Studies on Biofilm Growth, Attachment and Biokinetic Performance in Biofilters Packed with Macroporous Media

UNIVERSITY OF CINCINNATI

Date:______

I, ______, hereby submit this work as part of the requirements for the degree of: in:

It is entitled:

This work and its defense approved by:

Chair: ______

Studies on biofilm growth, attachment and biokinetic performance in biofilters packed with macroporous media

A thesis submitted to the Department of Research and Advanced Studies of the University of Cincinnati

in partial fulfillment of the requirements for the degree of

DOCTOR OF PHILOSOPHY In the Department of Chemical and Materials Engineering of the College of Engineering 2007

by Juan Jose Goncalves Rodrigues

BS, Universidade do Porto, Portugal (2000) BS, Universidad Simon Bolivar, Venezuela (1999)

Committee Chair: Dr. Rakesh Govind

ABSTRACT

The first part of this thesis encompasses fundamental studies on the attachment and growth of biofilms onto synthetic non adsorbing, macroporous solid foams aiming at supporting bioactive microorganisms in the removal of intricate hydrogen sulfide polluted airstreams in trickle bed columns at negligible pressure drop. A new theoretical model that predicts the performance of biofilters packed with non adsorbing, macroporous media was simultaneously developed based in the distribution of the fouled airstream within the porous media and around it, so that the geometric properties of the packing media can be chosen as to maximize the amount of air passing within the media where most microorganisms are located. During the experimental phase of this study, colonization of such non adsorbing, macroporous media with microorganisms was enhanced by the addition of positively charged, polymeric coatings which increase the attachment and spreading of the biofilms due to cell binding and electrostatic charge cancellation at physiological pH. Impedimetric tests using golden microelectrodes were applied separately to corroborate such results, and other cofactors reported in the Literature for the attachment of animal cells onto plastic surfaces were tested with the methodology. The use of such cofactors for biofilm attachment purposes and the impedimetric tests for the determination of the kinetics of the biofilm morphology development based in the transient change of observables such as resistance and capacitance, are the first attempts on such approaches to date. In the second part of this thesis, the macroporous, non adsorbing foams were replaced by adsorbing, reactive units of similar geometric properties but containing iron (III) (oxy)(hydr)oxides operating as adsorption towers and trickle bed biofilters. The abiotic H2S removal capability of such iron bearing media was found to be enhanced by dripping water down the bed, which allowed for complete elimination of the sulfide gas during the early acclimation period needed for bacteria to develop after such media had been seeded with activated sludge. The biological sulfide oxidation in the iron media biofilter was accomplished by the inorganic reduction of iron (III) species with H2S and following oxidation of iron (II) species into the original reactant by means of a bacterial strain never before reported to accomplish such functionality.

ACKNOWLEDGEMENTS

I would like to express my deepest gratitude to Dr. Rakesh Govind for his continuous academic and financial support, to all members of the Departments of Chemical and Materials Engineering and Civil and Environmental Engineering of the University of Cincinnati, to my relatives, friends, and to whomever has in some way, directly or indirectly, contributed to the conclusion of this dissertation.

ii TABLE OF CONTENTS

ABSTRACT i

ACKNOWLEDGEMENTS ii

LIST OF FIGURES vii

LIST OF TABLES xvi

I. ANALYSIS OF BIOFILTERS USING SYNTHETIC MACROPOROUS FOAM MEDIA

SUMMARY 1 I.I. INTRODUCTION 1 I.II. MODEL DEVELOPMENT FOR THE DESCRIPTION OF AIRSTREAM BIOTRICKLING FILTERS PACKED WITH MACROPOROUS SOLID FOAMS 9 I.III. MATERIALS AND METHOD 22 I.IV. RESULTS AND DISCUSSION 22 I.V. CONCLUSIONS 32 I.VI. SYMBOLS 32 I.VII. REFERENCES 35

II. ENHANCED BIOFILTRATION USING CELL ATTACHMENT PROMOTORS

SUMMARY 40 II.I. INTRODUCTION 41 II.II. MATERIALS AND METHODS 42 II.II.I. Coating batch experiments and coating selection 42

iii II.II.II. Continuous experiments 43 II.II.III. Analytical and quantification 43 II.III. RESULTS AND DISCUSSION 45 II.III.I. Overall biofilter performance 45 II.III.II. Section-based biofilter performance 49 II.III.III. Removal controlling mechanism and dimensionless analysis 55 II.III.IV. Biomass speciation and agar utilization 59 II.III.V. Biofilm coverage 64 II.III.VI. Pressure drop 68 II.IV. CONCLUSIONS 68 II.V. SYMBOLS 69 II.VI. REFERENCES 71

III. QUALITATIVE STUDY OF BIOFILM GROWTH AND METABOLIC ACTIVITY IN THIOSULFATE OXIDIZING BACTERIA USING IMPEDIMETRY

SUMMARY 73 III.I. INTRODUCTION 74 III.II. MATHEMATICAL DESCRIPTION OF THE BIOFILM GROWTH KINETICS AND THEIR ELECTRIC PROPERTIES MEASURED BY IMPEDIMETRY 76 III.III. MATERIALS AND METHODS 83 III.III.I. Impedance monitoring on gold electrode arrays 83 III.III.II. Analytical and quantification 85 III.IV. RESULTS AND DISCUSSION 85 III.IV.I. Bacteria characterization 85 III.IV.II. Transient behavior of the total (observed), media (calculated) and pseudo surface (calculated) resistances. 86 III.IV.III. Transient behavior of the total (observed), media

iv (calculated) and pseudo surface (calculated) capacitances. 96 III.IV.IV. Effect of pH on the transient behavior of the observed electrical properties. 104 III.IV.V. Effect of shaking the culture well on the transient observed electrical properties 106 III.V. CONCLUSIONS 106 III.VI. SYMBOLS 107 III.VII. REFERENCES 108

IV. ABIOTIC H2S UPTAKE IN IRON (III) (OXY)(HYDR)OXIDES AT STP CONDITIONS IN TRICKLE BEDS

SUMMARY 111 IV.I. INTRODUCTION 112 IV.II. MATERIALS AND METHODS 114 IV.II.I. Adsorbent/catalysts characterization 114 IV.II.II. Batch experiments 115 IV.II.III. Continuous experiments 115 IV.II.IV. Analytical and quantification 118 IV.III. RESULTS 118 IV.III.I. Preliminary catalyst characterization 118 IV.III.II. Dry continuous operation of packed beds 121 IV.III.III. Wet continuous operation of packed beds 125 IV.III.IV. Final catalyst characterization 126

IV.III.V. Kinetics of the H2S adsorption on IOPF 134 IV.III.V. Mass transfer limitations of the water layer in the wet operation of the IOPF and IOP beds 137 IV.IV. CONCLUSIONS 138 IV.V. SYMBOLS 139 IV.VI. REFERENCES 140

v V. INDIRECT H2S ABATEMENT USING BIOLOGICAL IRON OXIDATION BY ALICYCLOBACILLI ON IRON (III) FOAM, PACKED BIOREACTORS

SUMMARY 142 V.I. INTRODUCTION 143 V.II. MATERIALS AND METHODS 147 V.II.I. Adsorption experiments 147 V.II.II. Biological experiments 148 V.II.III.Analytical and quantification 149 V.III. RESULTS AND DISCUSSION 151 V.III.I. Dry and wet basis adsorption experiments 151 V.III.II. Biological operation 155 V.III.III. Biomass characterization 159 V.III.IV. Other operational considerations: Pressure drop, IOPF clogging and effect of carbon source on the accumulation and survival of biomass 161 V.IV. CONCLUSIONS 164 V.V. REFERENCES 165

vi LIST OF FIGURES

Chapter I

Figure I.1. Substrate concentration profile in a biofilm attached to non adsorbent media. 10 Figure I.2. Substrate concentration profiles for the three different situations described by Rittmann and McCarty [67]. Deep, Shallow and Fully penetrated biofilm. 20

Figure I.3. Gas flow distribution in the bioreactor as a function of the

superficial gas velocity vg for different foam pore sizes d p,o . Other design parameters are summarized in Table I.2. 23

2 Figure I.4. Effect of the coefficient Asvg on the pollutant removal efficiency E for a macroporous foam packed bed. Design and biokinetic parameters are summarized on Table I.2. 25 Figure I.5. Effect of gas superficial velocities v on the function A v 2 for a g s g

macroporous foam packed bed. Pore size d p,o effect. Design and biokinetic parameters are summarized on Table I. 2. 27 Figure I.6. Effect of gas superficial velocities on the function for a

macroporous foam packed bed. Packing factor Fpd effect. Design and biokinetic parameters are summarized on Table I. 2. 27 Figure I.7. Effect of gas superficial velocities on the removal efficiency E

of a pollutant in a bioreactor with the configuration and biofilm kinetic properties indicated in Table I. 2. 28 Figure I.8a. Comparison between predicted and experimental toluene concentration profiles in a polyurethane foam packed bioreactor as

presented by Moe and Irvine [31]. C g,i : 200 ppmv 29 Figure I.8b. Comparison between predicted and experimental toluene concentration profiles in a polyurethane foam packed bioreactor as

vii presented by Moe and Irvine [31]. C g,i : 50 ppmv 29 Figure I.9. Comparison between the experimental and predicted values for the

outlet concentration of a foam biofilter treating H2S polluted airstreams at EBRT: 4-20 sec and initial concentrations in the range

10-100 ppmv. 31

Chapter II

Figure II.1. Schematic of the biofiltration system to be used in this work 44

Figure II.2. Long term performance for the coated reactor removing H2S 46

Figure II.3. Long term performance for the uncoated reactor removing H2S. 46

Figure II.4. H2S removal performance for the coated and uncoated reactors over the first 4 days of operation. 47

Figure II.5. Section performance for the coated reactor removing H2S. Line represents 100% RE. 51

Figure II.6. Section performance for the uncoated reactor removing H2S. Line represents 100% RE. 51 Figure II.7. Contribution to of the lower reactor half to the observed

instantaneous H2S removal efficiency (RE) for the coated reactor. 54 Figure II.8. Contribution to of the lower reactor half to the observed

instantaneous H2S removal efficiency (RE) for the uncoated reactor. 54 Figure II.9. Removal efficiency (RE) for the bioreactor sections as a function of the dimensionless numbers Peclet (Pe) and Damkohler (Da). Clockwise from top left corner: Upper uncoated, Lower uncoated, Lower coated, Upper coated. Peclet numbers are: 2·106 (dotted circle), 4·106 (dotted triangle), 9·106 (half full circle) and 11·106 (star). 58 Figure II.10. SEM micrograph of the biomass grown on the PU foam after 125 days of reactor’s service. Picture was taken on media from the coated reactor, though qualitatively both reactors showed similar biomass imagery. 62

viii Figure II.11. Sulfur crystal sitting on top of the biomass in the bioreactors after 125 days of service. 62 Figure II.12. 16S rRNA bands separated through DGGE techniques from biomass samples taken from the bioreactors after service shutdown. Bands 1.1 to 4.2 correlate with Acidithiobacillus gene databank in 0.900-1.000. Reactor sections: AT: Upper uncoated, AB: Lower uncoated, BT: Upper coated, BB: Lower coated. 64 Figure II.13. Maximum calculated biofilm coverage for the uncoated reactor as a function of the Elimination Capacity EC. 66 Figure II.14. Maximum calculated biofilm coverage for the coated reactor as a function of the Elimination Capacity EC. 67

Chapter III

Figure III.1a. Qualitative sketch of the 10, 250µm gold electrodes and the large counter electrode in the substrate of the ECIS wells. The area for culture includes the microelectrodes, counter electrode and insulation. Electrical culture properties are measured atop the microelectrodes within a space closely located above them. 77 Figure III.1b. Representation of the three domains of the electrical circuit depicted in Figure 2. 77 Figure III.2. Equivalent RC circuit for the electrode-biofilm-culture media system representing the three domains in the culture wells atop the gold microelectrodes. 78 Figure III.3. 3D representation of the bacterial stack accumulating during the biofilm formation on the electrode surface. “j” and “k” represent the number of cells on the two dimensions of the plane of the electrode and in a parallel arrangement with respect to the electric flow,

whereas “Ns” represents the cells located perpendicularly to the electrode surface, and in series with respect to the electric flow. 80 Figure III.4a. Observed normalized total resistance of the PEI coated and

ix uncoated culture wells, with and without bacterial cells. Experimental conditions are summarized in Table III. 1. Resistance measurements were recorded at 400 Hz, since at this frequency the sensitivity between curves is maximum. 89 Figure II.4b. Calculated normalized media resistance of the PEI coated and uncoated culture wells, with and without bacterial cells. Experimental conditions are summarized in Table III. 1. Resistance measurements were calculated from data recorded at 400 Hz, since at this frequency the sensitivity between curves is maximum. 89 Figure III.4c. Calculated normalized pseudo surface resistance of the PEI coated and uncoated culture wells, with and without bacterial cells. Experimental conditions are summarized in Table III. 1. Resistance measurements were calculated from data recorded at 400 Hz, since at this frequency the sensitivity between curves is maximum. 90 Figure III.5a. Observed normalized total resistance of the PDL coated and uncoated culture wells, with and without bacterial cells. Experimental conditions are summarized in Table III. 1. Resistance measurements were recorded at 400 Hz, since at this frequency the sensitivity between curves is maximum. 92 Figure III.5b. Calculated normalized media resistance of the PDL coated and uncoated culture wells, with and without bacterial cells. Experimental conditions are summarized in Table III.1. Resistance measurements were calculated from data recorded at 400 Hz, since at this frequency the sensitivity between curves is maximum. 93 Figure III.5c. Calculated normalized pseudo surface resistance of the PDL coated and uncoated culture wells, with and without bacterial cells. Experimental conditions are summarized in Table III. 1. Resistance measurements were calculated from data recorded at 400 Hz, since at this frequency the sensitivity between curves is maximum. 93 Figure III.6. Observed normalized total resistance of the Col coated and uncoated culture wells, with and without bacterial cells. B1:

x bacteria from mother sludge with pH of 1.39; B2: bacteria from mother sludge with pH of 4.20. Resistance measurements were recorded at 400 Hz, since at this frequency the sensitivity between curves is maximum. 94 Figure III.7. Observed normalized total resistance of Col, PEI and PDL coated culture wells with bacterial cells. Experimental conditions are summarized in Table III. 1. Resistance measurements were recorded at 400 Hz, since at this frequency the sensitivity between curves is maximum. 95 Figure III.8a. Observed normalized total capacitance of PEI coated and uncoated culture wells, with and without bacterial cells. Other experimental conditions are summarized in Table III. 1. Resistance measurements were recorded at 40000 Hz, since at this frequency the sensitivity between curves is maximum. 97 Figure III.8b. Calculated normalized media capacitance of PEI coated and uncoated culture wells, with and without bacterial cells. Other experimental conditions are summarized in Table III. 1. Capacitance measurements were calculated from data obtained at 40000 Hz, since at this frequency the sensitivity between curves is maximum. 97 Figure III.8c. Calculated normalized pseudo surface capacitance of PEI coated and uncoated culture wells, with and without bacterial cells. Other experimental conditions are summarized in Table III. 1. Capacitance measurements were calculated from data obtained at 40000 Hz, since at this frequency the sensitivity between curves is maximum. 98 Figure III.9a. Observed normalized total capacitance of PDL coated and uncoated culture wells, with and without bacterial cells. Other experimental conditions are summarized in Table III. 1. Resistance measurements were recorded at 40000 Hz, since at this frequency the sensitivity between curves is maximum. 99

xi Figure III.9b. Calculated normalized media capacitance of PDL coated and uncoated culture wells, with and without bacterial cells. Other experimental conditions are summarized in Table III. 1. Capacitance measurements were calculated from data obtained at 40000 Hz, since at this frequency the sensitivity between curves is maximum. 100 Figure III.9c. Calculated normalized pseudo surface capacitance of PDL coated and uncoated culture wells, with and without bacterial cells. Other experimental conditions are summarized in Table III. 1. Capacitance measurements were calculated from data obtained at 40000 Hz, since at this frequency the sensitivity between curves is maximum. 100 Figure III.10. Observed normalized total capacitance of the Col coated and uncoated culture wells, with and without bacterial cells. B1: bacteria from mother sludge with pH of 1.39; B2: bacteria from mother sludge with pH of 4.20. Capacitance measurements were recorded at 40000 Hz, since at this frequency the sensitivity between curves is maximum. 101 Figure III.11. Observed normalized total capacitance of Col, PEI and PDL coated culture wells with bacterial cells. Experimental conditions are summarized in Table III. 1. Capacitance measurements were recorded at 40000 Hz, since at this frequency the sensitivity between curves is maximum. 101 Figure III.12a.CLFM micrograph of the mushroom/fibril like biofilm on a PEI coated well after 24 hours of incubation. Picture dimensions: 650 µm side, 120 µm depth. Adjacent panels show the perpendicular profile of the biofilm. Biofilm solid support bottom is located in the outermost edge of the adjacent panels. 102 Figure III.12b.CLFM micrograph of the mushroom/fibril like biofilm on an uncoated well after 24 hours of incubation. Picture dimensions: 650 µm side, 86 µm depth. Adjacent panels show the perpendicular

xii profile of the biofilm. Biofilm solid support bottom is located in the outermost edge of the adjacent panels. 102

