mean approach or Equation of state approach with the one evaluated by LW-AB approach (a) By using water contact angle (b) Using Formamide contact angle along with α-Bromonapthalene contact angle for Geometric mean approach

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3. Surface Characterisation: Paper IV Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001

60 (a)

by LW-AB by approach 10

γ 0

0 1020304050 γAB by Least Square fit

of Geometric mean approach

60 (b)

by LW-AB by approach 30

Total γ 20 30 40 50 60 γTotal by Least Square fit

of Geometric mean approach Figure 37. Correlation of (a) Acid-base component (b) total surface

energy when evaluate using either LW-AB approach or least square fit of Geometric mean approach to the 4 available contact angle values for each microbial surface

199

Chapter 4: Microbial adhesion on

Mineral Surfaces

4. Microbial Adhesion on Mineral Surfaces Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001

Adhesion of microorganisms to the surfaces is ever present in the natural environment and hence occurs between the microbe and mineral particle. Microbial adhesion occurs and is detrimental in wide range of areas from engineering- biocorrosion, biofouling, biodeterioration etc. to human health- plaque formation on tooth, biofilm formation on contact lenses, attachment of infectious microbes on implants etc (Fig. 4.2).

1. Transport 2. Initial Adhesion

Diffusion Convection

3. Attachment 4. Colonisation

Biofilm Micro- Polymer Fibrils colonies

Figure 4.1 Steps in the colonisation of surface by microorganisms

4.1 Microbial Adhesion Mechanism In general the biofilm formation at the solid-liquid interface occurs in the following sequence (Fig. 4.1). Transport of the cells to surface: Microbes can reach the surface by three different modes • Diffusive Transport is important in quiescent conditions only. Microbial cells exhibit non-negligible Brownian motion, about 15 µm/hr (van Loosdrecht, 1988), is observed under the microscope. This motion could account for random contact of microbes with the surface. Under quiescent conditions sedimentation of bacteria may also contribute to the microbial transport. • Convective Transport is the transport of the cells by liquid flow. Convective transport is several orders of magnitude faster than diffusive transport. This has significant contribution under turbulent flow. • Active Transport occurs where the microbes respond chemotactically to concentration gradient that may exist in the interfacial region. Such transport does not contribute significantly to the transport under turbulent flow conditions and for non-motile cells.

Initial Adhesion is a physico-chemical process and can be divided in two different stages, namely reversible and irreversible. Reversible adhesion is defined as deposition of the cells to a surface in such a manner that the cells still exhibit two-dimensional Brownian motion (only moving along the surface) and can be detached from the surface by shearing effect of water

201

4. Microbial Adhesion on Mineral Surfaces Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001 stream of cells own mobility. Irreversible adhesion no longer exhibits Brownian motion and cannot be removed by a moderate shear force

Firm Attachment: After the cells have been deposited on the surface, special cell surface structures (e.g. fibrils or polymers) may form a strong connection between cell and solid surface. Polysaccharides are essential for the development of the surface films

Surface Colonisation: When the firmly attached cells start growing and newly form cells stay attached to each other, micro colonies and then biofilm develops.

From the point of view of biobeneficiation, where the time interval between cell attachment and the mineral separation is short- of the order of minutes. The cells are transported to the mineral surface by convective currents and only "initial attachment" is the relevant mode of attachment, which needs be studied in detail. This is because the cells do not get much time to form either firm attachment or colonisation. Adhesion of microbial cells depends upon its electrostatic, van der Waals and acid/base interactions with the mineral surface. All these interactions are function of the microbial surface properties like, surface charge, surface hydrophobicity, the van der Waals component of microbial cell surface energy. Adhesion of microbial cells on the mineral surface causes the formation of biofilm and imparting its own surface properties to the mineral. Adhesion also leads to bio-catalysed oxidation or reduction of the mineral surface in the case of chemolithotrophic microorganisms, generation of surface-active bio-reagents or adsorption of metabolic products. All these processes lead to alteration in surface properties of the mineral. The possibility and extent of adhesion can be estimated using thermodynamic approach and colloidal approach. Microbial cell and mineral surface energy along with the surface charge are required as inputs (Fig. 4.2).

4.2 Factors Effecting Adhesion As a cell moved towards a solid surface, numerous factors come into play and affect the forces that determine whether adhesion will actually take place. Moreover these forces differ in strength and in the separation distance at which they influence the interaction. At separation distances of tens of nanometers, the interaction is a balance of attractive and repulsive forces between the two surfaces. Van der Waals interactions are usually attractive, occur between all adjacent surfaces and act over relatively long separation distances (>50 nm), but are relatively weak (Busscher et al., 1990). Electrostatic forces become significant at closer separation distances (10-20 nm). If the cell and surface have opposite charges then the electrostatic forces are attractive. On the other hand, most often with microbial cells and potential attachment surfaces, the net surface charges are negative, and therefore electrostatic repulsion occurs and may prevent the cell to approach the surface. However the repulsion forces decreases with an increase ionic strength of the medium e.g. in seawater there is enough electrolyte concentration to eliminate the electrostatic repulsion. As the cell

202

4. Microbial Adhesion on Mineral Surfaces Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001 Bacterial adhesion important in Mineral Bioprocessing 1) Biocorrosion 2) Biofouling & Biodeterioration Bio-beneficiation 3) Odontology (Oral cavity) 4) Thrombosis of biomaterial implants Bio-flotation Bio-flocculation 5) Blood platelet and Leukocyte adhesion (Coagulation) 6) Phagocytosis of bacteria Most important factors imparting selectivity (Immune-system) is selective adhesion of bacterial cells 7) Ophthalmology (Contact lens) on the mineral surface Adhesion of microbes selectively on solid surface Estimation of possibility and extent of adhesion

Thermodynamic approach Extended DLVO approach The interfacial free energy of the Balance of Lifshitz van der Waals attractive interacting surfaces i.e. microbe and force, Electrostatic forces and acid/base mineral is compared before and after interaction forces with separation distance

adhesion Gtotal=GLW+GEL+GAB ∆ γ γ γ Gadh= sb- sl- bl

GTotal Calculations Calculations vs ∆ Gadh Separation distance ∆ LW ∆ AB ( Gadh , Gadh )

γ LW γ AB γ LW γ + γ - γ sv , sv sv , sv , sv Zeta-potential sv γ LW γ AB γ LW γ + γ - γ bv , bv bv , bv , bv bv γ LW γ AB γ LW γ + γ - lv , lv lv , lv , lv

Equation Geometric mean Lifshitz van der Waals of state approach Acid/base approach

1 polar and 2 polar and 1 Liquid 1 non polar liquid 1 non polar liquid e.g. e.g. Water, e.g. Water, Formamide Water Methyleneiodide Methyleneiodide Contact Angles measurement and surface tensions

Figure 4.2 Theoretical estimation for the possibility and extent of microbial adhesion on solid surfaces. Approaches to convert contact angle data into solid surface energy.

moves closer to the surface (0.5 - 2 nm), another potential barrier to attachment is water that is absorbed to the cell or solid surface. Displacement of adsorbed water to allow closer approach of the two surfaces is energetically unfavourable. However, if either surface has apolar groups or patches, these can assist the exclusion of water by hydrophobic interactions (Busscher et al., 1990), which are likely to be important in many cases, either as the primary mechanism of adhesion or by facilitating close approach. Once the microbial cell overcomes the water barrier and the separation is on the order of <0.1 nm (Busscher et al., 1990) specific interactions, like hydrogen bonding, start playing between the cell and the surface. 203

4. Microbial Adhesion on Mineral Surfaces Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001

4.3 Thermodynamic Aspects of Adhesion: This approach compares the interfacial free energy of the interacting surfaces i.e. microbe and mineral, before and after adhesion. Adhesion of microbe on mineral surface causes formation of the new interface i.e. microbe-mineral at the expense of microbe-medium and mineral- medium interfaces. The comparison is expressed in terms of free energy of adhesion. ∆ = γ − γ − γ Gadh sm sl ml (4.1) where, γsm, γsl and γml are mineral-microbe, mineral-liquid and microbe-liquid interfacial free energies respectively. Like all systems in nature, this system also proceeds in the direction of lowering the total energy. Which means that adhesion of microbe will take place on the mineral when ∆Gadh is negative and adhesion is thermodynamically unfavourable when ∆Gadh is positive. ∆Gadh calculation needs the values of γsm, γsl and γml. γsl and γml are possible to be calculated by using contact angle data measured with standard liquids with known free energies. Different approaches to convert contact angle data to surface free energies are described previously in section 3.1 and Sharma et al. (1999). γsm is also obtained from γsl and γml by using the aforementioned approaches. Depending on the approach used in order to attain the γsm, γsl and γml values the ∆Gadh value is different.

Equation of state ( γ − γ )2 ( γ − γ )2 ( γ − γ )2 ∆G = sv mv − sv lv − mv lv (4.2) adh − γ γ − γ γ − γ γ 1 1.015 sv mv 1 1.015 sv lv 1 1.015 mv lv

For geometric mean approach and LW-AB approach the total free energy of adhesion can be divided in Lifshitz van der Waals component and acid/base component ∆ = ∆ LW + ∆ AB Gadh Gadh Gadh (4.3)

Geometric mean approach ∆ LW = − ( γ LW − γ LW )( γ LW − γ LW ) Gadh 2 mv lv sv lv (4.4) ∆ AB = − ( γ AB − γ AB )( γ AB − γ AB ) Gadh 2 mv lv sv lv (4.5)

LW-AB approach The Lifshitz van der Waals component is the same as the geometric mean approach but the acid base component is calculated by the following expression

∆ AB = + ( γ + − γ + )( γ − − γ − )− ( γ + − γ + )( γ − − γ − ) Gadh 2 mv sv mv sv 2 mv lv mv lv (4.6)

Free energy of adhesion of unadapted, pyrite-adapted and chalcopyrite- adapted P. polymyxa on pyrite and chalcopyrite mineral is calculated using LW-AB approach and is shown in Table 4.1. The Lifshitz van der Waals component is attractive for all the P. polymyxa on both pyrite and chalcopyrite but the acid/base component is highly repulsive, especially for chalcopyrite mineral. The total free energy of adhesion of P. polymyxa on both minerals is positive therefore, the

204

4. Microbial Adhesion on Mineral Surfaces Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001 thermodynamic approach predicts that there will be no adhesion of P. polymyxa on pyrite or chalcopyrite.

Table 4.1 Free energy of adhesion

2 System Free energy of Adhesion ∆Gadh (mJ/m ) LW AB Total ∆Gadh ∆Gadh ∆Gadh 1)Unadapted P.p. – Pyrite -2.46 6.68 4.22 2)Pyrite-adapted P.p. – Pyrite -1.78 8.51 6.72 3)Chalcopyrite-adapted P.p. – -1.95 7.00 5.05 Pyrite 4)Unadapted P.p. – -1.99 15.05 13.06 Chalcopyrite 5)Pyrite-adapted P.p. – -1.44 13.57 12.13 Chalcopyrite 6)Chalcopyrite-adapted P.p.- -1.58 11.3 9.71 Chalcopyrite

All the P. polymyxa strains are hydrophilic and especially the mineral adapted ones therefore the thermodynamics predicts repulsion from the mineral surface as the bacterial cells prefer to stay in the aqueous phase than to attach themselves to the mineral surface. The chalcopyrite mineral exhibits higher acid/base repulsion as compared to pyrite due to the fact that chalcopyrite have very low electron accepting character (γ+) and pyrite have comparatively higher electron accepting character. All the P. polymyxa have very high electron donating character (γ-) and hence they are relatively less repelled from pyrite. The maximum repulsion is between chalcopyrite mineral and unadapted P. polymyxa cells, its because both have very low γ-/γ+ ratio

4.4 Colloidal Aspects of Adhesion: Classical DLVO theory as described by Derjaguin, Landau, Verwey and Overbeek (Verwey and Overbeek 1948; Deryagin and Landau, 1941) includes (LW) Lifshitz van der Waals attractive forces and attractive or repulsive (EL) electrostatic force. LW is the force of attraction between neutral and chemically saturated molecules and was postulated by van der Waals in order to explain non-ideal gas behaviour. This force includes attractive force between permanent dipoles-permanent dipole, permanent dipole-induced dipole and induced dipole-induced dipoles. Electrostatic forces arise due to the double layer interactions between two particles and thus zeta-potential becomes important. Later Van Oss et al. (1986) added the acid/base contribution. The acid/base interactions are based on electron-donating and electron-accepting interactions between polar moieties in aqueous solutions. Microbial adhesion is described as a balance between attractive Lifshitz van der Waals force, repulsive or attractive electrostatic force and acid/base interaction force. GTotal(H)=GLW(H)+GEL(H)+GAB(H) (4.7) The decay of these forces depends on the geometry of interacting bodies and the type of force. Table III summarises the mathematical formulations used for the calculation of these forces. Apart from the knowledge of size of the interacting particles, different interaction forces need specific parameters for the calculations to be performed.

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4. Microbial Adhesion on Mineral Surfaces Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001

Table 4.2 Equations quantifying extended DLVO interactions

Lifshitz-van der Waals interaction energy- GLW(H)     Sphere-Sphere A  y y  x 2 + xy + x  1 (4.8) − + + 2ln     2 2  2  12 x + xy + x x + xy + x + y  x + xy + x + y    2πH    1+1.77     λ     Sphere-Flat plate ()+ +   (4.9) − A  2a H a −  H 2a  1   ln  12  H ()H + 2a  H   2πH   1 + 1.77     λ   λ=1000 Å Electrostatic interaction energy- GEL(H) 2 2 −κH πεa a ()ζ + ζ  2ζ ζ 1 + e − κ  Sphere-Sphere 1 2 1 2 1 2 ln + ln ()1 − e 2 H (4.10) + ζ 2 + ζ 2 − −κH  a1 a 2  1 2 1 e  − κ H  2ζ ζ 1 + e − κ  Sphere- Flat plate πε a ()ζ 2 + ζ 2 1 2 ln + ln ()1 − e 2 H (4.11) 1 2  ζ 2 + ζ 2 − −κ H   1 2 1 e 

Acid-Base interaction energy- GAB(H) []()− λ π λ∆ AB do H Sphere-Sphere a Gadh e (4.12) []()− λ π λ∆ AB do H Sphere-Flat plate 2 a Gadh e (4.13)

where H-Separation distance, a- Radius of solid particle, ζ- Zeta-potential, -1 10 2 ½ -1 κ- Double layer thickness (=0.328x10 (zi Mi) m ), A- Hamaker Constant, x= H/a1+a2, y= a1/a2, do- Minimum separation distance between 2 surfaces (1.57 Å), λ- Correlation length of molecules in liquid (≈ 6 Å)

The major problem in evaluating Lifshitz van der Waals interaction energy is that of evaluating Hamaker constant. Two methods are available for its evaluation. In London-Hamaker microscopic approach the constant is evaluated from the individual atomic polarisabilities and the atomic densities of the materials involved. The total interaction is assumed to be the sum of the interactions between all the interparticle atom pairs and is assumed to centre on a single oscillation frequency. The other method is the macroscopic approach of Lifshitz, in which the interacting particles and the intervening medium are treated as continuous phases. The calculations are complex, and require availability of bulk optical/dielectric properties of the interacting materials over a wide frequency range. The presence of a liquid dispersion medium, rather than vacuum (or air), between the particles notably lowers the van der Waals interaction energy. Therefore, constant A in the equation must be replaced by an effective Hamaker constant. Considering interaction of particle 1 and 2 in the medium 3 the effective Hamaker constant is A132=A12+A33-A13-A23 (4.14) The attarction between unlike phases is taken to be geometric mean of the attractions ½ of each phase to itself i.e. A12=(A11.A22) then the equation becomes ½ ½ ½ ½ A132=(A11 -A33 )(A22 -A33 ) (4.15) And if the particles are of the same material then ½ ½ 2 A131=(A11 -A33 ) (4.16)

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4. Microbial Adhesion on Mineral Surfaces Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001 Fowkes (1964) proposed the following equation to calculate the individual Hamaker constant of solid surfaces. 2 d A11=6πr γs (4.17) d Where, r represents intermolecular distance and γs represents the dispersion component of the surface free energy of the solid. According to Fowkes, in water and systems with the volume element such as metal atoms, CH2 and CH groups, which have nearly the same size, the value of 6πr2 can be taken as 1.44x10-18 m2. After evaluating the individual Hamaker constant the effective Hamaker constant can be calculated by using equation 15. The extended DLVO approach as developed by van Oss and co-workers can be considered a combination of the thermodynamic and classical DLVO approaches, as the Hamaker constants can be obtained from the Lifshitz-van der Waals interaction ∆ LW energy Gadh as calculated from contact angle measurements according to the equation 4.4. This gives the effective Hamaker constant. = − π 2∆ LW A 12 do Gadh (4.18)

Using the sphere-sphere equations 4.8, 4.10 and 4.12 the free energy of interactions, in kT units, were calculated for bacterial cell-bacterial cell, mineral- mineral and mineral-bacterial cell systems. The plots are presented in Fig. 4.3.

Bacteria-Bacteria system. The sum of van der Waals interactions, electrostatic interactions and acid/base interactions between bacterial cells themselves as a function of ionic strength (0.1 to 0.0001 M) and in the pH range 5 to 10 showed high repulsion as a function of separation distance (Fig. 4.3(b)). This means that aggregation of bacterial cells in the aqueous phase with pH 5-7 and ionic strength 0.01 to 0.0001 M is not possible. Although the van der Waals interactions are attractive but the acid/base interaction, which operate in a short range of about 50 Å, is highly repulsive. At large distance also the interaction is repulsive due to the electrostatic repulsion. The reason for this repulsion is the fact that bacterial cells are hydrophilic and hence they prefer to stay in aqueous phase rather than forming flocs.

Mineral-mineral system. The total interaction energy vs. separation distance curves for chalcopyrite-chalcopyrite is shown in Fig. 4.3a. The electrostatic repulsion is very high for the mineral system at pH 10 as the zeta-potential of the particles is highly negative (~ -35 mV) and hence flocculation is not possible on account of an energy barrier of about 800 kT. At pH 5 the zeta- potential of the particles is positive (~10 mV) and the energy barrier is about 50 kT. At pH 7 the particles are only slightly charged and in case of pyrite- chalcopyrite they are oppositely charged and hence flocculation is predicted.

Mineral-bacteria system Total interaction energy vs separation distance curves of chalcopyrite interaction with chalcopyrite-adapted P. polymyxa is shown in Fig. 4.3(c). The acid/base interaction between mineral-bacteria is highly repulsive but this force operates only at close distances of 50 Å. At pH values where the bacterial cells and mineral particles are oppositely charged the attractive electrostatic interactions cause the formation of secondary minima where adhesion of bacterial cells is possible on mineral surface.

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1000 (a) Chalcopyrite-chalcopyrite particle interaction

750 G T ota (p l H 1 0) 500

G(kT) 250 G (pH 5) EL G (pH 5) Total 0 25 50 75 100 125 150 175 G (pH 7) Separation distance(Å) Total -250 G (pH 5) AB

5) (pH

LW G 1000-500 Chalcopyrite adapted P.p.- Chalcopyrite adapted P.p

G interaction EL G ( Tota k=3.2 (b) 750 l 8X107 m-1)

) G

M 1 T 500 ota (k=

0 l 1.0

0 4 . X 108

0 m -1

= )

I (

B G(kT) A G G T 250 ota (k= l 3.2 8X1 08 m -1)

0 25 50 75 100 125 150 175 G (I=0.001M) LW Separation distance(Å)

-250 1000 Chalcopyrite - Chalcopyrite adapted P.p. interaction 750 (c)

G G AB (pH 500 Total 10)

250 Separation distance(Å) 0 050100150200 G(kT) 7) -250 G (pH LW G l Tota 5) (pH G otal -500 T

-750 G EL -1000

Figure 4.3 Interaction energy curves for (a) Mineral-mineral, (b) Bacteria-bacteria and (c) Mineral-bacteria system

At pH 10 both mineral and bacteria are highly negatively charged and hence electrostatic repulsion is repulsive. At pH 5 both the minerals are positively 208

4. Microbial Adhesion on Mineral Surfaces Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001 charged but bacterial cells are negatively charged (approximately same as at pH 10) and hence electrostatic interaction is highly attractive causing deepest secondary minima (~625-1000 kT). This predicts irreversible attachment. At pH 7 the mineral is only slightly charged hence the secondary minima is not very deep (~125-250 kT). Therefore, attachment is reversible. The mineral- bacteria interaction energy curves predict attachment of different P. polymyxa equally on both pyrite and chalcopyrite.

The thermodynamic approach predicts that there will be no attachment of bacterial cells on the mineral surface. The reason behind is the strong acid/base repulsion, which outweighs the van der Waals attraction. The other reason is the fact that electrostatic interactions are not considered at all in the thermodynamic approach. The DLVO approach gives a more realistic picture. It predicts repulsion between the individual bacterial cells, possibility of flocculation of mineral particles and attachment of bacterial cells on the mineral surface. Although the acid/base interaction is repulsive in the case of bacteria-mineral system but they are short-range interactions and due to attractive electrostatic interactions at longer distance a secondary minima is predicted at 25 Å and hence attachment in this minima. The changes in electrokinetic behaviour, FT-IR spectra and flotation behaviour of mineral after microbial interaction is supported by extended DLVO approach as it predicts microbial adhesion but not the thermodynamic approach. The DLVO approach predicts adhesion of all the three types of P. polymyxa on the both the minerals equally. This is actually observed in the flotation experiments Fig. 10, as the flotation recovery in presence of all the three P. polymyxa cells for both pyrite and chalcopyrite in absence of any collector is same. Even though the natural flotability of chalopyrite is high, after interaction with microbial cells it becomes same as pyrite. In presence of collector (Potassium isopropyl xanthate) the flotation recovery of chalopyrite remains the same but the recovery of pyrite is reduces. In this case the second factor comes into play where the interaction of the collector is different towards bacteria pre-conditioned chalcopyrite and pyrite.

4.5 Mineral-Microbe interaction: Experimental Studies Microbes when interacted with minerals attach themselves to the mineral surface, forming a biofilm. Direct evidence can be obtained by adsorption studies. Infrared spectroscopy gives the evidence of microbial attachment on mineral surface. Using this technique, mineral interaction with collector in presence and absence of biofilm is also studied. Adsorption of microbial cells on mineral surface manifests itself indirectly also by affecting the electrokinetic behaviour and flotation behaviour. Adsorption Studies. The adsorption of ferrous ions and So grown T. ferrooxidans cells onto chalcopyrite at pH 2 are shown in Fig. 4.4(a). The adsorption is observed to be very fast and reaches equilibrium within 10 minutes interaction in all the cases studied. The adsorption isotherms of T. ferrooxidans cells grown in ferrous ions and sulphur on pyrite and chalcopyrite at pH 2 are shown in Fig. 4.4(b). The isotherms display that the adsorption increases steeply at about an equilibrium cell density of 3x108 cells/ml until certain surface coverage (≈1x107 cells/cm2) is attained. Above this coverage, the adsorption isotherms increase linearly with increasing equilibrium cell concentration up to 1x109 cells/ml, above which equilibrium concentration, the isotherms tend to level off at an adsorption density of 1x108 cells/ml. This adsorption density corresponds to a full coverage of the surface

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4. Microbial Adhesion on Mineral Surfaces Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001 in a horizontal orientation while considering the geometrical cell dimensions (i.e., length x breadth: 1.65 x 0.35 µm).