Chapter IV

Figure IV.1. Schematic of the adsorption used in the present study for the

removal of H2S polluted airstreams in beds packed with IOPF and IOP materials. 116

Figure IV.2. H2S removal performance of columns packed with IOPF, IOP and IHG materials. A: dry IOPF. B: wet IOPF. C: dry IOP. D: wet IOP. E: 1 g IHG sample. F: 5 g IHG sample. Operating conditions are summarized in Tables IV.1 and IV.2. 122 Figure IV.3. Adsorption and desorption isotherms (where applicable) at STP

conditions for H2S on different iron containing materials. A: IOPF; B: IOP; C: IHG (1 g sample); D: IHG (5 g sample). Desorption isotherms were possible only in IOP packing. Other materials did not exhibit desorption characteristics. Operation conditions and isotherm fittings in Tables IV.1 to IV.3. 123 Figure IV.4. XRD spectra of the raw and spent IOPF and IOP materials after

treatment with H2S in wet continuous operation. A: raw IOPF; B: spent IOPF; C: raw IOP; D: spent IOP. Short dashed lines represent the location of the three most intense peaks of sulfur standards, whereas the long dashed lines represent those of hematite (α-

Fe2O3). 127 Figure IV.5a. 57Fe Mossbauer spectra at 77 K of the packing materials before and

after treatment with H2S in the wet mode continuous operation. A: raw IOPF; B: spent IOPF; C: raw IOP; D: spent IOP. Standards: Hem: hematite @ 6 K [13]; Pyr: pyrite @ 6 K [13]; Lep: lepidocrocite @ 140 K [14]; Ferr: ferrihydrite @ 70 K [14] and 75 K [15]. 130 Figure IV.5b. 57Fe Mossbauer spectra at 77 K of the packing materials before and

xiii after treatment with H2S in the wet mode continuous operation. E: raw IHG; F: spent IHGd; G: spent IHGw. 131 Figure IV.6. SEM micrographs of the raw and spent (after wet operation) IOPF and IOP solids. Top left: IOPF raw; top right: IOPF spent (bar: 100 μm). Center left: IOPF raw; center right: IOPF spent (bar: 10 μm). Bottom left: IOP raw; bottom right: IOP spent (bar: 200 μm). 133 Figure IV.7. Adsorption performance of a IOPF sample (6.18 g) saturated with

and atmosphere of H2S at STP conditions. Peaks represent spikes of

H2S. Continuous experiments carried out in the same sample. 134

Figure IV.8a. Maximum velocity of adsorption k1 for the IOPF material under an

atmosphere of H2S at STP conditions. Solid circles are single run experiments, whereas open circels are saturation runs. 136

Figure IV.8b. Half saturation constant k 2 for the IOPF material under an

atmosphere of H2S at STP conditions. Solid circles are single run experiments, whereas open circels are saturation runs. 136

Chapter V

Figure V.1. Schematic of the adsorption and subsequent biotrickling filter used

in the present study for the removal of H2S polluted airstreams. The column was operated as an adsorber both on a dry and wet basis first. Afterwards, the media was inoculated with activated sludge from a local water works facility to operate as a biotrickling filter. 148 Figure V.2. Microscopic picture depicting the IOPF structure and size of the macropores in the IOPF media. Bar represents 1 mm. 149 Figure V.3. Long term operation of the dry basis adsorption bed packed with IOPF media 152 Figure V.4. Long term operation of the wet basis adsorption bed packed with IOPF media 153

Figure V.5. Performance of the PU foam packed scrubber removing H2S with

xiv deionized water in a closed loop. Water pH was kept in the range 7- 9 by adding diluted NaOH. 154

Figure V.6. Performance of the PU foam packed scrubber removing H2S with deionized water in a closed loop. Water pH was let decrease naturally through the experiments. 154 Figure V.7. Long term performance of the IOPF biotrickling filter removing

H2S at the specified conditions. Iron oxidizing bacteria from the genus Allicyclobacillus was determined to have colonized the IOPF media after 80 days of operation. 156 Figure V.8. Overall performance of the IOPF packed biotrickling filter

removing H2S at different EBRT 157

Figure V.9. Relative H2S removal for the wet basis adsorption IOPF column for each of the sections of the bed. Each section equals one third of the total bed length. 157

Figure V.10. Relative H2S removal for the biological adsorption IOPF column for each of the sections of the bed. Each section equals one third of the total bed length. 158 Figure V.11. SEM micrographs showing Allicyclobacilli collected from the biomass grown on the IOPF bed after 80 days of biological operation. Bars represent, clockwise from top left, 2 µm, 0.5 µm, 0.5 µm and 0.5 µm. Top left picture shows a streptobacilli structure, compared to suspended cells on the other pictures. 161

Figure V.12. Performance of a IOPF packed biotrickling filter removing H2S for a system whose nutrient solution lacked agar or another organic carbon source. 163

xv LIST OF TABLES

Chapter I

Table I.1. Recent research on air treatment using PU foams as support media for immobilized biofilms. 6 Table I.2. Values of transport and kinetic parameters as well as reactor design variables used for the simulations. Kinetic values for toluene (Figures I.8a and I.8b) were assumed based on works reported in the Literature

[61, 70]. Kinetic values for H2S (Figure I.9) were obtained by fitting the present model (Equation I.17) to the experimental data collected in the foam biofilter. 24

Table I.3. Calculated values for the foam internal area Af , foam effective

contact area As , airstream pressure drop P / L , liquid layer

thickness l , biofilm thickness f and fraction of inlet airstream flowing inside the foam packing for the different experimental cases presented on Figures I.8a, I.8b and I.9. 30

Chapter II

Table II.1. Performance comparison between biofilters using different packing

media for the removal of H2S. 53 Table II.2. Modeling equations for the performance of the PU foam packed- biofilter [14] 56 Table II.3. Values used for the determination of the product of biofilm coverage and Monod’s velocity of reaction for the coated and uncoated reactors 57

Table II.4. Distribution of measured volatile solids and calculated θµmax product for the coated and uncoated reactors after 100 days of operation. 58 Table II.5. COD and TS pattern in the nutrients sump tank for the coated and uncoated reactors after 100 days of operation. 61

xvi Table II.6. Maximum kinetic constant values for Monod’s type reaction rates reported on studies of biofilters treating odors (biofilm coverage assumed to be unity). 65

Chapter III

Table III.1. Sludge properties and coatings used during the transient testing of the bacterial growth on coated and uncoated gold electrodes. Frequency scan tests conditions on the 10 electrodes are also included. Nomenclature for the coating used is also shown. 87

Table III.2. Initial values for the observed total resistance ( Rt ) and capacitance

(Ct ) for all data sets collected. Resistance values are measured at 400 Hz, whereas capacitance values are measured at 40000 Hz. Values in parenthesis represent the maximum calculated percentage error for data sets corresponding to the same experimental conditions and curves. 88 Table III.3. Calculated values of the linear regression of the type

Ct (t) a ln(t) b for all the tests carried out. Results obtained on 10 electrode wells. 105

Chapter IV

Table IV.1. Selected physicochemical and operation properties of the raw and

spent (dry and wet) catalysts used in this study for the H2S adsorption over airstreams. 117 Table IV.2. Elemental composition and performance of the “as received” and

spent materials tested before and after treatment with H2S in “dry” and “wet” conditions as shown in Table IV.1. 119 Table IV.3. Isotherm data fitting for the iron containing materials in equilibrium

with H2S at STP conditions. Data obtained from continuous dry bed

xvii experiments. 124

Table IV.4. Crystal structures detected by XRD with CuKα1 @ λ = 1.5406 Å. Species matched (θ ± 0.05) either two or all three (bold italic formulas) strongest peaks in the 2θ axis shown in ICDD libraries. Parentheses represent crystal symmetry and lattice, with bold italic values indicating matches against ICDD star quality PDF’s. (A: anorthic; BC: body centered; C: cubic; EC: end-centered: FC: face centered; H: hexagonal; M: monoclinic; O: orthorhombic; P: primitive; R: rhombohedral; RC: rhomb-centered T: tetrahedral) 129

Chapter V

Table V.1. Properties of the IOPF packing media and operation conditions for the dry and wet bed adsorption experiments, as well as the biological runs carried out after seeding of the IOPF with sludge from a local water works facility. 150 Table V.2. Maximum Elimination Capacity (EC) of some systems where explicit

indication of H2S removal performance is available, in processes

involving the chemical absorption of H2S with ferric ions and following oxidation of ferrous into ferric ions. 160

xviii

CHAPTER I

ANALYSIS OF BIOFILTERS USING SYNTHETIC MACROPOROUS FOAM MEDIA

SUMMARY

The removal efficiency of a pollutant being treated in a biofilter packed with macroporous units as biofilm media carriers can be maximized provided that more fouled air flows inside the unit pieces of the media rather than around it, since the internal specific area of the carrier is several orders of magnitude higher than outside it. A new model that accounts for the hydrodynamic distribution of a gas stream flowing inside a macroporous packed bed, more specifically open pore foams, is proposed. The model allows for the design of the carrier (pore size, media internal porosity, bed porosity and external bed resistance coefficient) in the presence or absence of outer layers such as a biofilm and a liquid film, so that a given pollutant removal efficiency can be attained or increased based on the amount of fouled gas flowing inside the media rather than around it, whilst the clogging effects typically observed in the field can be avoided. The model can also be used for the special case where the bed is packed with a monolithic open pore carrier. Predicted values for the biofilter performance both for a monolithic bed and a open pore packed bed are in good agreement with experimental results obtained in systems treating toluene and hydrogen sulfide under a wide range of Empty Bed Residence Times (EBRT) between 4-120 sec, and inlet concentrations between 10-200 ppmv.

I.I. INTRODUCTION

Air biofiltration and wastewater treatment at pilot, bench and full scales using immobilized biofilms have proven to be efficient techniques for the removal of organics and other pollutants such as Volatile Organic Compounds (VOC), Sulfur Reduced Compounds (SRC) and ammonia [1-7] as well as organic species quantified as Biological

Oxygen Demand (BOD), Chemical Oxygen Demand (COD), and Total Solids (TS) [8- 10]. The mechanism of pollutant removal in an air or wastewater biofilter is a combination of absorption and chemical oxidation of the pollutant material into a biofilm layer rich in bacteria and fungi where a biochemical reaction destroys the compounds. Such biofilm grows on either natural or synthetic support media, or both, and fills the equipment as in a packed bed reactor, or it can be attached to plastic or metallic disks spinning inside a closed vessel as in a Rotating Drum Contactor (RDC). The consortia that build up the biofilm are intrinsically available in natural bed media, such as peat and compost, or need be inoculated into the system when synthetic media are used, for instance, by acclimation with concentrated sludge from water treatment plants.

Unlike technologies such as adsorption, chemical oxidation, wet scrubbing, incineration or condensation, air biofiltration is a cost effective, simple purification process, and it is especially suited for air carrying low loads of contaminant. The main advantage of air biofiltration over other technologies is that no downstream separation is needed, since only non toxic gases such as carbon dioxide and vapor water are produced. Also, biofiltration can be carried out at room pressure and temperature, for which no extra energy supply is required. In addition, there are no chemical costs involved further than the administration of nutrients to the microflora in the system. Finally, biofiltration is an environmentally friendly process that mimics nature and reduces liabilities associated with chemicals and energy handling for the abovementioned unit processes in industry.

In order to ensure the biofilter performance, several operational variables such as contaminant loading flows and concentrations, external nutrient solutions supplied to the microflora, bed pH, media moisture content, temperature, biomass growth, etc. have to be properly monitored. Potential problems commonly encountered due to inadequate manipulation of such variables are clogging, air channeling and excessive pressure drop along the packed bed, acidification of the media, deactivating some specific bacterial breeds and decreasing the removal capacity of the system, etc. [4, 11-20].

One of the major constraints of concern in the operation of biofilters is the biomass growth control, since bed clogging and excessive gas phase pressure drop may arise, causing the biofilter operation to be halted for maintenance. Several techniques have been proposed to control excess biomass growth. Such techniques may be classified into three groups: chemical, physical and biological methods. Chemical methods aim at controlling the supply and chemical nature of nutrients. When excessive biomass in the system is not desired, reducing the load of nutrients into the system or replacing these nutrients by other slowly digested by the microflora may increase the rate of biomass decay and diminish the biofilm thickness. The opposite is observed when handling the constraints conversely [21-22], that is, an increase in the biomass accumulation is observed when the microflora is fed with more and more easily biochemically digested nutrients. In addition, physical methods are used to remove excess biomass. These methods are based on applying an intense shear stress on the biofilm by a water phase, such as in backwashing, on centrifugal forces, as in centrifugation, on crushing, or by ultrasound [21-24]. Finally, a biological method based in the use of predators to consume the biofilm has also been studied [25]. Biomass removal is difficult to control with this technology since predators, once placed on the media, cannot be forced to stay at specific locations and to devour biomass at specific rates.

Recent studies have precisely focused on the selection of media carriers that better handle clogging conditions. Compost, peat, soil, wood chips, bark, agricultural organic derivatives, activated carbon (AC), ceramic beads, perlite, celite, chicken manure, rubber latex, polyurethane (PU) foams, etc. are media that have received attention the most. From all of the aforesaid materials, PU foams have shown to be the ones that best optimize the overall performance of biofilters treating VOC and SRC in airstreams; that is, PU foam packed beds render high pollutant removal rates and maintain low pressure drops along the bed [1, 26-32]. The same conclusion is attained in packed bed filters and RDC treating wastewaters [8, 10, 33]. Previous attempts at improving the foam capabilities as biocarriers have been accomplished by including coatings of adsorbent materials such as AC or zeolites on the foam surfaces [27, 28]. Addition of magnetic particles, selective adsorbents or even nutrients into the polymeric structure of the foams,

are other techniques that may improve significantly the performance of these materials [10].

Media for biotreatment can be classified into two major groups; naturally bioactive media and manufactured synthetic media. Natural media such as compost, peat, bark, wood chips, animal manure, etc, have been widely used in biofiltration systems [19, 21, 34]. The main advantages of this type of materials are their low cost, broad availability, and the fact that inoculation is not needed since microflora is inherently encountered in natural media. However, they tend to compact due to biomass growth, and excess biofilm cannot be sloughed off. Therefore, clogging and high pressure drops are commonly observed in equipment operating with such matter [11, 24, 34, 35].

Synthetic media, on the other hand, can be manufactured to avoid compactness and excessive pressure drops, and to remain chemically inert to the species present in the system. Nevertheless, the closer the media approach ideal properties, the more expensive and the less available they are. Additionally, synthetic media need be inoculated and acclimated for microflora to grow within the system [36-39]. Typical synthetic media used in biofiltration encompass ceramics, activated carbon, plastic materials, polymeric foams, membranes, etc. [41-41].

Desirable media properties include, among others: high specific surface area, increasing therefore the contact area per unit bed volume between the species being transferred and biodegraded subsequently; high porosity, which allows for excess biomass to be sloughed off the system, preventing clogging, air channeling, and reducing the pressure drop along the bed; high rigidity, impeding the medium to compact when its density increases due to excessive biomass attached; good biofilm attachment on the synthetic media surface, which allows for rapid inoculation with external microflora; low gas phase pressure drop, which decreases operation costs, and low media cost per unit volume, which decreases initial design costs. The media should also be chemically inert to the species present in the system and pH changes, as well as resistant to operation temperature alterations [42- 45].

PU foams optimize the overall performance of equipment using immobilized biofilms for removal of VOC and SRC in airstreams [1, 15, 16, 28-32, 46, 47] for the removal of BOD and COD in wastewater in packed bed reactors and RDC [48], and for increasing the production rates of some enzymes and complex molecules using biofilms [49-55]. By optimum performance it is understood that the system attains maximum pollutant removal capacities and efficiencies, high bioprocessing production rates, and low pressure drops along the beds. Still, even though these authors have regarded foams as better biomedia carriers than other types of natural and synthetic media, their works do not address the optimization of PU foams themselves; i.e., pore size, porosity, core material other than PU, etc. Table I. 1 summarizes the most important remarks on recent works carried out to purify airstreams in equipment utilizing immobilized biofilms on PU foams.

Recently, Gabriel and Deshusses [1] and Gabriel et al. [56] reported high removal efficiencies for a field study where a municipally owned, full scale wet scrubber was packed with porous PU foam pieces and operated afterwards as a biofilter removing H2S, mercaptans and other organics. The original packing material encountered in the scrubber had a specific contact area several folds lower than the PU foam. Before retrofitting the full scale scrubber into a bioreactor, experimental studies were carried out in a pilot scale equipment to evaluate the technical feasibility of the conversion.

The performance of the pilot scale biofilter was evaluated at comparable pollutant concentrations and EBRT as those to be used in the scrubber, and both the original packing material and the PU foam were assessed. Contrary to the latter, the authors found that the original scrubber packing was unable to maintain a sufficient amount of biomass to yield H2S removal efficiencies under specifications. After the conversion took place, the authors reported removal efficiencies close to 100% for H2S despite the low EBRT in the equipment (2 sec) as opposed to typical equipment originally built for biofiltration purposes (10 to 60 sec) even at elevated average inlet concentrations (30 ppmv).

Table I. 1. Recent research on air treatment using PU foams as support media for immobilized biofilms.

Reference Equipment Pollutant removed Operation conditions Performance Remarks Ketone were better degraded Biotrickling filter Acetone More than 90% for an than aromatics, whose packed with Methyl ethyl Aromatics: ~ 10-30 ppm v intermittent (8 hr/d) removals were unstable. reticulated PU foam. ketone Acetone: 450 ppm [26] v pollutants loading, and up to even after long biofilter Media seeded with Toluene Methyl ethyl ketone: 12 ppm v 99% for a continuous operation. Fungi was de compost derived Ethylbenzene EBRT: 60 sec. pollutants input. predominant bioactive cultures. p-xylene species. Fungal biofilter showed n-butyl acetate, Biotrickling filter robustness towards sharply methyl ethyl Concentrations ranging from packed with PU foam Removal efficiency of 98% changing inlet VOC [69] ketone, methyl 15-45 ppm at fluctuating cubes. Media seeded v over a period of 94 days. conditions and had an propyl ketone, and rates. EBRT: 15 sec. with fungi. average removal efficiency toluene. of 95% under transient state. The authors highlight the Biotrickling filter Maximum average inlet H S and other Removal efficiencies up to removal capability of the [68] packed with PU foam 2 concentration of H S: 30 SRC. 2 95% for H S. system despite the low cubes. ppm . EBRT: 2 sec. 2 v EBRT. Robustness when loaded Biotrickling filter Removal efficiencies of 99% Concentrations ranging from with 530 ppm of pollutant packed with PU foam Methyl ethyl at steady inlet concentrations v [27] 100 to 530 ppmv. EBRT: 20 for one hour at different cubes, coated with ketone. of 106 ppmv over a period of sec. intervals, mainly due to the AC. 130 days. presence of AC Good performance at Biotrickling filter Concentrations ranging from Removal efficiencies of 90- transient high loads, due to packed with PU foam [28] Ammonia. 50-250 ppmv. EBRT: 23 to 47 98% within the the AC and zeolites. Carbon cubes, coated with sec. concentration range. fiber and rock wools AC and zeolites. performed better, however,

Table I. 1. Recent research on air treatment using PU foams as support media for immobilized biofilms (continued)

Authors Equipment Pollutant removed Operation conditions Performance Remarks Biotrickling filter PU foam yielded the highest packed with PU foam Removal efficiencies ranging removal rate and removal cubes, seeded with Loading rates ranging from [29] α-Pinene. from 50-90% during start-up capacity of α-pinene among fungi. Perlite, clay 25-40 g·m-3 ·hr-1 bed period of 60 days. the four types of media and compost also tested. tested. Nutrient supply had to be Removal efficiency of 99% restricted and biomass Biotrickling filter Maximum inlet concentration: over a period of 300 days. squeezed out from the media [32] packed with PU foam Toluene. 200 ppm . EBRT: 1-4 v Pressure drop less than 4 mm to maintain low biomass cubes. minutes. H2O. within the foam and prevent clogging.

The authors pointed out that these removal efficiencies were higher than other reported cases where longer contact times, i.e. lower superficial gas velocities and larger reactor volumes, were employed. Nevertheless, in experiments made with VOC and media other than foams, it has been reported that by increasing the gas velocity in the bioreactor, the removal efficiency of the system decreases considerably [57-59]. These observations can be explained as follows: When increasing the superficial gas velocity in the system, more gas will flow both through the foam pieces and around the foam pieces, while an increase in the gas phase pressure drop will be detected. This way, a larger fraction of the gas coming inside the bed will be exposed to the internal foam area, which is several orders of magnitude larger than the area outside the foam unit. Overall, the effective contact area between the gas and biofilm supported in the foam struts, which is the volumetric average of the amounts of air flowing both within and past the foam, will increase as well. Thus, assuming the distribution of the latter in the foam unit is uniform and that all struts are equally colonized by the microorganisms, more air will be in contact with the biofilm, which increases the pollutant removal efficiency. On the other hand, increasing the superficial gas velocity will bring about a reduction in the contact time available for the mass transfer process to occur, depleting the removal efficiency as a consequence. Both effects will counteract at different rates while varying the gas superficial velocity. Accordingly, there should exist a range of conditions (i.e. foam and bed porosity, pore size, and superficial gas velocity) under which increasing the gas superficial velocity will actually increase the removal efficiency until a maximum is attained.

For non porous packing material, however, these arguments do not hold and the pollutant removal efficiency should decrease monotonically with gas velocity. Further economical investigations [1, 60] revealed that up to $40,000 per scrubber per year could be attained if scrubbers were converted into biofilters at water works, which represents an estimated total savings of $3 billion worldwide.

I.II. MODEL DEVELOPMENT FOR THE DESCRIPTION OF AIRSTREAM BIOTRICKLING FILTERS PACKED WITH MACROPOROUS SOLID FOAMS

The equations that describe the hydrodynamics, substrate utilization and biomass growth in foam packed bed biotrickling filters are presented below. These equations are coupled to determine the effect of variables such as water and air loads, pollutant inlet concentration, bed height, foam pore size and porosity, etc. in the overall performance of the unit. A better understanding of the effect biomass growth and clogging on the removal efficiency of the system can be achieved based upon these equations.

In developing the biofilter performance equations, the following assumptions are considered: only one substrate is degraded in the system; Cartesian coordinates can be used as reference system (Figure I.1); advection is negligible in the biofilm; substrate transport occurs only due to diffusion in the x direction; no substrate accumulation or depletion occurs; substrate diffusivity in the biofilm is uniform and constant; no substrate partition takes place at the interface liquid-biofilm; there is no flux of substrate across the solid support. Therefore [16]:

2C (x, y) D f R (I.1a) f x 2 C f x f , C f ( f , y) Cl ( f , y) (I.1b) C (0, y) x 0 , f 0 (I.1c) x

Equation (I.1a) establishes that all the substrate diffusing into the biofilm is degraded by the microbial consortia, the rate of which can be expressed mathematically as an hyperbolic function as proposed by Monod:

kX C (x, y) R f f (I.2) C f K s C f (x, y)

Therefore:

2C (x, y) kX C (x, y) f f f (I.3) D f 2 x K s C f (x, y)

l f

C (y) g Non adsorbant support Cl (x, y)

C f (x, y)

Gas Liquid Biofilm

Figure I.1. Substrate concentration profile in a biofilm attached to non adsorbent media.

A mass balance in the liquid layer yields the following:

2C (x, y) l 0 (I.4a) x 2 x f , Cl ( f , y) C f ( f , y) (I.4b) 1 x , C ( , y) C ( , y) (I.4c) f l l f l H g f l

It can be further assumed that substrate transport in the gas phase is not a limiting step in the transfer mechanism, and that there exists complete mixing of the substrate in the gas

phase along the x direction (uniform pollutant concentration in the transversal direction for any given height of the biofilter.) Consequently:

Cg ( f l , y) Cg (x f l , y) (I.5)

The change in the advective flow of substrate along the bed equals the flow of substrate diffusing into the liquid layer from the bulk gas. Thus:

dC (y) v g A J (y) (I.6) g dy s g

From the conservation of mass:

As J g (y) As J l (y) As J f ( f , y) (I.7)

The substrate diffusive flux in the liquid phase, which is uniform in the direction, can be expressed in terms of Fick’s law:

C ( , y) C ( , y) J (y) D l f l D l f (I.8) l l x l x

Note that the derivatives are positive according to the coordinate system chosen. Combining Equations (I.6) through (I.8):

dC (y) C ( , y) v g A D f f (I.9) g dy s f x

Parvatiyar et al. [61] proposed that:

D J (y) J (y) J ( , y) f R (I.10) g l f f v C f g x f

Equation (I.10) is derived by using the observational definition of diffusivity and by assuming that the substrate concentration profile within the biofilm is linear (for a biofilm that typically has a thickness up to a few hundred microns) and approaches zero at the interface of the non adsorbing biofilm-support (no substrate flux exists into or out from the solid support) [61].

Combining Equations (I.2) and (I.10):

D f kX f C f ( f , y) J g (y) J l (y) (I.11) vg K s C f ( f , y)

The total substrate flux can also be expressed in terms of a mass transfer coefficient in the liquid phase as follows:

C (y) J (y) k (C ( , y) C ( , y)) k g C ( , y) (I.12) l l l f l l f l H f f

Combining Equations (I.11) and (I.12):

HD f kX f C f ( f , y) Cg (y) HC f ( f , y) (I.13) kl vg K s C f ( f , y)

Equation (I.13) can be differentiated to yield:

HD f kX f dCg (y) HdC f ( f , y) dCf ( f , y) kl vg K s C f ( f , y)

HD f kX f C f ( f , y) 2 dC f ( f , y) (I.14) kl vg (K s C f ( f , y))

Rearranging Equation (I.14) and combining Equations (I.2) and (I.7) through (I.10):

A D kX C ( , y) HD kX s f f f f dy HdC ( , y) f f dC ( , y) v 2 K C ( , y) f f k v K C ( , y) f f g s f f l g s f f HD kX C ( , y) f f f f dC ( , y) (I.15) k v (K C ( , y)) 2 f f l g s f f

This expression can be integrated along the longitudinal direction y to give:

C f ,o C f ,o C f ,o D f kX dC ( , y) D f kX dC ( , y) HK f f f HdC ( , y) f f f s k v C ( , y) f f k v K C ( , y) C f ,i l g f f C f ,i C f ,i l g s f f L A D kX s f f (I.16) 2 dy 0 vg

The reactor can be thought of a series of smaller reactors, the length of which is sufficiently small to assume that the biofilm kinetic properties k and X f , as well as the effective interfacial area As are relatively uniform along L . Thus, and introducing the definition of removal efficiency E , Equation (I.16) can be written as [61]:

C f ,i C f ,o As D f kX f L K s C f ,i C f ,i K s C f ,i E 2 ln ln ln (I.17) C f ,i Hvg C f ,i C f ,i C f ,o C f ,o K s C f ,o

D kX f f (I.18) Hkl vg C f ,i

Equation (I.17) is the removal efficiency for the bioreactor, and it is transcendental for the dependent variable C f ,o . This expression can further be simplified by assuming that

the substrate concentration at the boundary of the biofilm is approximately equal to that in the liquid at the boundary liquid-gas. For cases where there is an excess of substrate, this is, HK s Cg (y) , the pollutant biodegradation follows a zero order reaction rate, and Equation (I.17) can be rewritten as:

D f kX f LAs HK s E 2 ln(1 E) (I.19) C g,i vg C g,i

Further, whenever HK s C g (y) the second term of Equation (I.19) disappears, leaving and expression that can be physically interpreted in terms of the dimensionless numbers Damkohler ( Da ) and Peclet ( Pe ) as follows:

Da E (I.20a) Pe

Where:

kX L2 A Da f s (I.20b) Cg,i vg

Lv Pe g (I.20c) D f

Two conclusions are drawn from Equations (I.20a) to (I.20c): First, the removal efficiency of the reactor increases along with the dimensionless reaction time Da , which is the ratio of the time that the pollutant is allowed to be in contact with the biofilm (based on the gas convective time and the biofilm/pollutant diffusive time) divided by the time needed by the biofilm to oxidize the pollutant. On the other hand, the removal efficiency decreases when the axial gas advection is higher than the transversal diffusion of the pollutant into the biofilm, meaning that the pollutant does not have sufficient time

to enter the bulk of the biofilm while being carried along with the main gas stream. Secondly, it is clear from Equation (I.20a) that the removal efficiency E is inversely proportional to the inlet pollutant concentration C g,i , as intuitively expected. This is true since the available active sites of the biofilm for the removal of the excess pollutant are insufficient at higher pollutant concentrations. The relationship between Da , Pe and E is similar to that encountered in the Literature for most packed bed reactors involving the transfer of a species from a carrier stream into a second stationary phase where the species is momentarily or permanently retained, such as in catalytic plug flow reactors or sorption packed beds [62]. In general, unless the assumption that HK s C g (y) is fully met, Equation (I.20) will not be mathematically satisfied in a real system; however, Equation (I.20) gives a qualitative idea as to how the removal efficiency changes along with and .

The effective specific area As can be defined as a weighted average of both the internal and external foam piece specific areas, being the weighting factor equal to the percentage of the total gas flow flowing within and around the bed media. In other words:

v f v f As (1 b ) f Af 1 (1 b ) Ab (I.21) vg vg

Richardson et al. [63] provided an expression to calculate the internal foam specific area by assuming the foam to have a tetrakaidecahedral configuration:

12.979(1 0.97 1 f ) Af (I.22) d p 1 f

Typical values for the internal foam specific surface area are in the order of 104-105 m-1 for foams with plain pore sizes d p,o in the range 100-800 µm [63, 64]. Moe and Irvine

-1 [31], however, reported a manufacturer specified internal surface area Af of 620 m for

their PU foam with pore size and porosity of l125 µm and 86%, respectively, for a foam unit cube with a side length of 0.04 m. This number is intuitively much closer to the external unit foam piece area rather than the internal one, considering that for a solid cube -1 with the same dimension its external area Ab is 150 m , thus for a rugose one such area must be higher but in the same order of magnitude.

In order to determine the ratio of the gas flow within the foam to the total gas flow, it is assumed that the gas stream pressure drop within a foam unit equals the gas stream pressure drop around it. Then:

ΔP ΔP (I.23) L f L b

A modified model proposed by Smit and DuPlessis [64] was selected based upon experimental data collected in our experiments (data not shown). The pressure drop predicted by the model [64] is multiplied by a factor of 0.333 to fit most data collected in the lab using monolithic plastic and ceramic foam packing inside a tube through which air was flowing at velocities in the range 1-10 cm/sec. Such packing exhibited values in the following ranges: foam unit size between 1-3 cm, foam porosity f ,o between 70%-

90%, and average pore size d p,o between 250-300 µm, with macro pores well above 1000 µm. Thus [64]:

5 / 2 P 1 v 2 1 72 g f 1 (I.24) L f 3 0.57d p f Re

Where:

5 / 2 2 v (0.57d ) Re g f p (I.25) g f

4 1 2 2cos cos 1 (2 1) (I.26) 3 3 f

The pressure drop around the media can be determined from any correlation used to calculate the pressure drop along a stripping or absorption packed bed. The model of Robbins [65] was selected since it accounts for the effect of increasing the superficial liquid velocity in the bed:

L 0.1 P 2 C2L f f 2 C2L f 4 816.4 C1G f 10 0.4 (C1G f 10 ) (I.27) L b 20000

Where:

F ' G 986v' pd g 100.3 ' (I.28) f g 20 g

62.4 Fpd 0.1 L f Ll 'l (I.29) 'l 20

Equations (I.23), (I.24) and (I.27) are solved simultaneously to obtain vg and v f . The values for Fpd and C 2 have to be fitted from experimental data of the pressure drop in the bed packed with the media of consideration, both for the dry case (just a gas stream flowing along the bed; i.e. L f zero) to obtain Fpd , and then for the bed where the gas is flowing counter currently with respect to a downwards liquid flow to obtain C 2 ; C1 has been estimated to be independent from the type of packing and it has a constant value (see list of symbols) [65]. Such fitting is performed using the data of the packed bed operating without any bacteria in it. Once the calibration is made, Equation (I.27) can be used to predict the pressure drop of the bed for any other application where the media of interest is used to fill the reactor. Different values for are already available in the

Literature for several commercially available packing media [65].