1,00E+08 1,00E+09

1,00E+07 1,00E+08

1,00E+06 1,00E+07

1,00E+05 1,00E+06 Cells adsorbed (cells / cm2) Cells adsorbed (cells/m2) adsorbed Cells

1,00E+04 1,00E+05 0 20406080 Time (min) 1,00E+07 1,00E+08 1,00E+09 1,00E+10 1,00E+11 Equilibrium conc. (cells / ml)

Fig. 4.4a. Adsorption of ferrous and Fig. 4.4b. Adsorption isotherms of elemental sulfur grown Thiobacillus Thiobacillus ferrooxidans on minerls ferrooxidans on chalcopyrite at pH 2 with at pH 2. Open symbols are for different cell concentrations. Open symbols chalcopyrite and the closed one for with symbols discontinuous lines are for So pyrite (◊) sulfur grown., ( ) ferrous grown T.f. and close with full lines are for ions grown. ++ 8 9 Fe grown T.f. ( ) 4x10 cells/ml (∆) 4x10 10 cells/ml (Ο) 4x10 cells/ml

FT-IR spectroscopy of mineral powder. Diffuse reflectance FT-IR spectra of pyrite and chalcopyrite were recorded after its interaction with increasing amounts of Fe++ and So grown T. ferrooxidans at pH 2 and 7. The spectra show stronger amide I and II bands for minerals interacted with higher amounts of T. ferrooxidans hence giving evidence of increasing cell adhesion. The spectra were also recorded for pyrite and chalcopyrite after interaction with potassium isopropyl xanthate in presence and absence of T. ferrooxidans. Fig. 7 shown such spectra for pyrite interacted with Fe++ grown cells and xanthate. The pyrite mineral spectrum gets additional bands at 1177 and 1269 (C-O-C stretching), 1088 and 1025 (C=S stretching in the dixanthogen) cm-1 due to xanthate. And when xanthate is interacted with bacteria pre-conditioned pyrite the spectra shown amide I, II and the bands due to xanthate also. This shows that the mineral surface interacts with xanthate even after having a biofilm.

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++ Fig. 4.5 FT-IR spectras of pyrite interacted with Fe grown Thiobacillus ferrooxidans and Potassium Isopropyl Xanthate 1. Pyrite 2. Pyrite + Xanthate 3. Pyrite + T.f. 4. Pyrite + T.f. + Xanthate.

20 20 (a) (b) 10 10 pH pH 0 0 2345678910 2345678910 -10 -10 -20 -20 -30 -30

Zeta-potential (mV) Zeta-potential (mV) -40 -40 -50 -50

Fig. 4.6 Zeta-potential of (a) Pyrite (b) Chalcopyrite after interaction with ◆ × different Paenibacillus polymyxa cells. ( ) mineral alone, ( ) mineral interacted ▲ with chalcopyrite adapted cells, ( ) mineral interacted with pyrite adapted cells ■ and ( ) mineral interacted with wild cells.

20 (a) 20 (b)

10 10 pH pH

0 0 2345678910 2345678910 -10 -10

-20 -20

-30 -30 Zeta-potential (mV) Zeta-potential (mV) -40 -40

-50 -50

Fig. 4.7 Zeta-potential of (a) Pyrite (b) Chalcopyriteafter interaction with

different Thiobacillus ferrooxidans cells. (■) mineral alone, (●) mineral interacted with ferrous iron grown cells (▲) mineral interacted with sulfur grown cells.

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4. Microbial Adhesion on Mineral Surfaces Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001 Electrokinetic studies. The zeta-potential behaviour of pyrite before and after interaction with different types of P. polymyxa is presented in Fig. 4.6(a), and similarly for chalcopyrite in Fig. 4.6(b). The IEP of pyrite was observed to be pH ∼6.8. The IEP obtained for chalcopyrite was pH ∼7.4 which is higher when compared to the literature value of less than pH 3 (Healy et al., 1976, McGlashan et al., 1969). The shift in the IEP of minerals towards the IEP of the cells indicates specific adsorption of cells on the minerals. The IEP for both pyrite and chalcopyrite shifted to less than pH 2 when they are interacted with wild and pyrite adapted P. polymyxa. On the other hand, the IEP of pyrite shifted from pH ∼6.8 to ∼5.2 and of chalcopyrite from pH ∼7.4 to ∼3 when they are interacted with chalcopyrite adapted P. polymyxa. The IEP shift in case of chalcopyrite is observed to be much more pronounced than for pyrite, which can be due to higher affinity of chalcopyrite, adapted cells towards chalcopyrite mineral.

Fig. 4.7(a) shows the zeta-potentials of the mineral after interaction with ferrous ions grown T. ferrooxidans. The IEP's of both the sulfide minerals shifted to a lower pH values, i.e., pyrite pH 4.5 and chalcopyrite pH 4.1. In the presence of sulphur grown T. ferrooxidans (Fig. 4.7b), pyrite and chalcopyrite exhibited IEP's of pH 3.5 and 4.3 respectively. Although the shift in IEP´s does not correspond to full coverage of the cells on mineral, the sulphur grown cells cause more shift in the IEP of pyrite as compared to ferrous grown cells and for chalcopyrite the shift is similar in both the cases of the cells.

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Paper V Communicated to Colloids and Surfaces B: Biointerfaces

4. Microbial Adhesion on Mineral Surfaces: Paper V Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001

Adhesion of Paenibacillus polymyxa on chalcopyrite and pyrite: Surface thermodynamics and extended DLVO theory

P.K. Sharma, K. Hanumantha Rao Division of Mineral Processing, Luleå University of Technology SE-97187, Luleå, Sweden

Abstract The adhesion behaviour of Paenibacillus polymyxa bacteria on pyrite and chalcopyrite is examined by the surface thermodynamics and the extended DLVO theory approaches. In addition, the bacteria are adapted to pyrite and chalcopyrite minerals and the adhesion behaviour of these bacteria are also investigated. The significance of acid/base interactions in adhesion is assessed. The essential parameters needed for the calculations of interaction energy between bacteria and mineral are experimentally determined. The results illustrate that the bacterial surfaces are more energetic than the mineral surfaces and the bacteria acquired acid/base surface energy component during their adaptation to mineral. The extended DLVO approach is found to be more affective in predicting the adhesion behaviour than the expectations from thermodynamic approach. The thermodynamic approach yields no bacterial adhesion on minerals and this discrepancy is the result of inadequate description of electrostatic interactions. The adhesion predictions by the DLVO approach are able to partially explain the bioflotation results of pyrite and chalcopyrite. Extended DLVO shows that on account of high bacterial surface energy their aggregation is not feasible. But due to the hydrophobicity of pyrite and chalcopyrite, their aggregation is possible.

Introduction The advantages in using various microorganisms in mineral beneficiation and metal extraction processes have been emphasised by several authors [1-7]. Bio- leaching methods to treat copper, uranium and refractory gold ores, complex sulfides, manganese ore and industrial minerals have been developed and these processes have been adopted by certain industries. The microbes are capable to solubilize the valuable metals from the ore bodies by the oxidation of minerals. Although bioleaching is very effective for lean grade ores, the major hurdle for wider acceptance of this process is its very slow rate of the bio-oxidation step of minerals. The mechanism of bio-oxidation is still under debate but one of the mechanisms often quoted concerns the adhesion of bacteria on mineral thereby inducing the oxidation process [8,9]. The fundamental aspects concerning bacterial adhesion and its role in bioleaching contribute to the enhancement of leaching rates. In contrast to bioleaching, the biobeneficiation processes are very fast as they only concern with the modification of surface properties by the bacterial adhesion. Biobeneficiation covers the realm of bio-flotation and bio-flocculation. In both these processes, the concentration of a valuable mineral from ore bodies is accomplished while exploiting the differences in the surface properties. The selective adhesion of bacteria on minerals is required to alter the surface properties so as to achieve mineral separation either in flotation or in flocculation. Thus, the bacterial adhesion play a critical role in both bioleaching and biobeneficiation processes.

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Mineral Bioprocessing

Bio-beneficiation Bio-leaching

Bio-flotation Bio-flocculation

Factors imparting selectivity

Adhesion of microbes Interaction of adsorbed microbial cells with selectively on mineral surface added chemical reagents for flotation or flocculation

Prediction

Thermodynamic approach DLVO approach In this approach the interfacial free Microbial adhesion is described as a balance between energy of the interacting surfaces i.e. Lifshitz van der Waals attractive force, Electrostatic microbe and mineral is compared before forces and acid/base interaction forces with separation and after adhesion distance ∆ = γ −γ −γ Gadh sb sl bl G =G +G +G total LW EL AB

∆ GTotal Gadh ∆ LW ∆ AB Calculations Calculations vs ( Gadh , Gadh ) Separation distance

LW/AB LW/AB/+/- Equation approach approach of state γ LW γ AB γ LW γ + γ - γ sv , sv sv , sv , sv sv γ LW γ AB γ LW γ + γ - γ bv , bv bv , bv , bv bv γ LW γ AB γ LW γ + γ - lv , lv lv , lv , lv

Water, Water, Formamide Zeta-potential Water Methyleneiodide Methyleneiodide

Contact Angles measurement

Fig. 1 Factors influencing biobeneficiation and their prediction

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4. Microbial Adhesion on Mineral Surfaces: Paper V Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001 The separation of minerals in biobeneficiation is governed by two major factors, selective adhesion of microbial cells on mineral surface and the differential interaction of added chemical reagents with the microbe pre-conditioned mineral (Fig. 1). In order to fully understand and control the biobeneficiation processes, it is important to understand these two factors separately. The attachment of microbial cells on mineral surface is influenced by several properties, for example, surface charge, surface hydrophobicity, presence and configuration of surface polymers. Any theory that attempt to explain the bacterial adhesion must incorporate all these parameters. In general, the bacterial adhesion can be illustrated by the surface thermodynamics and by the extended DLVO theory of calculating the interaction energy as a function of separation distance [10]. These methods take into account Lifshitz van der Waals interactions, electrostatic interactions and hydrophobic/hydrophilic force interactions. These interactions are very well understood and formulated in mathematical equations. Apart from these interactions the microbial cells can have polymer interactions. The polymer layer on the microbial surface does not have a well defined boundary and polymers can extend in the liquid as much as 300 Å or more for gram negative bacteria [11-13]. Hence, polymer interaction can deviate the adhesion behaviour from the one as advocated by the above considerations. In this paper, an attempt has been made to predict and understand the adhesion of a heterotrophic bacteria, Paenibacillus polymyxa, on sulfide minerals through surface thermodynamics and extended DLVO theory. The resulting adhesion behaviour is examined in the bioflotation of sulfide minerals

Physico-chemical approaches of microbial adhesion Thermodynamic approach. In this approach the interfacial free energy of the interacting surfaces, i.e., microbe and mineral, is compared before and after adhesion. Fig. 2 shows that the adhesion of microbe on mineral surface leads to the formation of new interface i.e., microbe-mineral at the expense of microbe-medium and mineral- medium interfaces. The comparison is expressed in terms of free energy of adhesion: ∆ = γ − γ − γ Gadh sb sl bl (1) where, γsb, γsl and γbl are mineral-microbe, mineral-liquid and microbe-liquid interfacial free energies respectively. Like all systems in nature, this ml ml system will also proceed in the direction of M γ -γ lowering the total energy. Which means that adhesion of microbe will take place on L M γsl -γsl the mineral when ∆Gadh is negative and +γms adhesion is thermodynamically S S unfavourable when ∆Gadh is positive. ∆Gadh Fig. 2 Thermodynamics of calculation needs the values of γsb, γsl and bacterial adhesion γbl. The interfacial energies of γsl and γbl are possible to be calculated by using contact angle data measured with standard liquids with known free energies. Different approaches to convert contact angle data to surface free energies are described previously [14]. Detailed description of the methods can be found in the literature: equation of state approach [15,16], geometric mean approach [17] and Lifschitz van der Waals-acid base approach (LW-AB) [18-

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4. Microbial Adhesion on Mineral Surfaces: Paper V Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001 22]. In the present study LW-AB approach was used in order to calculate solid surface free energies and interfacial energies. The γsb is obtained from γsl and γbl by using the aforementioned approaches. Depending on the approach used in order to attain the γsb, γsl and γbl values, the ∆Gadh value is different.