In general, the foam porosity f decreases due to the biofilm and liquid layer presence. Both biofilm and liquid decrease the foam specific area and the velocity of the gas stream in the media for the same bed pressure drop. The reduction in the foam porosity and pore size with biofilm and liquid layers can be calculated as:

2 s 3.464( f l ) f 1 (1 f ,o ) (I.30) s d p d p,o 2( f l ) (I.31)

Where [63]:

s 0.971l 1 f ,o (I.32)

1 l 0.5498d p,o (I.33) 1 0.971 1 f ,o

It is important to highlight that the bed porosity b is not greatly affected by the presence of biofilm or water layers, since at typical values of b ranging from 0.5-0.7, the reduction in the bed porosity with bacterial and liquid films of a few microns in depth is almost negligible, unlike for the internal area of the foam where pores are of the same order of magnitude of the bacterial and liquid films. The liquid layer thickness can be estimated by applying a momentum balance over a flat surface:

3 l vl l 3 (I.34) Af l g

To estimate the biofilm thickness, the model of Rittmann and McCarty [66] may be used:

(As X f d f (y)) kX f C f (x, y) Y As d f (y) bAs X f d f (y) (I.35) t K s C f (x, y)

Equation (I.35) is merely a transient mass balance on the biomass contained within a differential volume of porous bed. The first term of the right hand side of Equation (I.35) is the contribution to the accumulation of biomass due to pollutant consumption and utilization by the bacteria in the biofilm, whereas the second one represents the reduction of the biomass volume due to decay, decomposition and slough off processes. The yield coefficient Y is defined as the mass of biofilm produced per mass of substrate consumed. At steady state:

YJ g (y) YJ l (y) bX f f (y) (I.36)

Equations (I.35) and (I.36) can be solved in what Rittmann and McCarty described as three different physical situations [66] as depicted in Figure I.2. There is a minimum substrate concentrations in the bulk gas C g,min (y) below which the cell decay rate exceeds the rate of new biomass formation. Within this range, no biofilm can be held.

There is also a deep substrate concentration in the bulk gas Cg,deep (y) so that substrate exists along the biofilm, reaching a zero concentration value just at the media support. In the extreme case where the biofilm is fully penetrated, the substrate concentration in the biofilm and liquid layer is uniform all the way through. All cases between deep and fully penetrated are called shallow biofilms.

When the substrate concentration at the gas bulk is minimum, Cg (y) Cg,min (y) , Rittmann and McCarty [66] suggested that the profile within the liquid layer is slightly uniform, since the flow of substrate is very low. Then:

Cg,min (y) HC f ,min ( f , y) (I.37)

Under steady state and combining Equations (I.35) and (I.37):

K sb Cl,min ( l f , y) Cl,min ( f , y) C f ,min ( f , y) (I.38) Yk b

l f Fully penetrated

Biofilm Gas shallow

Liquid deep

C(y)

Figure I.2. Substrate concentration profiles for the three different situations described by Rittmann and McCarty [67]. Deep, Shallow and Fully penetrated biofilm.

On the other hand, when the substrate concentration in the bulk phase attains the deep value:

Cg,deep (y) HC l,deep ( f l , y) (I.39)

From Fick’s law:

l J l,deep (y) l J l,deep (y) Cl,deep ( f l , y) Cl,deep ( f , y) C f ,deep ( f , y) (I.40) Dl Dl

Rittmann and McCarty solved Equation (I.35) empirically for the deep biofilm case as:

D f Cl,min ( f , y) D f HCg,min (y) J l,deep (y) 4.6 2 4.6 2 (I.41) Dl K s Dl K s

An algorithm for the simultaneous solution of Equations (I.3), (I.8) and (I.35) was proposed by the authors for shallow biofilms. The fitted numerical simulations to a single equation is:

3/ 2 Dl Cl ( f l , y) J l (y) l J l (y) J l,deep (y) 1 exp 1 (I.42) Dl Cl,min ( f , y)

By assigning values to Jl (y) , Cl ( f l , y) can be solved using Equation (I.42) provided that Cg.deep (y) HC l ( f l , y) Cg,min (y) . Then, Equation (I.36) can be used to determine the biofilm thickness f . For the case where HC l ( f l , y) Cg,deep (y) , Rittmann and McCarty [67] proposed the following empirical equation :

q J l,deep* (y) kX f Cl ( f l , y) Dl K s (I.43) As 2K s D f K s

1 2q D f kX f 2 2 2 l Dl 2K s D f (I.44) 2 kX f q 1 0.54 1 0.0121ln 1 2 l 1 8.325 ln 2K s D f 7.07

1 2q D f kX f 2 2 2 l Dl 2K s D f (I.45) 2 kX f q 1 0.54 1 0.0121ln 1 2 l 1 8.325 ln 2K s D f 7.07

q 0.75 0.25tanh(0.477 ) (I.46)

Once the biofilm and water layer thickness are determined, a new foam porosity f , foam pore size d p and effective specific area As have to be calculated and the substrate removal efficiency E corrected for these conditions.

I.III. MATERIALS AND METHODS

Continuous tests in one PU foam packed bed were carried out in order to test the H2S removal capability of the system and the feasibility to predict its performance using the model described in the previous section. The bed was filled up to an initial height of 20 cm with open-pore, foam cubes of 1.25 cm in side length, 8-12 pores per inch and a porosity of 98%. The EBRT of the air stream in the system was varied from 4 to 20 sec. A proprietary liquid nutrient solution was sprayed on the top surface of counter-current with respect to the air stream at a flux of 0.0025 L/min/cm2. The column was seeded with activated sludge from a local waste plant, and the continuous tests started after 10 days of operation, after the bacteria in the system had been already acclimatized. The inlet concentrations of H2S were varied in the range of 10-100 ppmv.

I.IV. RESULTS AND DISCUSSION

Figure I.3 shows the predicted gas flow distribution, i.e, gas flowing within the foam unit as a percentage of the total inlet gas flow, in a bed randomly packed with foam units as calculated from Equations (I.23), (I.24) and (I.27). Geometric and other parameters used for the simulation shown in this and other Figures are summarized in Table I. 2. Foam properties such as Fpd and C 2 were all obtained from fitting experimental data collected during dry and wet pressure drop measurements (data not shown) using plastic and ceramic foam packing with plain foam porosities f ,o , plain foam pore size d p,o , bed porosities b and foam pieces with unit dimensions similar to those shown in Table I. 2.

Values for the biofilm kinetic parameters such as k , X f , and Ks , are assumed based on

comparable reported values on the Literature in biofilters treating similar pollutants. It is clear from Figure I.3 that the amount of gas flowing through the media increases monotonically with the foam pore size for a fixed gas velocity vg . When the pore size increases, a larger gas volumetric flow is allowed to pass through the void spaces within the foam producing the same pressure drop as the gas flowing around the media, which is an operation condition sought after considering that the foam specific area is several orders of magnitude higher within the foam than outside it, and that the removal efficiency of the pollutant is proportional to the overall contact area between the colonized foam and the carrying gas. However, at very large pore sizes and high porosities, the specific area within the foam Af decreases itself as described by Equation (I.22). These two effects counteract, and as a consequence, for a given foam and bed porosity, and a fixed gas velocity , there should exist a pore size that maximizes the overall value of the effective specific area As .

100 2000 80 1500

60 1000

40 500

units [% total gas inlet] gas unitstotal [% dp,o= 100 m

Gas flowing within foam flowingGas within foam 20

0 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 v [m/sec] g

Figure I.3. Gas flow distribution in the bioreactor as a function of the superficial gas velocity vg for different foam pore sizes d p,o . Other design parameters are summarized in Table I. 2.

Table I. 2. Values of transport and kinetic parameters as well as reactor design variables used for the simulations. Kinetic values for toluene (Figures I.8a and I.8b) were assumed based on works reported in the Literature [61, 70]. Kinetic values for H2S (Figure I.9) were obtained by fitting the present model (Equation I.17) to the experimental data collected in the foam biofilter.

Figures Figures Figures Parameter I.3 I.3 I.4 and I.5 and I.8a and Figure I.9 I.7 I.6 I.8b

k [sec-1] - - 0.0025 - 0.000025 0.00005

-3 -6 -6 Ks [kg·m ] - - 1.0·10 - 0.005 2.0·10

b [sec-1] - - 0.0001 - 0.0001 0.0001

-3 X f [g·m ] - - 50 - 50 40

Y - - 0.5 - 0.5 0.2

-2 -1 -9 -9 -9 D f [m ·sec ] - - 1.0·10 - 0.7·10 1.0·10

-2 -1 -9 -9 -9 Dl [m ·sec ] - - 1.5·10 - 0.9·10 1.5·10

-1 -4 -4 -4 -4 -4 vl [m·sec ] 2.52·10 2.52·10 2.52·10 2.52·10 0 4.0·10

-5 -5 -5 -5 -5 C 2 9.45·10 9.45·10 9.45·10 9.45·10 - 9.45·10

-1 Fpd [ft ] 75 - 75 - - 30

d p,o [μm] - 100 50 - 1125 2886

f ,o 0.95 0.95 0.95 0.96 0.86 0.98

b 0.50 0.50 0.50 0.50 0 0.4-0.5

-1 Ab [m ] 600 600 600 600 - 600

L [m] - - 1 - 1 0.2

EBRT [sec] - - - - 120 4-20

H - - 0.416 0.416 0.224 0.416

Analogously, when the geometric properties of the foam such as foam pore size and foam porosity are fixed, the effective specific area As will also change with the superficial gas velocity vg since more or less gas will flow within the foam as described in Equation (I.21). When the gas velocity increases, so does the effective specific area of the bed , which translates in a higher removal efficiency. However, the contact time between the solid foam and the carrier gas diminishes and a negative effect in the pollutant removal is expected. Therefore, there is a gas velocity at which these counteracting effects maximizes the removal efficiency of the pollutant for a given configuration of foam and bed. As seen in Equation (I.17), for a given geometry of the foam and bed, and a given set of biofilm kinetic parameters, the removal efficiency will actually be a function of the

2 relationship Asvg , being As not only a function of the foam geometry but also a function of vg itself. This relationship happens to be a linear function, as depicted in Figure I.4.

100 Cg,i= 10 ppmv

80 20 30 40 60 50

[%] E 40

0 0 5e+4 1e+5 2e+5 2e+5 A /v 2 [sec2/m3] s g

2 Figure I.4. Effect of the coefficient Asvg on the pollutant removal efficiency E for a macroporous foam packed bed. Design and biokinetic parameters are summarized on Table I. 2.

The latter shows that for a given inlet concentration C g,i the removal efficiency will

2 increase along with Asvg .

2 The relationship between the coefficient Asvg and the foam geometry and gas velocity is depicted in Figures I.5 and I.6. It can be seen from Figure I.5 that values for the

2 relationship Asvg can be attained at three different gas velocities vg for some bed configurations. This is particularly true for beds with a high packing factor Fpd , i.e., high bed pressure drops for the same superficial gas velocity . In general, Fpd can be though of as a gas stream resistance coefficient whose magnitude is inversely proportional to the void fraction in the bed, b . Therefore, at higher values of more gas is flowing within the media. The local maxima attained at high velocities indicates that almost all the incoming gas is flowing within the foam, for which As attains its maximum value, equal to that of the internal foam piece Af . Past this point an increase in the superficial gas velocity will only reduce quadratically the ratio .

The aforementioned counteracting behavior on the effect of the gas superficial velocity on the pollutant removal by increasing the media effective area while decreasing the EBRT, is better seen in Figure I.7 for a biofilter with a typical geometric configuration and biokinetic parameters. The plotted curves shown in Figure I.7 give qualitative insights as to why a foam packed biofilter operating at very high superficial velocities and EBRT of just 1.6 sec had a remarkable performance, despite the short contact time between the pollutant and the biofilm carrier [68].

2e+5

2e+5 d = 100 m ] p,o

2 1e+5

[sec

2 200

A 300 5e+4 500 1000 1900 0 0 1 2 3 4 v [m/sec] g

2 Figure I.5. Effect of gas superficial velocities vg on the function Asvg for a macroporous foam packed bed. Pore size d p,o effect. Design and biokinetic parameters are summarized on Table I. 2.

2.0e+5 1.8e+5 1.6e+5

]

3 1.4e+5

2 1.2e+5 100 1.0e+5 90

[sec

2 80 g 8.0e+4 v 70

A 6.0e+4 60 4.0e+4 2.0e+4 -1 Fp,d= 50 ft 0.0 0 1 2 3 4 v [m/sec] g

Figure I.6. Effect of gas superficial velocities on the function for a

macroporous foam packed bed. Packing factor Fpd effect. Design and biokinetic parameters are summarized on Table I. 2.

From the aforementioned discussion, it can be recognized that the significance of the model herein presented lies in that, given a preset superficial velocity of a gas carrying a specific pollutant at a given initial concentration, the designer can determine the appropriate foam pore size and porosity so that the removal efficiency is maximized for a fixed bed height; in other words, the foam geometry can potentially be optimized in the design stage. Further, and unlike previous models, the impact of the biofilm and water layers on the reduction of the foam pore size and porosity, and therefore the reduction of

2 the value Asvg is accounted for. This way, and before the biofilter is operational, the foam material can be chosen so that the presence of biofilm will not clog up the pores of the foam, undermining the biofilter performance as it is commonly seen in the field.

100 C =10 ppmv g,i 80 50 40 60 30 20

[%] E 40

0 0 1 2 3 4 5 v [m/sec] g

Figure I.7. Effect of gas superficial velocities vg on the removal efficiency E of a pollutant in a bioreactor with the configuration and biofilm kinetic properties indicated in Table I. 2.

The model can be used to predict the performance of a biofilter packed with a monolithic macroporous foam media, as shown in Figures I.8a and I.8b, and for a biofilter packed with foam units where the bed porosity is different than zero, as shown in Figure I.9.

200

160

120

40 Tolueneconcentration [ppmv] 0 0.0 0.2 0.4 0.6 0.8 1.0 Reactor height [m] Model Experimental

Figure I.8a. Comparison between predicted and experimental toluene concentration profiles in a polyurethane foam packed bioreactor as presented by Moe and Irvine [31].

C g,i : 200 ppmv

10 Tolueneconcentration [ppmv] 0 0.0 0.2 0.4 0.6 0.8 1.0 Reactor height [m] Model Experimental

Figure I.8b. Comparison between predicted and experimental toluene concentration profiles in a polyurethane foam packed bioreactor as presented by Moe and Irvine [31].

C g,i : 50 ppmv

For the monolithic biofilter case, Figures I.8a and I.8b, the distribution of gas flow does not apply, and Equation (I.17) can be used directly by replacing As with Af . The predicted values for the concentration of the pollutant using the present model for the monolithic bed are in good agreement with the experimental results published by Moe and Irvine [31] in their reactor treating toluene. The biofilm kinetic parameters used for the simulation are in the same order of magnitude of those reported elsewhere for similar systems as in Table I. 2.

Table I. 3. Calculated values for the foam internal area Af , foam effective contact area

As , airstream pressure drop P / L , liquid layer thickness l , biofilm thickness f and fraction of inlet airstream flowing inside the foam packing for the different experimental cases presented on Figures I.8a, I.8b and I.9.

Parameter Figures I.8a and I.8b Figure I.9

[m-1] 46,708 127,071-150,553

-1 As [m ] 46,708 16,596-77,467

vg [m/sec] 0.008 0.010-0.050

P / L [Pa/m] 0.4-0.5 0.01-0.24

l [μm] 0 9-10

[μm] 5 1-8

Fraction of inlet air flowing within foam 100 12-50 packing [%]

Experimental results shown in Figure I.9 in the foam units, packed bed biofilter case were obtained in the lab as described in the materials and methods section for the reactor degrading H2S. Figure I.9 shows the experimental outlet concentrations of H2S versus the predicted ones for sets of data points obtained in the range of inlet H2S concentrations from 10-100 ppmv, and EBRT from 4-20 sec. Table I. 3 shows the corresponding calculated values for the specific areas for the foam and the effective specific area for the

systems illustrated in Figures I.8a, I.8b and I.9, as well as the gas phase head loss corresponding to each of these referenced works. The calculated gas phase pressure drops are very small for both systems compared to the maximum observed ones of 50 Pa/m for the case of the monolithic reactor [31] and 40 Pa/m for our experiments; however, such observed values are just upper limits, and they represent measurements determined using liquid manometers whose fluid is displaced just a few millimeters, which is difficult to read precisely, and should not be taken as accurate quantities. Also, it should be considered that due to foam compaction and uneven accumulation of biomass and loose debris in real systems, the pressure drop observed in the field will most likely be much higher than that predicted by Equation (I.24) which assumes uniform biofilm distribution and media rigidity. That the pressure drop is underestimated with Equation (I.24) and Equation (I.27) does not eliminate the flow distribution criterion indicated in Equation (I.23); also, it is assumed that the foam compaction and clogging with blocks of biofilm unevenly spread affects quantitatively the same way the calculation of the gas phase pressure drop both inside and outside the media.

100 90 80 70 60 50 Predicted 40 30 20

10 Predicted outlet concentration Predicted outlet [ppmv] 0 0 20 40 60 80 100

Experimental H2S outlet concentration [ppmv]

Figure I.9. Comparison between the experimental and predicted values for the outlet concentration of a foam biofilter treating H2S polluted airstreams at EBRT: 4-20 sec and initial concentrations in the range 10-100 ppmv.

I.V. CONCLUSIONS

In this paper, we have presented a model that couples biokinetics, hydrodynamics and geometry to allow for optimizing the foam configuration for treatment of airstreams in biotrickling filters using immobilized biofilms on these and other porous materials. The model can be used to predict the impact of biomass accumulation and clogging when inspecting the effect of pore size and porosity of the foam on the performance of the biotrickling reactor, and describes the air flow distribution in the system based on gas stream head loss arguments. The goodness of the model is that geometric parameters of the foam packing media such as porosity, pore size and foam unit size can be chosen so that some preset removal efficiency, gas phase head loss or biomass accumulation are attained. This way, the model presents a unique mathematical way to design macroporous packing media for biofiltration applications. The model can also be applied to monolithic beds with uniform porosity. Indeed, predicted results calculated with the model both for a monolithic biofilter and a packed bed foam biofilter are in good agreement with their experimental values, which involve a wide range of initial concentrations and EBRT for organic and sulfur reduced compound pollutants.

I.VI. SYMBOLS

-1 As : Effective specific area [m ]

-1 Ab : External foam specific area [m ] -1 Af : Internal foam specific area [m ] b : Decay coefficient [s-1] -3 C f : Substrate concentration in the biofilm [kg·m ]

-3 Cg : Substrate concentration in the gas [kg·m ]

-3 Cl : Substrate concentration in the liquid [kg·m ]

C1 : 0.000000074 [dimensionless]

C 2 : Empirical coefficient for each type of packing material, Equation (I.27) [dimensionless]

Da : Damkohler number, Equation (I.20a) [dimensionless] 2 -1 D f : Effective diffusivity in biofilm [m ·s ]

2 -1 Dl : Substrate diffusivity in liquid [m ·s ] d p : Operation foam pore size, with biofilm, water layer or both [m] d p,o : Plain foam pore size [m] E : Removal Efficiency [dimensionless] -1 Fpd : Packing factor [ft ] g : Gravity [10 m·s-2]

G f : Gas loading factor [dimensionless]

-1 -2 Gl : Gas mass velocity [lb·hr ·ft ] H : Dimensionless Henry’s law constant [dimensionless] -2 -1 J f : Diffusive flux in the biofilm [kg·m ·s ]

-2 -1 J g : Diffusive flux in the gas [kg·m ·s ]

-2 -1 J l : Diffusive flux in the liquid [kg·m ·s ] k : Maximum Monod’s velocity of reaction [s-1] -1 k l : Substrate liquid phase mass transfer coefficient [m·s ]

-3 Ks : Half saturation constant [kg·m ] l : Strut length [m] L : Reactor length [m]

L f : Liquid loading factor [dimensionless]

-1 -2 Ll : Liquid mass velocity [lb·hr ·ft ] Pe : Peclet number, Equation (I.20b) [dimensionless] ppmm: Parts per million, mass [dimensionless] ppmv: Parts per million, volume [dimensionless] P : Gas stream pressure drop within the foam [kg·m-2·s-2] L f P : Gas stream pressure drop in the bed [kg·m-2·s-2] L b

q : Dimensionless coefficient [dimensionless] R : Biodegradation rate [kg·m-3·s-1] C f Re: Reynolds number [dimensionless] t : Time [s] -1 v f : Superficial gas velocity through foam packing [m·s ]

-1 vg : Superficial gas velocity [m·s ]

-1 v'g : Superficial gas velocity [ft·s ]

-1 vl : Superficial liquid velocity [m·s ] -3 X f : Biofilm density [kg·m ] x : Axis tag [m] y : Axis tag [m] Y : Yield coefficient [dimensionless] Greeks : Factor Equation (I.18) : Tortuosity [dimensionless]

f : Biofilm thickness [m]

l : Liquid thickness [m]

b : Bed porosity with or without biofilm and water layer [dimensionless]

f : Operation foam porosity with biofilm, water layer or both [dimensionless]

f ,o : Plain foam porosity with neither biofilm nor water layer [dimensionless] : Dimensionless coefficient [dimensionless]

s : Strut thickness [m] -1 -1 g : Gas viscosity [kg·m ·s ]

-1 -1 l : Liquid viscosity [kg·m ·s ]

'l : Liquid viscosity [cP] -3 g : Gas density [kg·m ]

-3 'g : Gas density [lb·ft ]

-3 l : Liquid density [kg·m ]

-3 'l : Liquid density [lb·ft ] : Dimensionless coefficient [dimensionless] Subscrpits i : Inlet o : Outlet

I.VII. REFERENCES

1. Gabriel, D. and M. Deshusses. (2004) “Technical and economical analysis of the conversion of a full-scale scrubber to a biotrickling filter for odor control.” Water Science and Technology. 50(4) 309-318. 2. Shareefdeen, Z., Herner, B. and Wilson, S. (2002) “Biofiltration of nuisance sulfur gaseous odors from a meat rendering plant.” Journal of Chemical Technology and Biotechnology. 77(12) 1296-1299. 3. Cohen, Y. (2001) “Biofiltration - The treatment of fluids by microorganisms immobilized into filter bedding material: A review.” Bioresource Technology. 77(3) 257-274. 4. Cárdenas-González, B., Ergas, S., Switzenbaum, M. and N. Phillibert. (1999) “Evaluation of full-scale biofilter media performance.” Environmental Progress. 18(3) 205-211. 5. Chandravathanam, S. and Murthy, D. (1999) “Studies in nitrification of municipal sewage in an upflow biofilter.” Bioprocess Engineering. 21(2) 117-122. 6. Chitwood, D., Devinny, J. and F. Reynolds. (1999) “Evaluation of a two-stage biofilter for treatment of POTW waste air.” Environmental Progress. 18(3) 212- 221. 7. Cook, L., Gostomski, P. and Apel, W. (1999) “Biofiltration of asphalt emissions: Full-scale operation treating off-gases from polymer-modified asphalt production.” Environmental Progress. 18(3) 178-187 8. Jou, C. and G. Huang. (2003) “A pilot study for oil refinery wastewater treatment using a fixed-film bioreactor.” Advances in Environmental Research. 7(2) 463- 469. 9. Kwun, S. and C. Kim. (2002) “Enhanced nutrient removals using conventional anoxic biomechanic aerobic system for on-site wastewater treatment. Journal of Environmental Science and Health. Part A: Toxic/hazardous substances and environmental engineering. 37(5) 863-873. 10. Makarevich, A., Dunaitsev, I. and L. Pinchuk. (2000) “Aerobic treatment of industrial wastewaters by biofilters with fibrous polymeric biomass carrier.” Bioprocess Engineering. 22(2) 121-126.

11. Ramírez-López, E., Corona-Hernández, J., Dendooven, L., Rangel, P. and F. Thalasso. (2003) “Characterization of five agricultural by-products as potential biofilter carriers.” Bioresource Technology. 88(3) 259-263. 12. Delhoménie, M., Bibeau, L. and M. Heitz. (2002) “A study of the impact of particle size and adsorption phenomena in a compost-based biological filter.” Chemical Engineering Science. 57(24) 4999-5010. 13. Sheridan, B., Curran, T. and V. Dodd. (2002) “Assessment of the influence of media particle size on the biofiltration of odorous exhaust ventilation air from a piggery facility.” Bioresource Technology. 84(2) 129-143. 14. Hirai, M., Kamamoto, M., Yani, M. and M. Shoda. (2001) “Comparison of the biological H2S removal characteristics among four inorganic packing materials.” Journal of Bioscience and Bioengineering. 91(4) 396-402. 15. Govind R. and S. Narayan (2005). “Selection of bioreactor media for odor control.” Chapter 4 in “Biotechnology for Odor and Air Pollution Control.” Edited by Z. Shareefdeen and A. Singh. Springer Verlag, Germany. 65-100. 16. Goncalves, JJ. and R. Govind. (2005) “Simulation of biotrickling filters using novel foams for treating odors and volatile compounds.” Proceedings of the 2005 AIChE Fall Annual Meeting. Cincinnati, Ohio. 17. Shinabe, K., Oketani, S., Ochi, T., Kanchanatawee, S. and M. Matsumura. (2000) “Characteristics of hydrogen sulfide removal in a carrier-packed biological deodorization system.” Biochemical Engineering Journal. 5(3) 209-217. 18. Zhou, Q., Huang, Y., Tseng, D., Shim, H. and S. Yang. (1998) “A trickling fibrous-bed bioreactor for biofiltration of benzene in air.” Journal of Chemical Technology and Biotechnology. 73(4) 359-368. 19. Wang, Z. and R. Govind. (1997) “Biofiltration of isopentane in peat and compost packed beds.” AIChE Journal. 43(5) 1348-1356. 20. Smet, E., Chasaya, G., van Langenhove, H. and W. Verstraete. (1996) “The effect of inoculation and the type of carrier material used on the biofiltration of methyl sulphides.” Applied Microbiology and Biotechnology. 45(1-2) 293-298. 21. Delhoménie, M., Bibeau, L., Gendron, J., Brzezinski, R., and M. Heitz. (2003) “A study of clogging in a biofilter treating toluene vapors.” Chemical Engineering Journal. 94(3) 211-222. 22. Kennes, C. and M. Veiga. (2002) “Insert filter media for the biofiltration of waste gases - Characteristics and biomass control”. Reviews in Environmental Science and Biotechnology. 1(3) 201-214. 23. Aizpuru, A., Khammar, N., Malhautier, L. and J. Fanlo. (2003) “Biofiltration for the treatment of complex mixtures of VOC. Influence of the packing material.” Acta Biotechnologica. 23(2-3) 211-226. 24. Torkian, A., Dehghanzadeh, R. and M. Hakimjavadi. (2003) “Biodegradation of aromatic hydrocarbons in a compost biofilter.” Journal of Chemical Technology and Biotechnology. 78(7) 795-801. 25. Woertz, J., van Heiningen, W., van Eckert, M., Kraakman, N., Kinney, K. and J. van Groenestijn. (2002) “Dynamic bioreactor operation: effects of packing material and mite predation on toluene removal from off-gas.” Applied Microbiology and Biotechnology. 58(5) 690-694.

26. Qi, B. and W. Moe. (2006) “Performance of low pH biofilters treating a paint solvent mixture: Continuous and intermittent loading.” Journal of Hazardous Materials, B. 135(1-3) 303-310. 27. Li, C. and W. Moe. (2003) “Sequencing batch biofilter operation for treatment of methyl ethyl ketone (MEK) contaminated air.” Environmental Technology. 24(5) 531-544. 28. Kim, H., Xie, Q., Kim, Y. and S. Chung. (2002) “Biofiltration of ammonia gas with sponge cubes coated with mixtures of activated carbon and zeolite.” Environmental Technology. 23(8) 839-847. 29. Van Groenestijn, J. and J. Liu. (2002) “Removal of alpha-pinene from gases using biofilters containing fungi.” Atmospheric Environment. 36(35) 5501-5508. 30. Moe W. and R. Irvine. (2000) “Polyurethane foam medium for biofiltration. I: Characterization.” Journal of Environmental Engineering-ASCE. 126(9) 815-825. 31. Moe W. and R. Irvine. (2000) “Polyurethane foam medium for biofiltration. II: Operation and performance.” Journal of Environmental Engineering-ASCE. 126(9) 826-832. 32. Moe W. and R. Irvine. (2001) “Polyurethane foam based biofilter media for toluene removal.” Water Science and Technology. 43(11) 35-42. 33. Gaffney, A., Markov, S. and M. Gunasekaran. (2001) “Utilization of cyanobacteria in photobioreactors for orthophosphate removal from water.” Applied Biochemistry and Biotechnology. 91(3) 185-193. 34. Bibeau, L. and M. Heitz. (2000) “Biofiltration d'air pollué par du xylène sur un nouveau lit à base de cellulose.” Canadian Journal of Civil Engineering. 27(4) 814-828. 35. McNevin, D. and J. Barford. (2000) “Biofiltration as an odour abatement strategy.” Biochemical Engineering Journal. 5(3) 231-242. 36. Ibrahim, M., Mizuno, H., Yasuda, Y., Fukunaga, K. and K. Nakao. (2001) “Removal of mixtures of acetaldehyde and propionaldehyde from waste gas in packed column with immobilized activated sludge gel beads.” Biochemical Engineering Journal. 8(1) 9-18. 37. Paca, J., Koutsky, B., Maryska, M. and M. Halecky. (2001) “Styrene degradation along the bed height of perlite biofilter.” Journal of Chemical Technology and Biotechnology. 76(8) 873-878. 38. Chung, Y., Huang, C. and C. Tseng. (1996) “Operation optimization of Thiobacillus thioparus CH11 biofilter for hydrogen sulfide removal.” Journal of Biotechnology. 52(1) 31-38. 39. Chung, Y., Huang, C. and C. Tseng. (1996) “Biodegradation of hydrogen sulfide by a laboratory-scale immobilized Pseudomonas putida CH11 biofilter.” Biotechnology Progress. 12(6) 773-778. 40. Fitch, M., Neeman, J. and E. England. (2003) “Mass transfer and benzene removal from air using latex rubber tubing and a hollow-fiber membrane module.” Applied Biotechnology and Biochemistry. 104(3) 199-214. 41. Kim, B., Blaine, S. and L. Neilson. (1999) “Biofiltration of solvent vapors from munitions manufacturing operations.” CERL Technical Report, US Army Corps of Engineers, Construction Engineering Research Laboratory. 9-106.