Equation of state ( γ − γ )2 ( γ − γ )2 ( γ − γ )2 ∆G = sv bv − sv lv − bv lv (2) adh − γ γ − γ γ − γ γ 1 1.015 sv bv 1 1.015 sv lv 1 1.015 bv lv

For geometric mean approach and LW-AB approach the total free energy of adhesion can be divided into Lifshitz van der Waals component and acid/base component: ∆ = ∆ LW + ∆ AB Gadh Gadh Gadh (3)

Geometric mean approach ∆ LW = − ( γ LW − γ LW )( γ LW − γ LW ) Gadh 2 bv lv sv lv (4) ∆ AB = − ( γ AB − γ AB )( γ AB − γ AB ) Gadh 2 bv lv sv lv (5)

LW-AB approach. The Lifshitz van der Waals component is the same as the geometric mean approach but the acid base component is calculated by the following expression: ∆ AB = + ( γ + − γ + )( γ − − γ − )− ( γ + − γ + )( γ − − γ − ) Gadh 2 bv sv bv sv 2 bv lv bv lv − ( γ + − γ + )( γ − − γ − ) 2 sv lv sv lv (6)

Extended DLVO approach. Classical DLVO theory as described by Derjaguin, Landau, Verwey and Overbeek [23,24] includes (LW) Lifshitz van der Waals attractive forces and attractive or repulsive (EL) electrostatic force. The LW is the force of attraction between neutral and chemically saturated molecules and was postulated by van der Waals in order to explain non-ideal gas behaviour. This force includes attractive force between permanent dipole-permanent dipole, permanent dipole-induced dipole and induced dipole-induced dipoles. Electrostatic forces arise due to the double layer interactions between two particles and thus zeta-potential becomes important. Later the acid/base contribution was added by van Oss and co- workers [25]. The acid/base interactions are based on electron-donating and electron- accepting interactions between polar moieties in aqueous solutions. Therefore, microbial adhesion is described as a balance between attractive Lifshitz van der Waals force, repulsive or attractive electrostatic force and acid/base interaction force. GTotal(H)=GLW(H)+GEL(H)+GAB(H) (13) The decay of these forces depends on the geometry of interacting bodies and the type of force. Table 1 summarises the mathematical formulations used for the calculation of these forces.

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4. Microbial Adhesion on Mineral Surfaces: Paper V Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001 Table I. Interaction energy terms used in extended DLVO approach

Lifshitz-van der Waals interaction energy- GLW(H)     Sphere-Sphere A  y y  x 2 + xy + x  1 (7) − + + 2ln     2 2  2  12 x + xy + x x + xy + x + y  x + xy + x + y    2πH    1+1.77     λ       Sphere-Flat plate ()+ + (8) − A  2a H a −  H 2a  1   ln  12  H ()H + 2a  H   2πH   1 + 1.77     λ   λ=1000 Å 1.1 Electrostatic interaction energy- GEL(H) 2 2 −κH πεa a ()ζ + ζ  2ζ ζ 1 + e − κ  Sphere-Sphere 1 2 1 2 1 2 ln + ln ()1 − e 2 H (9) + ζ 2 + ζ 2 − −κH  a1 a 2  1 2 1 e  − κ H  2ζ ζ 1 + e − κ  Sphere- Flat plate πε a ()ζ 2 + ζ 2 1 2 ln + ln ()1 − e 2 H (10) 1 2  ζ 2 + ζ 2 − −κ H   1 2 1 e 

Acid-Base interaction energy- GAB(H) []()− λ π λ∆ AB do H Sphere-Sphere a Gadh e (11) []()− λ π λ∆ AB do H Sphere-Flat plate 2 a Gadh e (12) H-Separation distance, a- Radius of solid particle, ζ- Zeta-potential, κ- Double layer -1 10 2 ½ -1 thickness (=0.328x10 (zi Mi) m ), A- Hamaker Constant, x= H/a1+a2, y= a1/a2, do- Minimum separation distance between 2 surfaces (1.57 Å), λ- Correlation length of molecules in liquid (≈ 6 Å)

Apart from the knowledge of size of the interacting particles, different interaction forces need specific parameters for the calculations to be performed. The major problem in evaluating Lifshitz-van der Waals interaction energy is that of evaluating Hamaker constant. Two methods are available for its evaluation. In the first, i.e., London-Hamaker microscopic approach, the constant is evaluated from the individual atomic polarisabilities and the atomic densities of the materials involved. The total interaction is assumed to be the sum of the interactions between all the interparticle atom pairs and is assumed to centre on a single oscillation frequency. The other method is the macroscopic approach of Lifshitz, in which the interacting particles and the intervening medium are treated as continuous phases. The calculations are complex, and require availability of bulk optical/dielectric properties of the interacting materials over a wide frequency range. The presence of a liquid dispersion medium, rather than vaccum (or air), between the particles notably lowers the van der Waals interaction energy. Therefore, constant A in the equation must be replaced by an effective Hamaker constant. Considering interaction of particle 1 and 2 in the medium 3, the effective Hamaker constant is: A132=A12+A33-A13-A23 (14) The attarction between unlike phases is taken to be geometric mean of the ½ attractions of each phase to itself i.e., A12=(A11.A22) , then the equation becomes ½ ½ ½ ½ A132=(A11 -A33 )(A22 -A33 ) (15) And if the particles are of the same material, then ½ ½ 2 A131=(A11 -A33 ) (16)

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4. Microbial Adhesion on Mineral Surfaces: Paper V Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001 Fowkes [26] proposed the following equation to calculate the individual Hamaker constant of solid surfaces: 2 d A11=6πr γs (17) d where, r represents the intermolecular distance and γs represents the dispersion component of the surface free energy of the solid. According to Fowkes, for water and systems with the volume element such as metal atoms, CH2 and CH groups, which have nearly the same size, the value of 6πr2 can be taken as 1.44x10-18 m2. After evaluating the individual Hamaker constant, the effective Hamaker constant can be calculated by using equation 6.

Table II Surface energies (ergs/cm2) as obtained from literature [21,29,30]

Liquid γtotal γd/γLW γP/γAB γ+ γ- Water 72,8 21,8 51 25,5 25,5 Formamide 58 39 19 2,3 39,6 1-Bromonapthalene 44,4 44,4 ≈0 <0,1 <0,1 Methyleneiodide 50,8 50,8 ≈0 <0,1 <0,1

The extended DLVO approach as developed by van Oss and co-workers can be considered a combination of the thermodynamic and classical DLVO approaches, since the Hamaker constants are obtained from the Lifshitz-van der Waals interaction ∆ LW energy Gadh as calculated from contact angle measurements according to the equation 4. The equation 18 gives the effective Hamaker constant: = − π 2∆ LW A 12 do Gadh (18) The Hamaker constant was evaluated by two methods: 1) effective Hamaker constant was calculated using equation 18, and 2) Hamaker constant for bacteria was calculated using equation 17 and the γLW component, for mineral the value was used from literature. Effective Hamaker constant was calculated by equation 15. Evaluation of the electrostatic interaction term needs the surface potential values of the interacting particles at the interaction pH and at particular ionic strength, which inturn, dictates the value of κ (double layer thickness-1). The zeta-potential can be measured as a function of pH by methods based on electrophoresis, electro- osmosis and streaming potential techniques and this potential can be approximated to surface potential. ∆ AB Evaluation of acid-base interaction energy requires the knowledge of Gadh which can be calculated using equation 4 or 5 depending on the approach used.

Experimental methods Contact Angle measurement. Polar liquids of water and formamide, and apolar liquids of α-bromonapthalene and methylene iodide were used to measure the contact angles on mineral surfaces and bacterial lawns. The contact angles of these four liquids on bacterial lawns and solid sulfide surfaces were measured using the sessile drop technique with FIBRO 1100 DAT dynamic absorption tester. The surface energy components of the liquids are shown in Table II. Mineral surfaces. Pure solid mineral crystals were cut in order to attain small pieces with flat surfaces. These mineral pieces were polished successively on finer and finer polishing paper with water as a medium. The final paper used for polishing

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4. Microbial Adhesion on Mineral Surfaces: Paper V Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001 was #4000. Mineral pieces were mounted on the sample holder with the help of plasticine clay and then the contact angles were measured. In between measurements the mineral surfaces were freshly polished with paper #4000 and dipped in ethanol in order to prevent any oxidation of the surface, contact angle was measured within 5-10 minutes of polishing. Bacterial cells. Contact angles were measured on 800-900 bacterial layer thick lawns. The bacterial lawns were obtained by filtering a thick cell suspension through millipore filter [27,28]; the bacterial suspension was enough to completely distribute over the filter during the process of filtration in order to achieve a homogeneous thickness of the lawn. The Millipore filters were placed on 1% agar plate (10% vol./vol. glycerol in water) for about 15-30 minutes for homogenisation of water content in the lawn. The filter was then cut in square pieces and mounted on the sample holder plate with the help of double sided tape and allowed to air dry for 30- 60 minutes so that only bound water is present on the cell surface [29]. After that the contact angle was measured. Although the aforementioned authors suggest only about 60-70 layer thick lawns but these were found to be too thin to achieve a equilibrium state when the contact angles were measured, the reason is further explained in the results section of this paper.

Zeta-potential measurements. The zeta-potentials measurements of Paenibacillus polymyxa cells and sulfide minerals were made with a Laser Zee Meter (Pen Kem Inc., Model 501) equipped with a video system after 60 min conditioning at a specified pH value. The cells zeta-potentials were measured with a bacterial population of ~ 2x106 cells/ml and for mineral samples, a concentration of 1 g/l was used. All the measurements were made at constant ionic strength (I = 0.001) using KNO3. When the measurements with mineral samples in the presence of cells were conducted, the minerals were preconditioned with cells at the desired pH values.

Results Surface free energy of solids and bacteria. The contact angles measured with the four liquids on pyrite and chalcopyrite, and the calculated surface energy components using LW-AB approach are presented in Table III. The surface energies of 44.1 and 38.1 mJ/m2 for pyrite and chalcopyrite respectively show that both the mineral surfaces are equally energetic. If we compare only the γAB part then chalcopyrite surface seems less polar than pyrite surface. But, chalcopyrite have higher electron donating (γ-) part as compared to pyrite but because chalcopyrite have very low electron accepting part (γ+) therefore, the γAB became low. The bacterial surfaces are found to be more energetic than mineral surfaces, and hence more hydrophilic, principally due to very high electron donating characteristic. The unadapted P. polymyxa cells surface became more energetic after adaptation to pyrite and chalcopyrite minerals. The cells surface energy of unadapted cells (29 mJ/m2) increased to 57,3 and 62,2 mJ/m2 for pyrite and chalcopyrite adapted cells respectively. The increase in surface energy is primarily due to fact that mineral- adapted cells acquired the γ+ component and hence γAB also became higher. The results suggest that the bacterial secretions are more when they are grown in the presence of minerals and the cell surfaces have higher density functional groups after adaptation. Although the surface energy of pyrite and chalcopyrite mineral is higher than unadapted P. polymyxa which, is because of the fact that γAB component for them is 219

4. Microbial Adhesion on Mineral Surfaces: Paper V Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001 small. But unadapted cells have much higher γ- component but because of very low value of γ+, the γAB value becomes small and, hydrophilicity depends on both γ+ and γ- interaction with water.

Hamaker constants. The Hamaker constants are evaluated using the two methods as described before. The results are presented in Table IV. The values of effective Hamaker constant obtained by the two methods are similar for all the systems. Method 1 calculates the Hamaker constant based on the Lifshitz van der ∆ LW Waals component of the free energy of adhesion ( Gadh ) which, in itself considers the fact that interaction is taking place in water instead of vaccum. So effective Hamaker constant is obtained by using equation 18. On the other hand, method 2 evaluates the individual Hamaker constants for the two interacting particles and then incorporates the affect of water by the equation 14. In this method Hamaker constant for bacterial cells was calculated using equation d 17 and γs but, the Hamaker constant for mineral was taken from literature. The literature value for pyrite mineral as shown in Table IV is higher than chalcopyrite and hence, the effective Hamaker constant evaluated for the case in which pyrite is interacting with bacterial cells is higher than in the case of pyrite. Zeta-potential. The surface of a 20 bacterial cell is charged due to the presence Pyrite mineral Chalcopyrite mineral of functional groups such as carboxyl (- 10 Unadapted P.p. COOH), amino (-NH2) and hydroxyl (- Pyrite-adaped P.p. Chalcopyrite-adapted P.p.

OH), originating from the cell wall V) 0

m 34567891011 ( components of lipopolysaccharides, l pH -10a lipoprotein and bacterial surface proteins. ti en The zeta-potentials of the three types of P. -20t polymyxa as a function of pH are shown in a-po t

Fig. 3. The zeta-potential behaviour of -30e adapted and unadapted cells is same, the Z cells are negatively charged throughout the -40 pH range with an iep of less than pH 2.5. The results in Fig. 3 show an iep's of about Fig. 3 Zeta-potential behaviour as a pH 6.8 and 7.4 for pyrite and chalcopyrite function of pH respectively. Free energy of Adhesion Free energy of adhesion of unadapted, pyrite- adapted and chalcopyrite-adapted P. polymyxa on pyrite and chalcopyrite mineral is calculated using LW-AB approach and is shown in Table V. The Lifshitz van der Waals component is attractive for all the P. polymyxa on both pyrite and chalcopyrite but the acid/base component is highly repulsive, especially for chalcopyrite mineral. The total free energy of adhesion of P. polymyxa on both minerals is positive therefore, the thermodynamic approach predicts that there will be no adhesion of P. polymyxa on either pyrite or chalcopyrite. All the P.polymyxa strains are hydrophilic and especially the mineral adapted ones therefore the thermodynamics predicts repulsion from the mineral surface as the bacterial cells prefer to stay in the aqueous phase than to attach themself to the mineral surface. The chalcopyrite mineral exhibits higher acid/base repulsion as compared to pyrite due to the fact that chalcopyrite have very low electron accepting character (γ+) and pyrite have comparatively higher electron accepting character. All the P.polymyxa have very high electron donating character (γ-) and hence they are

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4. Microbial Adhesion on Mineral Surfaces: Paper V Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001 relatively less repelled from pyrite. The maximum repulsion is between chalcopyrite mineral and unadapted P.polymyxa cells, its because both have very low γ-/γ+ ratio.

Table III Contact angle and surface free energy data for mineral and bacterial cells.

Contact Angle θ(o) Surface energy (mJ/m2) Water Formamide Diiodomethane α-Bromonapthalene γLW γ+ γ- γAB Pyrite mineral 70 50.35 35.2 27.5 41.9 0.11 10.5 2.23 Chalcopyrite 70 57.16 48.83 Asymmetric drops 37.6 0.006 15.4 0.59 mineral Unadapted 40.81 56 66.68 51.84 28.6 0.001 59.1 0.48 P.polymyxa Pyrite- 19.85 18.42 71.8 56.66 26.6 4.73 49.8 30.7 adapted P.polymyxa Chalcopyrite- 15.85 21.05 70.6 55.6 27.1 6.78 45.3 35.1 adapted P.polymyxa

Table IV The calculated values of Hamaker constants for different systems.

* LW 2 1.1.1 System H11(bacteria) H22(mineral) ∆G (mJ/m ) H132(Method 2) H(Method 1) Pyrite-Unadapted P.p. 38.4x10-21 J 120x10-21 J -2.4597 14.09x10-21 J 2.29x10-21 J Pyrite-Pyrite adapted 38.4x10-21 J 120x10-21 J -1.7825 12.6x10-21 J 1.66x10-21 J P.p. Pyrite-Chalcopyrite 38.4x10-21 J 120x10-21 J -1.9567 12.9x10-21 J 1.82x10-21 J adapted P.p. Chalcopyrite- 39.1x10-21 J 33x10-21 J -1,9942 3.05x10-21 J 1.85x10-21 J Unadpated P.p. Chalcopyrite-Pyrite 39.1x10-21 J 33x10-21 J -1.4457 2.73x10-21 J 1.34x10-21 J adapted P.p. Chalcopyrite- 39.1x10-21 J 33x10-21 J -1.5869 2.81x10-21 J 1.47x10-21 J chalcopyrite adapted P.p. * Literature values (Lins et al. 1995)

Table V. Free energy of adhesion.

2 System Free energy of Adhesion ∆Gadh (mJ/m ) LW AB Total ∆Gadh ∆Gadh ∆Gadh Unadapted P.p. – Pyrite -2.46 6.68 4.22 Pyrite-adapted P.p. – Pyrite -1.78 8.51 6.72 Chalcopyrite-adapted P.p. - Pyrite -1.95 7.00 5.05 Unadapted P.p. – Chalcopyrite -1.99 15.05 13.06 Pyrite-adapted P.p. – Chalcopyrite -1.44 13.57 12.13 Chalcopyrite-adapted P.p.- Chalcopyrite -1.58 11.3 9.71

DLVO approach. Using the sphere-sphere equations 7, 9 and 11 the free energy of interactions, in kT units, were calculated for bacterial cell-bacterial cell, mineral-mineral and mineral-bacterial cell systems. These results are presented in Figs. 4, 5 and 6. Bacteria-bacteria system. The sum of van der Waals interactions, electrostatic interaction and acid/base interactions between bacterial cells themselves as a function of ionic strength (0.1 to 0.0001 M) and in the pH range 5 to 10 showed high repulsion at closest approach (Fig. 4). This means that aggregation of bacterial cells in the aqueous phase with pH 5-7 and ionic strength 0.01 to 0.0001 M is not possible. Although the van der Waals interactions are attractive but the acid/base interaction, which operate in a short range of about 50 Å, is highly repulsive. At large distances

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4. Microbial Adhesion on Mineral Surfaces: Paper V Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001 also the interaction is repulsive due to the electrostatic repulsion. The reason for this repulsion is the fact that bacterial cells are hydrophilic and hence they prefer to stay in aqueous phase rather than forming flocs. Mineral-mineral system. The total interaction energy vs. separation distance curves for pyrite-pyrite, chalcopyrite-chalcopyrite and pyrite-chalcopyrite are shown in Figs. 5a, 5b and 5c respectively. For the three systems at low ionic strength (I=0.0001 M) the energy barrier is very high for flocculation to take place but as the ionic strength is increased the barrier decreases and at I= 0.01 M the energy barrier is only about 10-15 kT where the flocculation is possible. The electrostatic repulsion is very high for the mineral system at pH 10 as the zeta-potential of the particles is highly negative (~ -35 mV) and hence flocculation is not possible on account of an energy barrier of about 800 kT. At pH 5 the zeta- potential of the particles is positive (~10 mV) and the energy barrier is about 50 kT. At pH 7 the particles are only slightly charged and in the case of pyrite-chalcopyrite, they are oppositely charged and hence flocculation is predicted. When the size of the particle increases from 1 µm to about 5 µm, even though the van der Waals attraction increases but the electrostatic repulsion also increases. Therefore, the total interaction energy curve shows an energy barrier of 175 kT for 5 µm particles. But for particles of 1 µm size the energy barrier is low (25-30 kT). Mineral-bacteria system. Total interaction energy vs. separation distance curves of pyrite and chalcopyrite interaction with different P. polymyxa is shown in Figs. 6a and 6b respectively. The acid/base interaction between mineral-bacteria is highly repulsive but this force operated only at close distances of 50 Å. At pH values where the bacterial cells and mineral particles are oppositely charged the attractive electrostatic interactions cause the formation of secondary minima where adhesion of bacterial cells is possible on mineral surface. At pH 10 both mineral and bacteria are highly negatively charged and hence electrostatic interaction is repulsive. At pH 5 both the minerals are positively charged but bacterial cells are negatively charged (approximately same as at pH 10) and hence electrostatic interaction is highly attractive causing deepest secondary minima (~625- 1000 kT). This predicts irreversible attachment. At pH 7 the mineral is only slightly charged hence the secondary minima is not very deep (~125-250 kT). Therefore, attachment is reversible. The mineral-bacteria interaction energy curves predict attachment of different P. polymyxa equally on both pyrite and chalcopyrite.

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4. Microbial Adhesion on Mineral Surfaces: Paper V Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001

1000 Unadapted P.p.- Unadapted P.p.

750

G G (k= EL Total 3.28X1 7 0 m-1) 500

) G M

T 1 ( otal k=1

0 .04

0 X

. 108 m -

0 1) =

G(kT)

( G 250 B T (

A ot k=

al 3.28 G X 108 m -1)

0 25 50 75 100 125 150 175

G (I=0.001M) Separation distance(Å) LW -250 1000 Pyrite adapted P.p.-Pyrite adapted P.p.

G 750 EL G T (k=3 otal .28X1 7 0 m-1)

500 G Tot (k= al 1.04 X108 m-1) G(kT)

250 B G

A Tot (k= G al 3.28 X1 8 0 m- 1) 0 25 50 75 100 125 150 175 G LW Separation distance(Å) -250 1000 Chalcopyrite adapted P.p.- Chalcopyrite adapted P.p G EL G ( Tota k=3.2 750 l 8X107 m-1)

) G

M 1 T 500 ota (k= 0 l 1.0

0 4X . 108

0 m -1

= )

(

B G(kT) A G 250 G To (k tal =3. 28X 108 m-1 ) 0 25 50 75 100 125 150 175 G (I=0.001M) LW Separation distance(Å)

-250

Fig. 4 Free-energy vs separation distance curves for bacteria-bacteria system in pH range 5 to 10.