42. Hartikainen, T., Martikainen, P., Olkkonen, M. and J. Ruuskanen. (2002) “Peat biofilters in long-term experiments for removing odorous sulphur compounds.” Water, Air and Soil Pollution. 133(1-4) 335-348. 43. Shroeder, E. (2002) “Trends in application of gas phase bioreactors.” Reviews in Environmental Science and Biotechnology. 1(1) 65-74. 44. Kennes, C. and F. Thalasso. “Waste gas biotreatment technology.” Journal of Chemical Technology and Biotechnology. 72 (1998) 303-319. 45. Wani, A., Branion, R. and A. Lau. (1998) “Degradation kinetics of biofilter media treating reduced sulfur odors and VOC’s.” Journal of the Air & Waste Management Association. 48(12) 1183-1190. 46. Hori, K., Yamashita, S., Ishi, S., Kitagawa, M., Tanji, Y. and H. Unno. (2001) “Isolation, characterization and application to off-gas treatment of toluene- degrading bacteria.” Journal of Chemical Engineering of Japan. 34(9) 1120-1126. 47. Thalasso, F., Razo-Flores, E., Ancia, R., Naveau, H. and E. Nyns. (2001) “Pressure-drops control strategy in a fixed-bed reactor.” Journal of Hazardous Materials. 81(1-2) 115-122. 48. Yang, Y., Tada, C., Miah, M., Tsukahara, K., Yagishita, T. and S. Sawayama. (2004) “Influence of bed materials on methanogenic characteristics and immobilized microbes in anaerobic digesters.” Materials Science and Engineering. Part C: Biomimetic and Supramolecular Systems. 24(3) 413-419. 49. Jianlong W. (2000) “Production of citric acid by immobilized Aspergillus niger using a rotating biological contactor (RBC).” Bioresource Technology. 75(3) 245- 247. 50. Rodríguez, S., Longo, M., Cameselle, C. and Sanromán, A. (1999) “Production of manganese peroxidase and laccase in laboratory-scale bioreactors by Phanerochaete chrysosporium.” Bioprocess Engineering. 20(6) 531-535. 51. Rodríguez, S., Rivela, I., Muñoz, A. and A. Sanroman. (2000) “Ligninolytic enzyme production and the ability of decolourisation of Poly R-478 in packed bed bioreactors by Phanerochaete chrysosporium.” Bioprocess Engineering. 23(3) 287-293. 52. Chiou, T., Wang, Y. and H. Liu. (1998) “Utilizing the macroporous packed bed for insect cell/baculovirus expression.” Bioprocess Engineering. 18(2) 91-100. 53. Daraktchiev, R., Beschkov, V., Kolev, N. and T. Aleksandrova. (1997) “Bioreactor with a semi-fixed packing: anaerobic lactose to lactic acid fermentation.” Bioprocess Engineering. 16(2) 115-117. 54. Feijoo, G., Dosoretz, C. and J. Lema. (1995) “Production of lignin peroxidase by Phanerochaete chrysosporium in a pecked bed bioreactor operated in semi- continuous mode.” Journal of Biotechnology. 42(3) 247-253. 55. Amin, G. (1994) “Continuous production of glutamic acid in a vertical rotating immobilized cell reactor of the bacterium Corynebacterium glutamicum.” Bioresource Technology. 47(2) 113-119. 56. Gabriel, D., Cox, H. and M Deshusses. (2004) “Conversion of full-scale wet scrubbers to biotrickling filters for H2S control at publicly owned treatment works.” Journal of Environmental Engineering. 130(10) 1110-1117. 57. Corsi, R. and L. Seed. (1995) “Biofiltration of BTEX: Media, substrate, and loading effects.” Environmental Progress. 14(3) 151-158.

58. Jorio, H., Bibeau, L. and M. Heitz. (2000) “Biofiltration of air contaminated by styrene: Effect of nitrogen supply, gas flow rate, and inlet concentration.” Environmental Science and Technology. 34(9) 1764-1771. 59. Lu, C., Lin, M. and I. Wey. (2001) “Removal of pentane and styrene mixtures from waste gases by a trickle-bed air biofilter.” Journal of Chemical Technology and Biotechnology. 76(8) 820-826. 60. Gabriel, D. and M. Deshusses. (2003) “Retrofitting existing chemical scrubbers to biotrickling filters for H2S emission control.” Proceedings of the National Academy of Science USA. 100(11) 6308-6312. 61. Parvatiyar, M., Govind, R. and Bishop, D. (1996) “Biodegradation of toluene in a membrane biofilter”. Journal of Membrane Sciences. 119(1) 17-24. 62. Bird, R., Stewart, W. and E. Lightfoot. (2006) “Transport Phenomena.” Second Edition. Wiley. 63. Richardson, J. Peng, Y. and D. Remue. (2000) “Properties of ceramic foam catalyst supports: pressure drop.” Applied Catalysis A: General. 204(1) 19-32. 64. Smit, G. and J. DuPlessis. (1999) “Modeling of non-Newtonian purely viscous flow through isotropic high porosity synthetic foams.” Chemical Engineering Science. 54(5) 645-654. 65. Robbins, L. (1991) “Improved pressure drop prediction with a new correlation.” Chemical Engineering Progress. May 1991 87-91. 66. Rittmann, B. and McCarthy, B. (1980) “Model of steady-state biofilm kinetics.” Biotechnology and Bioengineering. 22 2343-2357. 67. Rittmann, B. and McCarthy, B. (1978) “Variable-order model of bacterial-film kinetics.” Journal of Environmental Engineering-ASCE. 104 889-900. 68. Gabriel, D. and M. Deshusses. (2003) “Performance of a full-scale biotrickling filter treating H2S at a gas contact time of 1.6 to 2.2 seconds.” Environmental Progress. 22(2) 111-118 69. Moe, W. and B. Qi. (2004) “Performance of a fungal biofilter treating gas phase solvent mixtures during intermittent loading.” Water Research. 38(9) 2259-2268. 70. Mars, A., Prins, G., Wietzes, P., de Koning, W. and D. Janssen. (1998) “Effect of trichloroethylene on the competitive behavior of toluene-degrading bacteria.” Applied Environmental Microbiology. 64(1) 208–215.

CHAPTER II

ENHANCED BIOFILTRATION USING CELL ATTACHMENT PROMOTORS

SUMMARY

The use of positively charged polymers to enhance attachment of bacteria to surfaces, for enhancing biofiltration of H2S has been investigated. Preliminary batch experiments on polyurethane (PU) foam cubes submerged in a bacterial broth for 40 days showed that a 25 mg/L polyethyleneimine (PEI) aqueous solution yielded the highest solids attachment to the PU foam. Based on this result, two columns operating as biotrickling filters packed with PU foam cubes, one with cubes coated with a solution of 25 mg/L of PEI (coated reactor) and the other containing just plain PU foam cubes (uncoated reactor) were tested in order to determine the effect of the coating on the performance of the reactors operating at Empty Bed Residence Times (EBRT) ranging from 6 to 60 sec and inlet H2S 3 concentrations up to 235 ppmv (inlet overall loads of up to 44 gH2S/m bed/hr). After acclimation was achieved, both reactors showed overall removal efficiencies (RE) mostly in the range of 90-100% over a period of 125 days. The lower and upper halves of the 3 coated reactors exhibited elimination capacities (EC) of 63, 77 gH2S/m bed/hr, respectively, and retention of Volatile Solids (VS) of 42 and 30 mg VS/cube, while the 3 uncoated reactor gave lower and upper half values of 27 and 40 gH2S/m bed/hr for the EC and 23 and 24 mg VS/cube for the VS retention. The experimental data was fitted to a model which included the airflow distribution through and around the foam packing media including the water and biofilm layer thickness, and the best fit for the product of media coverage and maximum Monod’s biodegradation velocities were 6.69x10-4 and 2.56x10-4 sec-1 respectively, for the coated and uncoated reactors. Denaturing Gradient Gel Electrophoresis (DGGE) performed on the collected biomass showed that the predominant bioactive species in both reactors belonged to the genus Acidithiobacillus, with no differentiation within each reactor itself.

II.I. INTRODUCTION

Biofilters for treating odors and/or volatile organic compounds (VOCs) have mainly relied on either naturally bioactive media, such as compost, wood chips, etc., or synthetic media. While naturally bioactive media suffers from settling due to decomposition, eventually requiring replacement, synthetic media biofilters require extended acclimation times for initial establishment of biofilms on the media surface. This requires synthetic media biofilters to emit odors and VOCs during the acclimation period, which can extend for several weeks. Attempts to reduce this acclimation time have included using small particulate media or foam, in which any biomass added during seeding is caught due to small pass-through passage or high tortuosity. However, such biofilters eventually suffer from clogging due to inability to allow biomass to slough off from the surface of the media, thereby resulting in excessive gas pressure drop.

Several studies have argued the selection of the most adequate media according to the degree they satisfy properties such as high interfacial contact area, low density, rigidity, high porosity and water retention capacity, chemical resistance to a wide range of pH values, and appropriate bacterial attachment [1,2]. PU foam has received considerable attention due to its ability to capture biomass during seeding and thereby reduce acclimation time. PU foam filters have been successfully used for treating H2S, wherein the biomass yield is low due to the absence of carbon source and hence have not suffered from clogging issues [3-8]. However, emission of odors during the acclimation period is still a major issue with PU foam biofilters used for treating H2S from wastewater treatment plants.

Other attempts to improve the start-up performance of PU foam biofilters includes modifying their material properties by either coating or mixing with adsorbents like activated carbon or zeolites [4, 5]. In this paper, the use of positively charged polymers to reduce the initial acclimation time and improve biofilter efficiency has been studied. Most synthetic media using plastic materials offer a negatively charged surface. Anchorage-dependent cells, especially under starvation conditions, do not produce

sufficient quantities of positively charged extracellular matrix proteins, thereby adhering weakly to the plastic surface. Pre-coating of the plastic surface with extracellular matrix proteins, such as collagen, fibronectin, laminim, etc. or synthetic polymeric cations have been shown to improve cell attachment. For instance, Vancha [9] showed that after coating culture plates with PEI, Poly-d-Lysine (PDL) and Collagen as individual coating agents at different diluted concentrations, several cell lines attached more firmly and spread more widely after a few days of incubation, compared to cells deposited on uncoated culture dishes. Interestingly, no studies have been found dealing with the potential environmental applications of these polymeric cofactors and bioadhesives on the treatment of airstreams or wastewater using immobilized biofilms.

II.II. MATERIALS AND METHODS

II.II.I. Coating batch experiments and coating selection

Batch experiments were carried out using open pore, black PU foam cubes of 2.54 cm in side length, 8-12 pores-per-inch (PPI) and porosity of 98%, submerged in a beaker containing 4000 mL of a nutrient solution with the following composition [g compound per 1000 g of deionized water]: Na2HPO4, 1.2; KH2PO4, 1.8; MgSO47H2O, 0.1;

(NH4)2SO4, 0.1; CaCL2, 0.03; FeCl3, 0.02; MnSO4, 0.02 and agar, 1.5. The medium was inoculated with 2.50 g of a microbial blend used for BOD5 analysis (B.O.D. Seed, Bio- systems Int. IL). The batch was bubbled with 15 sccm of a 5% v/v H2S in nitrogen source (Matheson, IL) as well as air while stirring gently. Before submersion, 6 different cationic solutions were used to coat the PU foam cubes, 8 cubes per solution, while other 8 cubes were submerged without any previous treatment. The aqueous solutions were PEI (Supelco, PA) at 25, 50 and 100 mg/L, PDL (Sigma, MO) at 50 and 100 mg/L, and collagen from calf skin (Sigma, MO) at 3 g/L. The coating was accomplished by submerging the cubes in a beaker containing the solution and stirring gently for 20 minutes. The cubes were then removed and allowed to dry by natural convection under a stream of room temperature air. The increase in mass attached to the cubes was

monitored for 40 days by weighing one of the eight cubes at a time every 5 days after drying at 105°C overnight.

II.II.II. Continuous experiments

A schematic of the experimental set-up for the continuous tests is shown in Figure II.1.

Compressed air (room temperature and pressure, 0.1% relative humidity, 30 ppmV H2O) from the lab air piping system entered two transparent PVC packed bed reactors (90 cm length, 10 cm diameter) filled up to an initial height of 60 cm (initial bed porosity of 53%) with open-pore, white PU foam cubes of 1.25 cm in side length, with 8-12 PPI and a porosity of 98%. The Empty Bed Residence Time (EBRT) of the air stream in the system was varied from 6 to 60 sec. A liquid nutrient solution, as described above, was sprayed on the top surface of counter-current with respect to the air stream at a flux of 0.061 GPM/ft2 (0.0025 L/min/cm2). Both reactors were equipped with 4 sampling ports separated 30 cm from each other, and each sampling port was connected to a solenoid valve operated by a computer program to allow controlled sampling. One of the columns was packed with PU foam cubes coated with the cofactor (PEI solution at 25 mg/L) selected from the batch experiments as described above (Coated reactor, or just “Coated”) and the other column was packed with plain PU foam (Uncoated reactor, or just “Uncoated”) for control and comparison purposes. The two columns were seeded with a 1:1 mixture of secondary sludge from Cincinnati’s Mill Creek WWTP and the nutrient solution. The inlet concentrations of H2S varied up until 235 ppmv.

II.II.III. Analytical and quantification

H2S concentrations were measured in real time by means of an inline electrochemical sensor (iTrans, Industrial Scientific Corp., PA) able to read concentrations of up to 500 ppmv at an inlet flow of 0.8-1.0 STPL/hr. The PU foam PPI and its pore size were determined by an imaging analysis software (ImagePro 4.0, MediaCybernetics, MD) using a light microscope. In order to determine the accumulation or depletion of the agar and organics in the nutrients solution, Chemical Oxygen Demand (COD) was determined

over a period of two consecutively nutrients solution make-up additions by using a spectrophotometer (DR2000, Hach, CO) after digesting the samples with dichromate at 150°C for 2 hours. Scanning Electron Microscope (SEM) and Energy Dispersive X-ray Spectroscopy (EDS) were used to observe the biomass attached to the foams and to analyze the chemical composition of spots of interest.

Sensor and Data Acquisition Mass Flow Controller

Biofilter

Recycling pump Hydrogen Sulfide

Compressed Air

Nutrient Tank

Figure II.1. Schematic of the biofiltration system to be used in this work

Upon completion of the continuous experiments, VS were determine in each half of both reactors by weighing 3 samples containing the aforementioned biomass after drying them at 105°C overnight and 500°C for 4 hours, subsequently. Additionally, DGGE of 16S rRNA biomass fragments was performed on samples containing a 1:1 biomass and sterile nutrients solution mixture obtained from each half of both reactors. The biomass was retrieved by squeezing out and washing the foam cubes with deionized water, and letting the extracted biomass settle for about 2 hours. The sampling for the VS and DGGE analyses were performed on biomass as extracted from the foam without any further purification method, other than those required by DGGE protocols itself.

II.III. RESULTS AND DISCUSSION

II.III.I. Overall biofilter performance

The removal efficiency of H2S for the coated and uncoated reactors is shown in Figures II.2 and II.3, respectively, for an operating period lasting 125 days. Both plots depict the instantaneous H2S removal efficiency for each operating load expressed in units of 3 gH2S/m bed/hr. In addition, the EBRT is shown as horizontal lines along the plots. It is clear from Figures II.2 and II.3 that both reactors had removal efficiencies ranging mostly in 90-100%. After acclimation of the seeded activated sludge during the first week, the inlet H2S load was increased at the same EBRT until reaching removal efficiencies higher than 80-90%. The coated reactor responded better and faster to changes of concentration with similar hydrodynamics than the uncoated reactor, whose performance decreased substantially after 10 operating days due to suspected biomass detachment. It is unclear what the reasons for this detachment were, since it was found that the nutrients solution being recirculated in the system was trickled down from the top at the same rate for both reactors during the long term test. High removal efficiencies were thereafter attained in the uncoated reactor but at much lower inlet H2S concentrations compared with the 3 coated reactor. As a result, loads higher than 20 gH2S/m bed/hr, were almost completely removed in the coated reactor by day 45, whereas similar values were attained in the uncoated reactor only around day 60.

The EBRT was decreased in the coated reactor after day 45 at a similar inlet H2S concentration, resulting in a higher load at lower contact time, which yielded removal efficiencies under the preset lower limit of 90%. In general, this approach was applied to both reactors, where the EBRT was manipulated either at the same inlet concentration or at an adjusted concentration so that the load would be the same but the pollutant removal would depend upon different controlling mechanisms. Furthermore, the EBRT was varied repeatedly 11 to 13 times for the uncoated and coated reactors, respectively, in order to ensure the repeatability of the treatment performance.

80 100 60

[%] 50 80

60 RE

bed/hr] 40

3 Inlet 60 Outlet

S/m

2 40 RE [%] 30 EBRT [sec] 40 20 EBRT [sec] 20

S flux S[gH flux

2 20

S removal efficiency 10

H 0 0 0 0 25 50 75 100 125

Elapsed time [d]

Figure II.2. Long term performance for the coated reactor removing H2S.

80 100 60

50 80 [%]

60 RE

bed/hr]

3 40 60 Inlet

S/m

2 40 Outlet 30 RE [%] EBRT [sec] 40 20 [sec] EBRT 20

S load [gH load S

2 20

S removal efficiency efficiency removal S 10

H 0 0 0 0 25 50 75 100 125

Elapsed time [d]

Figure II.3. Long term performance for the uncoated reactor removing H2S.

In general, both reactors showed good robustness and removals over 90% when changing the EBRT, though the uncoated reactor was able to sustain an EBRT as low as 6 seconds, compared to 9 for the uncoated one. The elimination capacity of the coated reactor is

3 considerably higher than the uncoated one, since loads as high as 55 gH2S/m bed/hr 3 could be maintained within the threshold of 80% RE as opposed to 35 gH2S/m bed/hr in the uncoated reactor.

The effect of the coating on the reactor performance was particularly important during the acclimation stage. Rapid acclimation of the microflora in the reactor is important in the practical implementation of synthetic media biofilters. Typically, during the acclimation period, activated carbon adsorbers have to be used in series with these biofilters to ensure that complaints do not arise during start-up, and this increases the investment cost of the treatment system substantially. The coated reactor achieved almost 100% RE after three days of operation, while the uncoated reactor exhibited intermittent periods of high treatment efficiency. The collected data was plotted as in Figure II.4 which shows the performance of both reactors during the first four days. It is seen that even at higher H2S loads the coated reactor performed better than the uncoated one at lower loads.

bed/hr]

S/m 6 Coated

2 Uncoated

S [gH load

2 2

Inlet H 0 0 20 40 60 80 100 H S Removal Efficiency [%] 2

Figure II.4. H2S removal performance for the coated and uncoated reactors over the first 4 days of operation.

The observed effect of the PEI coating in the attachment and rapid acclimation of bacteria in our continuous system has also been reported in still and agitated batch cultures, where polystyrene microcarriers were coated with positively charged molecules to speed up the adsorption of some types of cells and proteins [10]. In general, the authors report that by adding the positive film onto the supporting surface, the negative charge from glycoproteins and phospholipids pertaining to the suspended cell is then cancelled and both entities driven to bind by ionic forces. The authors report higher attachment rates when using not only the positively charged polymer, but also organic material (collagen, fibronectin, laminin) referred as cell attachment factor, either individually or in combination with a positively charged polymer. These organic cell attachment factors possess specific domains made of some aminoacid sequences that recognize similar structures in the incoming cells and bind them covalently or by means of ionic forces. In our study, the fresh agar added with each nutrient solution makeup is believed to have acted as the organic cell attachment factor. Clapper [10] reported that a combination of both a positively charged polymer with a cell attachment factor enhances the attachment of cells at a rate faster than when using only one of these elements at a time, or none of them.

Moreover, the authors report that when covalently binding the positively charged polymer or the cell attachment factor to the cell carrier, or both, the rate of external cell adhesion is faster than when these compounds are just adsorbed. This is a consequence of the hydrophilic condition of the positively charged molecule, required to dissolve and form a film in the suspended culture. In our study, the PEI coating was physically adsorbed with no further enhancement. Therefore, by covalently bonding the PEI coating with the PU foam it is likely that a faster and more homogeneous bacterial spreading could have been accomplished. Nevertheless, respectable results were observed during our study of the biofilters. Methods for binding the coatings and cell attachment factors onto plastic surfaces are reported in the literature [10, 11] and in general require that both substances have chemically reactive groups able to form radicals when excited with external sources of energy such as ultraviolet or heat. These molecules, though reactive, must keep the functional group that gives them the ability to cancel the cell charge and to

bind amino acid domains in the incoming cell. The use of porous material (pore size ranging from 1-1000 µm) made of collagen for the immobilization of suspended cells, has also been successfully reported in stirred tanks [12]. Prior to these findings, Swann [13] had already proposed methods for condensing the positively charged polymer of PEI, and for strengthening the stability and adhesion of the resulting coating onto mineral surfaces such as vermiculite, with positive results regarding external cell attachment.

Since both coated and uncoated reactors were added nutrients solution with the same composition, including the type and concentration of organic agar, the difference in performance is attributed to the positively charged PEI coating. Even though the coated reactor operated at higher H2S inlet loads than the uncoated reactor, which ultimately promoted a more prominent biomass growth in the former and more activity towards the degradation of such pollutant, the coating agent was responsible for faster acclimation and therefore a stronger capacity for afterwards handling higher inlet H2S loads, as opposed to the uncoated reactor.

II.III.II. Section-based biofilter performance

Due to fast accumulation of biomass in the foam, both beds compacted steadily throughout days 60-70, attaining a reduction of the bed volume of 25% for the uncoated reactor and 33% for the coated one. The highest compaction in the latter is a consequence of the higher accumulation and growth of biomass promoted by the coating factor and the higher amounts of substrate fed to the bioactive films. Consequently, and in order to be able to calculate and compare the performance of the reactors in terms of bed volume, both reactors were thought as of having two halves, the first one consisting of the lower 20.32 cm, and the following one on top, having a height of 20.32 cm for the coated reactor and 25.4 cm for the uncoated one. Even though this approach considered the top section as having more packing media, the rapid compaction and the lack of significant amounts of biomass within the top layers of packing due to slough off from the trickled nutrient solution, it was assumed to even out the conditions of both sections. In this study,

all data herein presented considers this subdivision, and all calculations account for the fact that the EBRT and both bed and foam void porosities were transient variables.

Figures II.5 and II.6 show the performance of the coated and uncoated reactors lower and upper halves, respectively, for all collected data over 125 days. The inlet load for each half versus the removed load is depicted in each diagonal section of the plot for one reactor at a time. One of the advantages of this approach is to compare the performance of both reactors with data that reflects the critical load and critical removal efficiencies of the system. The maximum removal achieved for the upper and lower halves were 40 and 3 3 27 gH2S/m bed/hr for the uncoated reactor, and 63 and 77 gH2S/m bed/hr for the coated one. Gabriel and Deshusses [3] reported an overall maximum removal capacity of 105 3 gH2S/m bed/hr in a retrofitted full-scale biofilter operating at EBRT close to 2 sec., packed with PU foam cubes. Even though the authors claim the system exhibited the highest reported H2S removal capacity for a biofilter operating at such fast airstreams space velocities, maximum inlet H2S concentrations reached only 30 ppmv with peaks at

60 ppmv. Thus, it could not be established, even less understood, whether the controlling resistance for the pollutant removal in such system was kinetically or mass transfer controlled. Reasons for such performance could be the proper design of the existing full scale scrubber, the ratio of the foam cube size to the column diameter (1:45 compared to 1:8 in this study), and a system whose controlling mechanism was the axial advection of the pollutant rather than the biomass ability to biodegrade higher concentrations of H2S.

In this study, although higher concentrations of H2S enter the system in the lower sections of the reactor, both upper halves exhibited higher removal capacities. Both maxima at the upper halves were observed after the bed had achieved a slow compaction rate, at a height that did not differ for more than 1% of the final value; that is, these results may not be blamed on the higher height of the upper halves at the initial stages of the experiments.

3 H2S load upper half [gH2S/m bed/hr]

100 80 60 40 20 0 0 100

bed/hr]

3 lower half 3 20 upper half 80

S/m

40 60

60 40

80 20

S removal lower half [gH S removal lower

S removal upper Shalf [gH removal upper

100 0 2

H H 0 20 40 60 80 100

H S load lower half [gH S/m3bed/hr] 2 2

Figure II.5. Section performance for the coated reactor removing H2S. Line represents 100% RE.

3 H2S load upper half [gH2S/m bed/hr]

70 60 50 40 30 20 10 0 0 70

bed/hr]

3 10 60 3 lower half

S/m

upper half S/m

2 20 50 2

30 40

40 30

50 20

60 10

S removal lower half S[gH removal lower

S half [gHremoval upper

70 0 2

H H 0 10 20 30 40 50 60 70

H S load lower half [gH S/m3bed/hr] 2 2

Figure II.6. Section performance for the uncoated reactor removing H2S. Line represents 100% RE.

Therefore, we hypothesize that our observations are the result of the higher concentration of fresh nutrients and agar trickled down from the top, with the bacterial anchoring effects described previously on the upper section microflora. These results are congruent with our observation of higher compaction on the coated bed due to more biomass content.

Figures II.7 and II.8 represent the contribution of the lower section for each reactor to the observed removal for all 125 operation days, regardless that the observed overall removal was less than the threshold of 80%. As expected, while increasing the inlet H2S load, the contribution of the lower section decreases due to the higher bed volume required to oxidize higher amounts of pollutant, when the upper section plays its part. This result is better seen in the uncoated reactor; however, the coated reactor shows high contributions of the lower section even at elevated H2S loads. This is particularly true for high inlet concentrations and EBRT, where the pollutant is exposed to the more abundant biomass in the coated reactor. Although not shown in Figure II.7, the tendency of lower section 3 contribution decreases with inlet H2S load starting at 60 gH2S/m bed/hr for the coated reactor. On the whole, the contribution of the lower section of the reactors to the observed overall removal is almost complete for the coated reactor until a load of 45 3 3 gH2S/m bed/hr, compared to only 10 gH2S/m bed/hr in the uncoated reactor.

Table II.1 compares the H2S removal performance of biofilters packed with different types of media; however, most of these studies (excluding ours) were carried out at much higher EBRT, and the reported results do not make reference to other operational problems. In particular, some of these studies have been carried out using natural media, which ultimately suffers the effects of rough acidic conditions, chemical decomposition, loss of the physical structure of the media, clogging and excessive pressure drop.

Table II.1. Performance comparison between biofilters using different packing media for the removal of H2S.

Active packing Bacterial Maximum EC Reference EBRT 3 Comments material source [gH2S/m bed/hr] [17] Activated carbon 3-21 sec. Activated 181 EC observed at RE of 94%. Ratio of AC maximum H2S adsorption (AC) sludge capacity of biologically seeded to virgin AC: 45:1. Spent AC employed in physical and chemical sorption can be afterwards seeded with sludge to increase the lifespan and EC of the bed considerably. [18] PU foam 9-28 sec. Previously 165 Ratio of liquid to air flows was below 0.01 and maintained in most acclimatized cases in the range 0.001-0.005 (in this study 0.007). The authors activated reported a marked performance depletion at ratios higher than sludge 0.006. Also, the nutrients pH was kept in between 3.0-4.0 and the sulfate concentration controlled at low levels. Reactor only operated for 56 days. [19] Peat 29-426 Indigenous 130 EC observed at RE of 95%. Air taken from a waste water pumping sec. consortia station. Other VOC and sulfur reduced gases not measured during encountered in situ experiments. Decomposition of packing material noticed. in peat Liming of peat required. [16] Compost and hog 38 sec. Activated 120 EC observed at RE of 31% (Based on Figure 4 of printed source.) fuel either alone or sludge Perlite added to active media in a ratio 4:1 to avoid clogging. Media in a 1:1 mixture. needed be limed. Foul gas pressure of 149 Pa/m. [3] PU foam 1.6-2.2 Activated 105 Maximum inlet H2S concentration averaging 30 ppmv with peaks at sec. sludge 60 ppmv. Results are for full scale studies. Pilot scale studies yielded RE of just 29% at inlet H2S concentration of 64 ppmv. This study PU foam (Coated 6-60 sec. Activated 77 EC observed at RE of 99% and EBRT 9 sec.. Maximum EC for the with PEI solution, sludge (Coated, upper upper half of the coated reactor observed at RE: 90% and EBRT of and Uncoated or half) 10 sec. plain PU foam) [20] Peat 114-266 Thiobacillus 55 EC observed at RE of 64%. sec. thioparus (Based on Figure 5 of printed source.) [21] Calcium alginate 28 sec. Thiobacillus 23 EC observed at EBRT of 70 sec. and RE of 82%. (Based on Figure beads thioparus 6 of printed source.) [22] Plastic Pall 18-24 sec. Activated 22 EC observed at EBRT of 24 sec. and RE of 100%. (Based on Figure Rings sludge 4 of printed source.)

100

S removal for the

coated reactor [%] 20

total H

Contribution of lower Contributionsection of lower to observed 0 5 10 15 20 25 30

H S load [gH S/m3bed/hr] 2 2

Figure II.7. Contribution to of the lower reactor half to the observed instantaneous H2S removal efficiency (RE) for the coated reactor.

100

S removal for the

uncoated reactor [%]uncoated 20

total H

0 Contribution of lower section of Contribution tolower observed 0 5 10 15 20 25 30

H S load [gH S/m3bed/hr] 2 2

Figure II.8. Contribution to of the lower reactor half to the observed instantaneous H2S removal efficiency (RE) for the uncoated reactor.

II.III.III. Removal controlling mechanism and dimensionless analysis

To clarify the interplay of mass transfer and biodegradation kinetics, all experimental data collected was compared using two dimensionless numbers that account for the ratio of mass transfer controlling mechanisms, i.e., diffusion versus advection, specifically

Peclet number (Pe), and the biomass ability to chemically oxidize the H2S, namely Damkohler (Da), as defined in Table II.2. The Damkohler number includes the product of the surface coverage by the biofilm and the maximum velocity of reaction of the Monod’s type, product which was determined fitting all collected data to a proposed model that quantifies the performance of a biofilter packed with highly porous media, degrading a single pollutant [14]. The model considers the effect of the biomass accumulation and nutrient layer in the clogging and air distribution within the media and in the biofilter. This way, an effective contact area is used that comprises a weighted average of the specific area of the media, both internal and external, being the weighting factor the amount of air flowing inside and past the packing. Values used for the quantification of the parameters of equations shown in Table II.2 are summarized in Table II.3. Results for these fittings are summarized in Table II.4, along withy the measured VS per each reactor section.

Figure II.9 shows a compilation of these two dimensionless numbers for each section of the two reactors. The sections belong to, clockwise from the top left corner, the upper uncoated, lower uncoated, lower coated and upper coated half reactors. It is clear from Figure II.9 that for small values of the Peclet number, the system is kinetically controlled. When the Peclet number is small, the contact time between the polluted stream and the biomass, the diffusion of the pollutant into the biomass, or both are maximized. This translates into low convective and diffusive mass transfer resistance, for which the pollutant removal would depend on the activity of the biomass towards the biodegradation of the H2S. When the Peclet number is small, a minor change on the Damkohler number increases the removal efficiency greatly, and this tendency is seen in

Table II.2. Modeling equations for the performance of the PU foam packed-biofilter [14]

Variable Equation Description Removal Efficiency RE C C A D X L HK C C HK C Performance equation assumes a linear E g,i g,o s f max f s ln g,i ln g,i ln s g,i profile of the pollutant concentration inside C v2C C C C HK C g,i g g,i g,i g,o g,o s g,o the biofilm due to its thin thickness. D X f max f klvgCg,i

kl Dl / l Effective specific area of the v v The ratio of the air velocities within and A f (1 ) A 1 f (1 ) A media s v b f f v b b around the foam makes the weighting factor g g for the effective specific area. Criterion to determine specific ΔP ΔP The pressure drop in each side will depend area of the media on the geometry of the foam piece and bed; L f L b i.e, pore size, porosity, size of the foam unit, reactor diameter, etc. Biofilm thickness d C (x, y) Unsteady state biofilm thickness. Different f Y max g b dt HK C (x, y) f f algebraic equations arise depending on the s g concentration of the pollutant in the airstream at steady state [23]. Water layer thickness Derived from a momentum balance over a 3 Lvl l 3 flat surface. Af L g Damkholer number X L2 A Dimensionless time of reaction. Da max f s Cg,ivg Peclet number Lv Ratio of diffusive to convective mass transfer g Pe resistances. D f

all sections studied. Conversely, at higher Peclet numbers, both mass transfer and kinetics mechanisms are important in the biodegradation of the pollutant, and sharper changes in the Damkohler number are required for the reactors to attain similar removal efficiencies than when operating at smaller Peclet values. The advection and diffusion mass transfer resistances are more evident in the lower section of the uncoated reactor (top left) since at a Peclet number of 9·106 a considerable increase in the Damkohler number from 1 to 7 does not improve significantly the removal efficiency, increasing from around 20% to only 50%. In general, it can be said that the reactors are kinetically controlled when the Peclet number is 2·106 and both mass transfer and kinetically controlled for higher values. When comparing the uncoated and coated reactors (top versus bottom plots of

Figure II.9) both reactors show the same rate of change in the removal efficiency of H2S with respect to the Damkohler number for the same Peclet; this is, the differences in the biomass activity and accumulation on both reactors due to the polymeric cofactor do not influence the physical mechanism of pollutant removal.