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4. Microbial Adhesion on Mineral Surfaces: Paper V Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001

150 At pH 5 and for partical size of 2 microns G EL G (k= Tota 3.28x 7 l 10 m-1) 100

G (k Total =1. 50 04x1 8 of ionic strength 0 m-1) G(kT)

G (k= Tota 3.28x 8 l 10 m-1)

0 25 50 75 100 125 150 175

Separation distance(Å)

G G LW AB 1000-50 At partical size of 2 microns and I=0.001M, k=1.04X108 m-1

750 G T ota (p l H 1 0) 500

G(kT) 250 G (pH 5) EL G (pH 5) Total

0 25 50 75 100 125 150 175

G (pH 7) Separation distance(Å) Total of pH As a function As a function -250 G (pH 5) AB 5) (pH LW

G -500200 At I=0.001 M, k=1.04X108 and pH 5

G 150 (2 G EL µm p To (5 art tal µ icle m s) par ticle s) 100

G (2 µ Total m pa 50 rticles)

G(kT) G (1 µm Total particles) 0

)25 50 75 100 125 150 175 s e l c Separation distance(Å) i t r -50 a p

µ m G (2 m particles) µ AB 2 (

W L

As a function of particle size of size As particle a function -100 G Fig. 5a Free energy vs separation distance curves for chalcopyrite-chalcopyrite particle system.

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4. Microbial Adhesion on Mineral Surfaces: Paper V Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001

150 At pH 5 and partical size of 2 microns G E ( L I=0 .00 G (k= 1 Total 3.28X107 -1 M) m ) 100

of ionic strength of ionic strength G 50 T (k otal =1.0 4X1 8 G(kT) 0 m- 1) G (k=3.28X108 m-1) Total 0 25 50 75 100 125 150 175 G (I=0.001M) Separation distance(Å) LW G (I=0.001M) AB 1000-50 At partical size of 2 microns and I=0.001M, k= 3.28X108 m-1

750 G To ( tal pH 10 500 )

G(kT) 250 G (pH 5) G (pH 5) EL Total

0 25 50 75 100 125 150 175 ) 5

As a function of pH As a function H Separation distance(Å) p G (pH 7) ( Total

-250 W L G G (pH 5) AB -500 200 At pH 5 and I=0.001M, k= 3.28X108 m-1

G 150 E (2 G L µm To (5 pa tal µm rtic pa les rtic ) les) 100

G (2 µm 50 Total particles)

G(kT) G (1 µm Total particles) 0 25 50 75 100 125 150 175 Separation distance(Å) -50 G (2 µm particles) AB

G (2 µm particles)

size As a function of particle LW -100 Fig. 5b Free energy vs separation distance curves for pyrite-pyrite particle system.

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4. Microbial Adhesion on Mineral Surfaces: Paper V Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001

150 At pH 5 and for partical size of 2 microns G EL G To (k=3.2 tal 8x107 -1 m ) 100

of ionic strength (k Total =1.0 50 4x108 m-1) G(kT) G (k= Total 3.28x1 8 0 m-1) 0 25 50 75 100 125 150 175 Separation distance(Å)

G G LW AB 1000-50 At partical size of 2 microns and I=0.001M, k=1.04X108 m-1

750 G To (p tal H 9) 500

G(kT) 250 G (pH 5) EL G (pH 5) Total 0 25 50 75 100 125 150 175

As a function of AspH a function As a function Separation distance(Å)

) G (pH 7) Total -250 5 H

p ( G (pH 5) W AB L

G -500200 At I=0.001 M, k=1.04X108 and pH 5

G 150 (2 G EL µm p To (5 art tal µ icle m s) par ticle s) 100

G (2 µ Total m pa 50 rticles)

G(kT) G (1 µm Total particles) 0 25 50 75 100 125 150 175 ) s le c Separation distance(Å) ti r a -50 p

m G (2 µm particles) µ AB 2 (

As a function of particle size size Asof particle a function W L -100 G Fig. 5c Free energy vs separation distance curves for chalcopyrite-pyrite particle system.

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4. Microbial Adhesion on Mineral Surfaces: Paper V Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001

1000 Pyrite - Unadapted P.p. system 750 G AB G ( Total pH 500 10)

250 Separation distance(Å)

0 25 50 75 100 125 150 175

G(kT) -250 G (pH 7) G Total LW 5) (pH G -500 Total

-750 G EL -10001000 Pyrite - Pyrite adapted P.p. system

750 )

H H G

( B T

500 ota (pH A l 10) G 250 Separation distance(Å) 0 25 50 75 100 125 150 175 G(kT) G (pH 7) -250 Total

-500 G (pH 5) Total -750

G (pH 5) EL -10001000 Pyrite - Chalcopyrite adapted P.p. system

750 G AB G 500 To (pH tal 10)

250 Separation distance(Å) 0 05010015020G 0 LW -250G(kT) G (pH 7) ) Total H 5 -500 (p al G G Tot EL -750

-1000

Fig. 6a Interaction of pyrite mineral with different

Paenibacillus polymyxa at different pH.

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4. Microbial Adhesion on Mineral Surfaces: Paper V Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001

1000 Chalcopyrite - Unadapted P.p. system 750 G ( G Tota pH 500 AB l 10)

250 Separation distance(Å) 0 050100150200

G(kT) -250 G LW (pH 5) GTotal -500

-750 G G (pH 7) EL Total

-10001000 Chaclopyrite - Pyrite adapted P.p. system

750 G AB 500 G To (pH tal 10) 250 Separation distance(Å) 0 050100150200 G(kT) G pH 7) -250 LW ( GTotal

5) -500 (pH al G Tot -750

G EL -1000 1000 Chalcopyrite - Chalcopyrite adapted P.p. system

750 G AB G (p 500 Total H 10)

250 Separation distance(Å) 0 050100150200

G(kT) 7) -250 G (pH LW G l Tota 5) (pH G otal -500 T

-750 G EL -1000 Fig. 6b Interaction of chalcopyrite mineral with

different Paenibacillus polymyxa at different pH.

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4. Microbial Adhesion on Mineral Surfaces: Paper V Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001

100 (a) 90

80 70

60 50

% Recovery 30

20 10

No Cells 105 106 107 108 -1 Cell density ( Cells ml ) 100 (b) 90 80

70 60

50 40

30 % Recovery 20

10 0 No Cells 106 107 108 Cell density (cells ml-1) 100 (c) 90 80

70 60

50 40

% Recovery 30 20

10 0 No Cells 105 106 107 108 Cell density (cells ml-1)

Fig. 7 Flotation of minerals in the presence of (a) Unadapted (b)pyrite-adapted and (c) chalcopyrite-adapted Paenibacillus polymyxa cells. ( ) pyrite, (■) pyrite + 0.01 mM xanthate, (Ο) chalcopyrite, (●) chalcopyrite + 0.01 mM xanthate. 229

4. Microbial Adhesion on Mineral Surfaces: Paper V Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001 Discussion The thermodynamic approach predicts that there will be no attachment of bacterial cells on the mineral surface. The reason behind is the strong acid/base repulsion, which outweighs the van der Waals attraction. The other reason is the fact that electrostatic interactions are not considered at all in the thermodynamic approach. The DLVO approach gives a more realistic picture. It predicts repulsion between the individual bacterial cells, possibility of flocculation of mineral particles and attachment of bacterial cells on the mineral surface. Although the acid/base interaction is repulsive in the case of bacteria-mineral system but they are short-range interactions and due to attractive electrostatic interactions at longer distance a secondary minima is predicted at 25 Å. And hence attachment in this minima. In the previous work [14] where flotation of pyrite and chalcopyrite was performed in the presence of different P. polymyxa, the results clearly showed the attachment of bacterial cells on minerals. This is predicted by DLVO approach but not by thermodynamic approach. The flotation results as summarised from our earlier work in Fig. 7 show a possibility of selective flotation of chalcopyrite from pyrite. When the flotation is performed in presence of unadapted P. polymyxa and chalcopyrite-adapted P. polymyxa, selectivity is achieved but not in presence of pyrite-adapted P. polymyxa. The DLVO approach predicts adhesion of all the three types of P. polymyxa on the both the minerals equally. This is actually observed in the flotation experiments, as the flotation recovery in presence of all the three P. polymyxa cells for both pyrite and chalopyrite in absence of any collector is same. Even though the natural floatability of chalopyrite is high, after interaction with microbial cells it becomes same as pyrite. In presence of collector (potassium isopropyl xanthate) the flotation recovery of chalcopyrite remains the same but the recovery of pyrite reduces. In this case the second factor comes into play where the interaction of the collector is different towards bacteria pre-conditioned chalcopyrite and pyrite.

Conclusions The flotation results show that unadapted and mineral-adapted Paenibacillus polymyxa do attach on the mineral surface and hence change the flotation behaviour. The thermodynamic approach did not predict the attachment and showed that there will be repulsion between bacteria and mineral on account of highly repulsive acid/base interactions. But the extended DLVO approach was effective in predicting the attachment behaviour of different P. polymyxa on pyrite and chalcopyrite as it considered the electrostatic interactions also and which were attractive. The extended DLVO approach also predicted stable microbial suspension in water on account of highly repulsive acid/base and electrostatic repulsion. The possibility of homo- and hetero-aggregation of fine pyrite and chalcopyrite particles was also shown by extended DLVO approach. Attachment behaviour as predicted by DLVO approach was able to partially explain the previously obtained bioflotation results for pyrite and chalcopyrite. The explanation was only partial because of the fact that extended DLVO approach only could explain the attachment behaviour of bacterial cells on the mineral but, the selective interaction of collector with bacteria pre-conditioned mineral made it possible for the selective flotation of chalcopyrite from pyrite.

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4. Microbial Adhesion on Mineral Surfaces: Paper V Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001 References 1. R.W. Smith, M. Misra, J. Dubel, Miner. Engg., 4 (1991) 1127-1141. 2. N. Deo, K.A. Natarajan, Miner. Engg. 10 (1997) 1339-1354. 3. N. Deo, K.A. Natarajan, Int. J. Miner. Process, 55 (1998) 41-60. 4. N. Deo, K.A. Natarajan, Miner. Engg. 11 (1998) 717-738. 5. M.K. Yelloji Rao, K.A. Natarajan, P. Somasundaran, In: Mineral Bioprocessing, Eds. R.W. Smith and M Mishra, Miner. Met. Mater. Soc., 1991, 105-120. 6. N.N. Lyalikova, L.L. Lyubavina, Fundamentals and Biohydrometallurgy, Eds. R.W. Lawrence, R.M.R. Branion and H.B. Ebner, Elsevier, New York, 1986, 403- 406. 7. M. Mishra, S. Chen, In: Mineral bioprocessing, Eds. D. Holmes and R. W. Smith, Miner. Met. Mater. Society., 1995 8. H. Tributsch, Proceedings: International biohydrometallurgy symposium, Elsevier, Amsterdam, 1999, 51-60 9. W. Sand, T. Gehrke, P.G. Joza, A. Schippers, Proceedings: International biohydrometallurgy symposium, Elsevier, Amsterdam, 1999, 27-50 10. R. Bos, H.C. van der Mei, H.J. Busscher, FEMS Microbiol. Rev., 23 (1999) 179- 230 11. S.E. Paradis, D. Dubreuil, S. Rioux, M. Gottschalk, M. Jacques, Infect. Immun. 62 (1994) 3311-3319. 12. V. Williams, M. Fletcher, Appl. Environ. Microbiol. 62 (1996) 100-104 13. B.A. Juckers, A.J.B. Zehnder, H. Harms, Environ. Sci. Technol. 32 (1998) 2909- 2915 14. P.K. Sharma, K. Hanumantha Rao, Min. Met. Proc. 16 (1999) 35-41 15. A.W. Neumann, R.J. Good, C.J. Hope, M. Sejpal, J. Coll. Inter. Sci., 49 (1974) 291-304 16. A.W. Neumann, O.S. Hum, D.W. Francis, W. Zingg, C.J. van Oss, J. Biomed. Materl. Res. 14 (1980) 499-509 17. D.K. Owens, R.C. Wendt, J. Appl. Pol. Sci. 13 (1969) 1714-1747. 18. C.J. Van Oss, M.K. Chaudhury, R.J. Good, Adv. Coll. Inter. Sci. 28 (1987) 35-64 19. C.J. Van Oss, M.K. Chaudhury, R.J. Good, Chem. Rev. 88 (1988) 927-941 20. C.J. Van oss, Coll. Surf. A 78 (1993) 1-49 21. C.J. Van Oss, Interfacial forces in aqueous media, Marcel Dekker, 1994 22. C.J. Van Oss, Coll. Surf. B 5 (1995) 91-110 23. E.J.W. Verwey, J. Th. G. Overbeek, Theory of the stability of lyophobic colloids, Elsevier, Amsterdam, 1948 24. B.V. Deryagin, L. Landau, Acta. Physicochem., URSS, 14 (1941) 633-662. 25. C.J. Van Oss, R.J. Good, M.K. Chaudhury, J. Coll. Inter. Sci. 111 (1986) 378-390. 26. F.M. Fowkes, J. Phy. Chem. 66 (1964) 382. 27. H.J. Busscher, A.H. Weerkamp, H.C. Van der Mei, A.W.J. van pelt, H.P. de jong, J. Arends, App. Environ. Microbiol. 48 (1984) 980-984. 28. H.C. Van der Mei, M. Rosenberg, H.J. Busscher, Chapter 10 In: Micribial cell surface analysis: Structural and physicochemical methods, Eds. Mozes, N., Handley, P.S., Busscher, H.J., Rouxhet, P.G., VCH pub., 1991, 263-287. 29. H.C. van der Mei, R. Bos, H.J. Busscher, Coll. Surf. B: Biointerfaces 11 (1998) 213-221. 30. M.N. Bellon-Fontaine, N. Mozes, H.C. van der Mei, J. Sjollema, O. Cerf, P.G. Rouxhet, H.J. Busscher, Cell. Biophys. 17 (1990) 93-106.

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Chapter 5. Biobeneficiation: Bioflotation

5. Biobeneficiation: Bioflotation Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001

Two major areas, which are making advances in minerals bioprocessing are bio-leaching and bio-beneficiation. Bioleaching can be defined as a hydrometallurgical dissolution process assisted by microorganisms for the recovery of metals from their ores/concentrates. Major activity has been in bioleaching of sulphide minerals and chemolithotrophic bacteria have been used for the bio-leaching process. Over the past three decades bioleaching has come a long way and is now economically competitive, many processes have been commercialised and are in use. Whereas, biobeneficiation is relatively a new area and a new term which, has recently has been defined as “bio-beneficiation involves the selective removal of undesired mineral constituents from an ore through interaction with microorganisms, enriching the solid residue with respect to the desired mineral phase” (Natarajan, 1998). Microbe-mineral interaction results in several significant consequences of relevance in mineral biobeneficiation namely, • Adhesion of microorganisms to mineral surfaces, for longer duration of interaction results in biofilm formation • Alteration of mineral surface properties just because of the fact that microbial cells impart their surface properties to the mineral • Bio-catalysed oxidation and reduction reactions. This occurs where chemoautolithotrophic bacteria are involved with minerals which can acts as their substrates • Adsorption/chemical interaction of metabolic products, resulting in surface modification due to dissolution of mineral constituents, biometal accumulation etc. • Finally biobeneficiation is brought about by either of the following reasons 1. Alteration of surface chemistry of minerals 2. Generation of surface-active chemicals 3. Selective dissolution of mineral phases in an ore matrix 4. Sorption, accumulation and precipitation of ions and compounds

Role of Thiobacillus ferrooxidans (T.f.) in biobeneficiation Bacterial conditioning with T.f. can produce significant surface modification on sphalerite and galena and hence on their flotation characteristics (Yelloji Rao et al., 1992). The natural flotability of both sphalerite and galena is enhanced by pretreatment with sulphuric acid due to formation of elemental sulphur on the surfaces. After interaction with T.f. the flotability of galena is markedly decreased due to the fact that the elemental sulphur of oxidised to sulphate which is soluble. In the case of sphalerite this phenomena is not observed, sphalerite depression is only possible at very high cell densities, possibly due to excessive cell and cell product attachment. T.f. has also been used in the processing of high sulphur coal, where the sulphur is in the form of pyrite. T.f. is used at biosurface modifier to increase the pyrite surface hydrophilicity and hence during froth flotation pyritic sulphur rejection is significant without effecting the coal recovery as a float (Attia et al., 1993; Atkins et al., 1987; Attia and Elzeky, 1985). The mechanism of the pyrite depression was studied in detail using AC impedance spectroscopy by Mishra et al. 1996. Kawatra and Eisele studied the pyrite depression of pyrite flotability using T.f. and yeast (Saccharomyces cerevisiae) and concluded that both the microorganisms depress

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Role of sulphate reducing bacteria Unlike T.f., sulphate-reducing bacteria (SRB) of the type Desulfovibrio are anaerobic heterotrophs. They reduce sulphate to sulphides at an optimum pH range of 6-7. H2S and other metal sulphides are generated guring the growth of these organisms. These bacteria are very useful for oxidised ores, which can be effectively sulphidised in the presence of SRB. Flotation of cerussite is improved by 20-25% after pretreatment with SRB (Townsley et al., 1987). Besides, they were also found to be effective desorbent of xanthate coatings from mineral surfaces, this observation is of significance in differential flotation process wherein desorption of the collector coating from a mineral surface before addition of a new type of collector is important.

Role of Mycobacterium phlei Mycobacterium phlei which is a Gram-positive, rod shaped prokaryotic cell has been observed to be useful as a flocculent for phosphate slimes and hematite (Smith et al., 1991). The cell walls contain glycolipids, phospholipids and free lipids and free long chain fatty acids have been isolated from this organism. Accumulation of polar groups at the surface imparts a high negative charge and a large number of apolar groups confer hydrophobicity to the surface of these cells. The bacterial cells could adhere to mineral surfaces such as hematite, which could be efficiently floated after bacterial conditioning under controlled conditions. At mildly acidic pH values, the cells flocculated hematite but not quartz, substantial settling of hematite fines could be observed within few minutes after bacterial interaction. These bacteria have also been used for selective flocculation of fine coal and hence used in desupharisation of coals (Misra at al., 1993; Raichur, 1996). The bacterial cells selectively adhere to coal particls; they do not adhere to pyrite and other ash-forming minerals present in the coal matrix. Optical micrographs of the flocs shoa that the coal particles are held in the aggregate by adheiosn and bacterial bridging and this is attributed to hydrophobic interactions.

Role of Paenibacillus polymyxa (P.p.) P.p. which is a Gram-positive, rod, heteroprophic bacteria have been demonstrated to be useful in the biobeneficiation of iron ore and processing of bauxite (Deo, 1998). Desiliconisation of calcite, alumina and iron oxide (Deo and Natarajan, 1997) and selective separation of silica and alumina from iron ore (Deo and Natarajan, 1998a) was reported using the bacterial cells. It also showed capability to degrade collectors like dodecyl amine, diamine, isopropyl xanthate and sodium oleate (Deo and Natarajan, 1998b), which can be used for stripping the residual collector from the mineral surface and can solve environmental problems associated with mineral processing industry.

Flotation Tests The single mineral flotation tests were carried out in a Hallimond tube using 1 g of mineral samples. The mineral samples were first conditioned with

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Bioflotation studies The flotation responses of minerals after interaction with P. polymyxa are shown in Fig. 5.1, and after interaction with T. ferrooxidans are shown in Fig. 5.2. The natural flotabilities of pyrite and chalcopyrite are found about 10% and 48% respectively. In presence of 10-5 M isopropyl xanthate, the recoveries of pyrite and chalcopyrite are 62 and 74% respectively. The flotation results after interaction with wild, pyrite adapted and chalcopyrite adapted P. polymyxa are presented in Figs. 5.1(a), (b) and (c) respectively. The effect of the interaction of wild cells is noticeable until a cell density of 107 cells/ml, after which flotation behaviour of mineral with or without collector is the same, i.e., recovery increases drastically to about 80 to 90% at cell density of 108 cells/ml which is thought to be due to an excessive frothing caused by an increase in cell population. Natural flotability of pyrite increased from 10 to 20% for cell density of 106 to 107 cells/ml and for chalcopyrite it decreased from 48 to 25%. In presence of the collector, the recovery of chalcopyrite is nearly constant at 70-75% but the pyrite is depressed gradually up to 2x106 cells/ml and then it increased. Therefore a selective flotation of chalcopyrite from pyrite was possible at a wild cell density of 2x106 cells/ml. Interaction of minerals with pyrite adapted P. polymyxa caused depression of both pyrite and chalcopyrite even in the presence of collector as shown in Fig. 5.1(b). The difference in recovery of chalcopyrite and pyrite is about 15%, the highest recovery for chalcopyrite was observed to be 50% at 4x106 cells/ml. Even at higher cell densities no excessive frothing was observed and hence the flotation recoveries were in the range of 27 to 40%. In absence of collector, the flotation behaviour of both minerals was similar and the recoveries were about 15%. Flotation results after interaction with chalcopyrite adapted cells are shown in Fig. 5.1(c). The natural flotability of both the minerals is same about 25% recovery, but in presence of the collector the recovery of chalcopyrite is constant at 65 % to a cell density of 3x106 cells/ml and pyrite is depressed to about 30% recovery. Therefore, a selective flotation of chalcopyrite from pyrite is possible in a large range of cell density, between 6x104 and 3x106 cells/ml. This shows that after interaction with chalcopyrite adapted cells, the collector adsorption on chalcopyrite is not hindered much, but adsorption on pyrite is prevented to a large extent. The flotation results using ferrous and sulphur grown T. ferrooxidans with increasing cell concentration and at two different xanthate concentrations is shown in Figs. 5.2(a) and 5.2(b) respectively. The flotation recoveries of chalcopyrite and pyrite at 1x10-4 M xanthate concentration are respectively 85% and 38% compared to 65% and 22% recoveries at a lower xanthate dosage (1x10-5 M).

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100 (a) 100 90 (a) 80 90 70 80 60 50 70 40 60

% Recovery 30 50 20 10 40 0 5 6 7 8 No Cells 10 10 10 10 (%) Recovery 30 Cell density ( Cells ml-1) 100 20 (b) 90 10 80 70 0 60 5,0x106 1,0x107 1,5x107 2,0x107 2,5x107 3,0x107 50 Cell Density (cells/ml) 40 100 30 (b) % Recovery 20 90 10 80 0 No Cells 106 107 108 70 Cell density (cells ml-1) 100 60 (c) 90 50 80 70 40 60 Recovery (%) Recovery 30 50 40 20 % Recovery 30 10 20 10 0 6 7 7 7 7 7 0 5,0x10 1,0x10 1,5x10 2,0x10 2,5x10 3,0x10 No Cells 105 106 107 108 Cell Density (cells/ml) Cell density (cells ml-1) Figure 5.1 Flotation of minerals in Figure 5.2 Flotation of minerals in the the presence of (a) Wild (b)pyrite presence of (a) ferrous ions grown and adapted and (c) chalcopyrite (b) Sulfur grown Thiobacillus adapted Paenibacillus polymyxa ferrooxidans at two different xanthate cells. ( ) pyrite, (■) pyrite + 0.01 concentrations. (■) pyrite + 0.01 mM mM xanthate, (Ο) chalcopyrite, xanthate, ( ) pyrite + 0.1 mM xanthate, (●) chalcopyrite + 0.01 mM (●) chalcopyrite + 0.01 xanthate, (Ο) xanthate. chalcopyrite + 0.1 mM xanthate

The preconditioning of minerals with bacterial cells prior to the addition of collector reduced the flotability of pyrite and chalcopyrite. The reduction in the floatability is found to be dependent on cell concentration. The flotation is little affected at 7.0x104 cells/ml and complete depression of pyrite flotation occurred at a cell population of 3.3x107 cells/ml. The sulphur grown cells completely depressed the pyrite flotation when compared to the ferrous iron grown cells. At 1x10-5 M and 1x10- 4 M xanthate concentrations, the ferrous grown cells reduced the pyrite recovery from 40% to 20% and 10% respectively, whereas no flotation resulted in the presence of sulphur grown cells. The presence of cells at a higher xanthate concentration (1x10-4 M), irrespective of their growth conditions, has little influence on chalcopyrite flotation. The ferrous iron and sulphur grown cells reduced chalcopyrite recovery (88%) to only about 70% and 80% respectively. Thus, T. ferrooxidans cells can be 248

5. Biobeneficiation: Bioflotation Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001 used as a depressant for pyrite during chalcopyrite flotation. Since bacterial cells get energy by oxidising iron and sulphur, the cells might have strongly adsorbed on pyrite surface and xanthate ions cannot replace the adsorbed cells. In the case of chalcopyrite, the surface copper ions are in a way toxic to the cells and these sites are left unoccupied by the cells so that the adsorption of xanthate takes place on these sites by complexation. Alternatively, the affinity between the xanthate and copper ions is so great that the xanthate ions replace the adsorbed cells on chalcopyrite.

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Chapter 6: Conclusions

6. Conclusions Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001

Thiobacillus ferrooxidans bacteria, which are chemolithotrophs and are associated with sulphide ores, could be grown in the presence of higher sulphide mineral content as compared to heterotroph Paenibacillus polymyxa bacteria.

The adaptation to minerals induced alterations in the physico-chemical properties of the microbial cell surface. The iso-electric point of Thiobacillus ferrooxidans shifts towards higher pH value after adaptation to minerals and the Paenibacillus polymyxa became more hydrophilic without showing any change in the electrokinetic behaviour.

The bacterial surfaces became more energetic after adaptation to minerals. The increase in the surface energy is due to the increased density of functional groups of metabolite products on the surface, which is evidenced by the FT-IR spectra. The surfaces acquired the acid/base surface energy component during adaptation and this component was decisive for microbial adhesion.

Among the different approaches that are followed to evaluate the surface energy of bacteria and minerals, the LW-AB approach correlated well with other physico- chemical characteristics of the microbial cells and was able to explain more explicitly the affinity of Paenibacillus polymyxa towards the adherence to organic solvents, in particular to chloroform.

From detailed analysis it was found that the Geometric mean and Equation of state approaches evaluate similar surface energy values if same liquid contact angles are used. Both these approaches are not internally consistent since the surface energy values varied with the change in liquid contact angle used. The LW-AB approach evaluated consistent results even after changing the apolar liquid contact angle. Due to the inherent mathematical instability, the use of Water, Formamide and Methyleneiodide/Bromonapthalene is prescribed. The electron-donating characteristic evaluated using LW-AB approach could differentiate between Gram-positive and Gram-negative bacteria.

The extended DLVO approach was effective in predicting the adhesion behaviour than the expectations from thermodynamic approach. Contrary to experimental observations, the thermodynamic approach yields no bacterial adhesion on minerals and this discrepancy is the result of inadequate description of electrostatic interactions. The adhesion predictions by the extended DLVO approach are able to explain the bioflotation results of pyrite and chalcopyrite.

Selective flotation of chalcopyrite and depression of pyrite flotation was achieved in the presence of either Thiobacillus ferrooxidans or Paenibacillus polymyxa. However, theoretical calculations cannot account this selective behaviour since the interactions between collector and mineral in the presence of bacteria are difficult to characterise

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7. References Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001

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7. References Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001 Sharma, P.K., Hanumantha Rao, K., Forssberg, K.S.E., Natarajan, K.A., 2001, Surface chemical characterisation of Paenibacillus polymyxa before and after ada ptation to sulfide minerals, International Journal of Mineral processing, 62 (1- 4), 3-25 Silverman, M.P., and D.G. Lundgren, 1959. Studies on the chemoautotrophic iron bacteria Ferroobacillus ferrooxidans.1. An improved medium and a harvesting procedure for securing high yields. J. Bacter. 77: 642-647. Skinner, F.K., Rotenberg, Y., Neumann A.W., 1989, Contact-angle measurements from the contact diameter of sessile drops by means of a modified axisymmetric drop shape-analysis, J. Colloid. Interface Sci., 130(1), 25-34 Sleytr, U.B., Messner, P., In: Crystalline bacterial cell surface layers, Eds., Sleytr, U.B., Messner, P., Springer Verlag, Berlin, 1988, 160 Sleytr, U.B., Sara, M., Pum, D., Schuster, B., 2001, Characterisation and use of crystalline bacterial cell surface layers, Progress Sur. Sci., 68, 231-278 Smith, R.W., Misra, M., Dubel, J., 1991, Minerals bioprocessing and the future, Miner. Engg., 4, 1127-1141 Smyth, C.J., Jonsson, P., Olsson, E., Söderlind, O., Rosengren, J., Hjertén, S., Wadström, T., 1978, Differences in hydrophobic surface characteristics of porcine enteropathogenic E. coli with or without K88 antigen as revealed by hydrophobic interaction chromatography, Infect. Immun., 22, 462-472 Stenström, T.A., 1989, Bacterial hydrophobicity, an overall parameter for the measurement of adhesion potential to soil particles, Appl. Environ. Microbiol., 50(1), 142-147. Stenström, T.A., Kjelleberg, S., 1985, Fimbriae mediated nonspecific adhesion of Salminella typhymurium to mineral particles, Arch. Microbiol. 143, 6-10. Townsley, C.C., Atkins, A.S., Davis, A.J., 1987, Suppression of pyritic sulfur during flotation tests using the bacterium thiobacillus-ferrooxidans, Biotechnol. Bioeng. 30(1), 1-8 Tokekawa, S., Vozumi, N., Tsukagisni, N, Udaka, S., 1991, J. Bacteriol., 173, 6820 Twardowski, J., Anzenbacher, P., 1994. Raman and IR spectroscopy in biology and biochemistry, Ellis Horwood, London and Polish Scientific Publishers, Warsaw. Van der Mei, H.C., Weerkamp, A.H., Busscher, H.J., 1987, A comparison of various methods to determine hydrophobic properties of Streptococcal cell-surfaces, J. Microbiol. Meth., 6(5), 277-287 Van der Mei, H.C., Leonard, A.J., Weerkamp, A.H., Rouxhet, P., Busscher, H.J., 1988a, Surface-properties of streptococcus-salivarius hb and nonfibrillar mutants - measurement of zeta-potential and elemental composition with x-ray photoelectron-spectroscopy, J. Bacteriol., 170(6), 2462-2466 Van der Mei, H.C., Leonard, A.J., Weerkamp, A.H., Rouxhet, P., Busscher, H.J., 1988b, Properties of oral streptococci relevant for adherence - zeta potential, surface free-energy and elemental composition, Colloid Surf., 32(3-4), 297-305 Van der Mei, H.C., Genet, M.J., Weerkamp, A.H., Rouxhet, P.G., Busscher, H.J., 1989a, A comparison between the elemental surface compositions and electrokinetic properties of oral streptococci with and without adsorbed salivary constituents, Arch. Oral. Biol., 34(11), 889-894 Van der Mei, H.C., Brokke, P., Dankert, J., Feijen, J., Rouxhet, P.G., Busscher, H.J., 1989b, Physicochemical surface-properties of nonencapsulated and encapsulated coagulase-negative staphylococci, Appl. Environ. Microbiol., 55(11), 2806-2814

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Publications during the Present Study (Oct. 1997 – Dec. 2001) Surface Studies Relevant to Microbial Adhesion and Bioflotation of Sulphide Minerals, P.K. Sharma, 2001

Publications

1. Paper I: Sharma, P.K., Hanumantha Rao, K., Forssberg, K.S.E., Spectroscopic characterisation of Thiobacillus ferrooxidans cell surface components grown under different conditions, In: Biohydrometallurgy "Fundamentals, Technology and Sustainable Development", Eds: Ciminelli, V.S.T., Garcia Jr., O., IBS, Brazil, Elsevier, Amsterdam, 2001. 2. Paper II: Sharma, P.K., Hanumantha Rao, K., Forssberg, K.S.E., Natarajan, K.A., 2001, Surface chemical characterisation of Paenibacillus polymyxa before and after adaptation to sulfide minerals, International Journal of Mineral processing, 62 (1-4), 3-25 3. Paper III: Sharma, P.K., Hanumantha Rao, K., Surface characterisation of bacterial cells relevant to mineral industry, Communicated to International Journal of Mineral Processing 4. Paper IV: Sharma, P.K., Hanumantha Rao, K., Analysis of different approaches for evaluation of surface free energy of bacterial cells by contact angle goniometry, Communicated to Advance in Colloids and Interface Science 5. Paper V. Sharma, P.K., Hanumantha Rao, K., Adhesion of Paenibacillus polymyxa on chalcopyrite and pyrite: surface thermodynamic and extended DLVO approaches, Communicated to Colloids and Surfaces B: Biointerfaces 6. Paper VI: Das, A., Rao, K.H., Sharma, P.K., Natarajan, K.A., Forssberg, K.S.E., 1999, Surafce chemical and adsorption studies using Thiobacillus ferrooxidans with reference to bacterial adhesion to sulfide minerals, International Biohydrometallurgy Symposium (IBS) 1999, Spain, Proceeding part B, Chapter 2, 697-708. 7. Paper VII: Sharma, P.K., Hanumantha Rao, K., 1999, Role of a heterotrophic Paenibacillus polymyxa bacteria in the bioflotation of some sulfide minerals, Minerals & Metallurgical Processing, 16(4), 35-41 8. Paper VIII: Sharma, P.K., Das, A., Hanumantha Rao, K., Forssberg, K.S.E., 1999, Thiobacillus ferrooxidans interaction with sulfide minerals and selective chalcopyrite flotation from pyrite, In: Advances in Flotation Technology, Eds. Parekh, B.K., Miller, J.D., SME, 147-165. 9. Paper IX: Sharma, P.K., Hanumantha Rao, K., Natarajan, K.A., Forssberg, K.S.E., 2000, Bioflotation of sulfide minerals in the presence of heterotrophic and chemolithotrophic bacteria, XXI IMPC, Rome, Proceeding part B, B8a-94 to B8a- 103. 10. Sharma, P.K., 1999, Physico-chemical characterisation of microbial cell surface and bioflotation of sulfide minerals, Licenciate thesis, Division of Mineral Processing, Luleå University of Technology, Luleå, ISSN: 1402-1757.

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