Table II.3. Values used for the determination of the product of biofilm coverage and Monod’s velocity of reaction for the coated and uncoated reactors

Variable Value Fluid properties Liquid viscosity [Pa/sec] 0.001 Liquid density [kg/m3] 1000 Air viscosity [Pa/sec] 0.0000185 Air density [kg/m3] 1.1614 2 H2S diffusivity in water [m /sec] 0.0000000015 2 H2S diffusivity in biofilm [m /sec] 0.0000000010 H2S Henry’s constant [1] 0.416 Geometry and operation Liquid velocity through foam [m/sec] 0.0004 PU foam porosity [%] 98 PU foam pore diameter [m] 0.002886 Bed porosity [%] 0.53-0.33 Specific area around foam [m-1] 600 -1 Packing factor Fpd [24] [ft ] 30 Biokinetic properties Biofilm density [kg/m3] 40 Decay rate [sec-1] 0.00001 3 Half saturation constant [kg/m ] HCg,i Yield [kg biomass/kg H2S] 0.2

Table II.4. Distribution of measured volatile solids and calculated θµmax product for the coated and uncoated reactors

Uncoated Uncoated Coated Coated upper lower upper Lower VS per cube 23.59 23.32 30.36 41.71 [mg/cube] θµmax 0.000109 0.000256 0.000669 0.000228 [sec-1]

1.0 1.0

0.8 0.8

0.6 0.6

0.4 0.4

0.2 0.2

0.0 0.0 0 1 2 3 4 5 6 7 0 2 4 6 8 10 12 14 1.0 1.0

[1=100%]

RE 0.8 0.8

0.6 0.6

0.4 0.4

0.2 0.2

0.0 0.0 0 2 4 6 8 10 12 14 16 18 0 2 4 6 8 10 12 14 16 18

Da [1]

Figure II.9. Removal efficiency (RE) for the bioreactor sections as a function of the dimensionless numbers Peclet (Pe) and Damkohler (Da). Clockwise from top left corner: Upper uncoated, Lower uncoated, Lower coated, Upper coated. Peclet numbers are: 2·106 (dotted circle), 4·106 (dotted triangle), 9·106 (half full circle) and 11·106 (star).

In both reactors, for low to medium EBRT (6-23 sec in the coated reactor and 9-24 sec in the uncoated one) the H2S removal efficiency decreases along with inlet concentration, more markedly for the uncoated reactor even at higher EBRT than the coated one. For

high EBRT ranging between 40-60 sec., removal efficiencies in excess of 85% are typical in both systems. Thus, at high airstream space velocities, the controlling mechanism for the oxidation of the pollutant is the rate of diffusion of the H2S into the biofilm as opposed to the advection along the bed, whereas at low velocities the oxidation capability of the biomass limits the degradation of the pollutant. Data points on the high EBRT range located below the threshold of 80% RE belong to the early acclimation period (Figures II.2 and II.3). In the case of the uncoated reactor, the observed performance decline after a few days past startup is attributed to biomass slough off, as discussed earlier. The coated reactor was able to handle airstreams at EBRT as low as 6 sec. compared with 9 sec in the uncoated reactor. Moreover, most data shown in the coated reactor as in Figure II.2 is within the preset operation of RE between 90-100%. Increasing the inlet pollutant concentration at the same EBRT or increasing the EBRT at an adjusted inlet pollutant concentration yields the same inlet load; however, depending upon the controlling removal mechanism, a different reactor behavior is obtained. In particular, our results show that very high concentrations of H2S (up to 235 ppmv) can be effectively removed by the biomass but mostly whenever sufficient contact time is given to the phases for complete mass transfer. Thus, our results show that reporting reactor performances in terms of just bed volumetric loads does not have much meaning unless other operating variables are presented.

II.III.IV. Biomass speciation and agar utilization

In order to determine whether enough organics from the agar were appropriately consumed, and the possible detachment of biomass from the PU foam, COD quantification was performed in samples of the nutrients solution being recycled. Since some water is evaporated due to the very low relative humidity of the incoming air available from the lab piping (less than 0.1%) the concentration of both organic and inorganic material was expected to increase even though their absolute amounts remained unchanged. Therefore, accumulation or depletion rates for the species were corrected for the rate of water evaporation in the nutrient tank. The measurements were carried out in between the period of two consecutive nutrients solution addition to avoid external

supply of organic species. Results for the COD and Total Solids (TS) are summarized in Table II.5. For both coated and uncoated reactors, the rate of increase of COD concentration in the nutrient solution was lower than the rate of evaporation in the tank, i.e. 75% and 90%, respectively. This shows that the equilibrium between biomass growth and detachment, and use of organics from the nutrients was inclined to the metabolic expenditures of agar, as well as attachment and growth of more biomass. This organic consumption was slightly higher in the coated reactor. The daily rate of TS accumulation, on the other hand, was much higher than the evaporation rate of the nutrients tank, and it was even for both reactors at around 70% above the evaporation rate. It is worth noticing that during these measurements, the coated reactor was working at an inlet H2S load about twice as high as that of the uncoated reactor. This means that the higher amounts of sulfur and sulfates produced by higher H2S conversions on the coated reactor were balanced by a higher consumption of nutrients and organics from the solution. Sulfur and sulfate entrapment in the biomass of the coated reactor was not believed to have happened in a relevant matter, even though few spots of yellowish material were seen in the foams after service shutdown. Actually, higher amounts of yellowish spots were seen in the foam of the uncoated reactor.

Figures II.10 and II.11 show the structure of the biomass on the colonized PU foam and a sulfur crystal spotted on it after the reactors were shut down. EDS performed on this spot revealed a composition of the crystal consisting of 85% sulfur and 15% oxygen, in atomic scale.

The contents of the nutrient solution tank were also qualitatively inspected during the course of the experiments and particularly the first stages of the reactors service. Brownish, greenish and reddish colors were predominant during the first 2-3 weeks of operation, due to the presence of different species containing iron in the predominant oxidation state according to the operation pH.

Table II.5. COD and TS pattern in the nutrients sump tank for the coated and uncoated reactors after 100 days of operation.

Elapsed time Tank volume Daily change COD [mg/L] Daily change Solids Daily change [d] [mL] in volume in COD [%] concentration in solids [%] [%] [g/L] Uncoated reactor 100 1720 0 963 0.1274 101 1560 10 1044 8 0.1683 32 102 1400 11 1107 6 0.1869 11 103 1200 17 1253 13 0.2845 52 104 1000 20 1480 18 0.3713 31 Coated reactor 100 1720 0 1423 0.2210 101 1400 23 1797 26 0.3364 52 102 1160 21 2071 15 0.4541 35 103 840 38 2417 17 0.7583 67 104 640 31 2868 19 1.0238 35

Figure II.10. SEM micrograph of the biomass grown on the PU foam after 125 days of reactor’s service. Picture was taken on media from the coated reactor, though qualitatively both reactors showed similar biomass imagery.

Figure II.11. Sulfur crystal sitting on top of the biomass in the bioreactors after 125 days of service.

The nutrients pH decreased steadily from 4.72 and 3.85 for the coated and uncoated reactors, respectively, to values consistently lower than 2.00 after about 60 days of

operation. Even though the initial pH of the nutrients was slightly higher in the coated reactor tank, final values of 1.13 and 1.22 for the coated and uncoated reactor tanks, respectively, were attained after the reactors service. It must also be pointed out that the contents of the tanks were just washed away 4 times over the reactors operation, while all solids and soluble sulfates being produced were accumulated in the tanks in between this procedure. Upon reaching a pre-established minimum tank level, new make-up nutrient solution was added keeping the exhausted contents of the tank. This way, even though the nutrients solution stock had a pH of 6.6-6.8, the nutrients inside the reactor quickly reached a value lower than 2.0, for which the acidic conditions of the bacterial environment were not perturbed. Furthermore, the nutrients consumption is minimized as opposed to a scheme of one pass nutrients flow. Due to the high activity and biomass accumulation on the foam, the low solubility of H2S at acidic conditions was not a limiting factor for its biodegradability.

Samples of the biomass attached in each half of each section were squeezed out the foam for biomass quantification and bacterial speciation using 16S rRNA DGGE. As mentioned previously, the “as extracted” biomass was collected without any purification method other than those that the DGGE protocols themselves establish. Results of the bacterial speciation are shown in Figure II.12 indicating the most prominent rRNA bands on the denaturing gel panel. The four samples are named AB, AT, BB and BT. The first letter, A and B, denotes reactors A (uncoated) and B (coated), whereas the second letter, B and T, represents, respectively reactor bottom and top. All bands correlate with more than 90% exactitude stored fragments of bacteria belonging to the strain Acidithiobacillus ssp. This finding is in accordance with the new classification of the genus Thiobacillus made in the early 1980’s, where species were differentiated according, among other criteria, to the acidity of alkalinity of the surroundings. Also, even though different amounts of biomass accumulation were observed in each half of each reactor, all sections were populated by the same bacteria. This is an indication that although the combination of cofactors and extra-cellular organic material decrease repulsive electrostatic charges among a varied initial bacteria consortia, and spreads biomass more firmly and easily,

there is no effect on the survival of specific species of bacteria under the operation conditions of this study.

Figure II.12. 16S rRNA bands separated through DGGE techniques from biomass samples taken from the bioreactors after service shutdown. Bands 1.1 to 4.2 correlate with Acidithiobacillus gene databank in 0.900-1.000 Reactor sections: AT: Upper uncoated, AB: Lower uncoated, BT: Upper coated, BB: Lower coated.

II.III.V. Biofilm coverage

A value of 0.0025 sec for µmax was chosen for our experimental conditions by fitting field data on similar PU biofilters treating H2S [3] and assuming coverage equal to unity. This value is within the range of reported Monod’s kinetic coefficients for similar systems, as shown in Table II.6. This way, the value of θ can be deconvoluted from the fitted product

θµmax to obtain the PU surface coverage by the biofilm, as depicted in Figures II.13 and

II.14. During the application of our model to the fitting and determination of θµmax, it was assumed that the Monod’s half saturation constant parameter, Ks, was equal to the

Table II.6. Maximum kinetic constant values for Monod’s type reaction rates reported on studies of biofilters treating odors (biofilm coverage assumed to be unity).

Reference Maximum kinetic Inlet concentration and Pollutants treated Constant carrier material 3 [16] 6.23 gS/kgbed/d 53 ppmv H2S only. Bed density assumed 0.5 g/cm (Compost and hug fuel) [21] 1.36 gS/kgbed/d 46 ppmv H2S only. (Ca-alginate beads) Pseudomona putida CH11 Neutral pH. [25] 5.52 gS/kgbed/d 84.7 ppmv H2S, methyl mercaptan, dimethyl mercaptan and (Peat biofilter) methanethiol. [15] 5.00 gS/kgbed/d 55 ppmv H2S, methyl mercaptan and methanethiol. (Peat) -1 [26] 0.011 sec. 200-600 ppmv Toluene. Membrane made of polysulfone. Membrane reactor -1 [27] 0.000063 sec. 78 ppmv 2 stage biofilter degrading H2S, methyl mercaptan, (Compost and wood chip) dimethyl mercaptan and methanethiol. -1 This study 0.000109-0.000669 sec. Up to 235 ppmv H2S only. Bulk density of the PU foam is around 100 2836-22849 gS/kgbed/d PU foam (bulk density 25 times smaller than the bulk density of soil. kg/m3)

maximum solubility of the pollutant at equilibrium with its gas phase concentration, which does not necessarily hold for most biomass. Thus, high values of θµmax were expected.

The values of θ increase along with the elimination capacity EC due to the expansion of the biofilm onto the foam surface as more bacteria are created while they oxidize more substrate. When the elimination capacity reaches values of around 20 and 50 3 gH2S/m bed/hr for the uncoated and coated reactor halves, respectively, the biofilm coverage has increased four folds to theoretically complete supporting media coverage. The fact that a larger EC is required for the coated reactor for its media to be completely covered is an indication that the biomass grown there is more kinetically active, since high removals are attained with less biomass. A coverage higher than 1 is an agreement with previous observations that mature biofilms have already evolved into mushroom like and fibrils entities, which posses a surface area higher than that of the plane support upon which they are attached [28].

1 lower half upper half

0.1

fraction of surface 0.01

0.001 1 10 100

EC [gH S/m3bed/hr] 2

Figure II.13. Maximum calculated biofilm coverage for the uncoated reactor as a function of the Elimination Capacity EC.

lower half 1 upper half

0.1

fraction of surface fraction 0.01

0.001 1 10 100

EC [gH S/m3bed/hr] 2

Figure II.14. Maximum calculated biofilm coverage for the coated reactor as a function of the Elimination Capacity EC.

An additional observation can be drawn from the data summarized in Table II.6. For a coverage of unity, when converting the values of the calculated µmax in units of gS/kgbed/d our values are three to four folds higher than other reported ones. This is a consequence of the low packing density of the PU foam compared with natural soil, which has a density two folds higher than the former. Even correcting for the density of the packing material, our results show a system up to two folds more kinetically active than those reported in natural media. When comparing the relative ratios of µmax to VS contained in each section of each reactor, both reactors exhibit different behaviors. This ratio is higher for the upper section of the coated reactor, whereas is higher in the lower section of the uncoated one. This means that the amount of bioactive biomass to total biomass is higher whenever the foam was treated with the cofactor and fed with fresher sludge. The attachment of more bacteria from the sludge on the top section of the trickled down, coated foam biofilter is once again believed to have promoted this observation. In particular, even though the uncoated reactor exhibited the same amount of VS accumulated on their media, the biomass in its lower section contained more catalytically active bacteria, perhaps because of higher concentrations of incoming substrate. The

distinction between active and inactive yet alive biomass is common in biotechnology, where some bacterial strains have been reported to grow, however they are not able to utilize some substrates at the same rate of other similar species contained in such biomass.

II.III.VI. Pressure drop

During the long term experiments, the foul gas exhibited a pressure drop ranging from 42-59 Pa/m for both reactors, even though a considerable accumulation of biomass occurred and compaction of about 33% was observed. These values compare satisfactorily to others found in the Literature. Hirai [15] reported pressure drops ranging from 60-304 Pa/m in biofilters treating H2S. Minimum values for the pressure drop were obtained for beds packed with porous ceramic, whereas soil exhibited the largest foul gas compression. Wani [16] reported a pressure drop of 149 Pa/m in airstreams treating odors in a biofilter using natural media.

II.IV. CONCLUSIONS

The pollutant removal efficiency and capacity of two PU foam cubed, packed bed biotrickling filters degrading H2S as sole substrate from airstreams were tested (EBRT and inlet H2S concentrations ranging from 6-60 sec and 10-235 ppmv, respectively.) One of the beds was packed with PU foams coated with a positively charged solution of 25 mg/L of PEI (coated reactor) in order to extrapolate results published elsewhere that claim faster and stronger biomass attachment onto plastic surfaces when coating such materials with the aforesaid cationic solutions. During the first 3 days of operation, the coated reactor exhibited higher removal efficiencies than the one packed with untreated

PU foam (uncoated reactor) even at higher inlet H2S concentrations. While maintaining a preset limit of minimum removal efficiency of 90% during the 125 days of continuous operation, the coated reactor was able to operate at inlet H2S loads of up to 44 3 3 gH2S/m bed/hr, compared with 21 gH2S/m bed/hr in the uncoated one. The coated

reactor exhibited both higher VS attachment and kinetic activity expressed as the calculated Monod’s maximum velocity of reaction.

The better performance observed on the coated reactor when compared to the uncoated one is believed to have been promoted solely by the positively charged polymeric coating. The anchoring and spreading capabilities of the agar used as carbon source might have also contributed to the biomass growth and colonization of the foam, yet the effect is thought as to be even in both reactors, given the fact that equivalent concentrations and compositions of nutrients, including agar, were fed to both reactors.

Both reactors were mostly mass transfer controlled, being the advection time the controlling parameter for removal of the pollutant. The biomass accumulated in both reactors showed high kinetic activity towards degrading high concentrations of H2S, whenever enough time for contact between the airstreams and the biomass was given.

The anchoring and spreading effect of the positively charged polymer and agar did not favor the survival of different bacterial species in both reactors or within each one. DGGE analysis carried out in different sections of both reactors after their continuous service was stopped showed predominant colonization by Acidithiobacillus ssp, in accordance with the acidic conditions in which both reactors operated (nutrients solution pH lower than 2.0.)

II.V. SYMBOLS

-1 As : Effective specific area [m ]

-1 Ab : External foam specific area [m ]

-1 Af : Internal foam specific area [m ] b : Decay coefficient [s-1] 3 C f : Substrate concentration in the biofilm [kg/m ]

3 Cg : Substrate concentration in the gas [kg/m ]

Da : Damkohler number [1] 2 D f : Effective diffusivity in biofilm [m /s]

2 Dl : Effective diffusivity in water [m /s] 3 EC: Removal capacity [kgH2S/m bed/hr] -1 Fpd : Packing factor [18] [ft ] g : Gravity [10 m/s2] H : Dimensionless Henry’s law constant [1] k l : Substrate liquid phase mass transfer coefficient [m/s]

3 Ks : Half saturation constant [kg/m ] L : Reactor length [m] Pe : Peclet number [1] P : Gas stream pressure drop within the foam [kg/m2/s2] L f P : Gas stream pressure drop in the bed [kg/m2/s2] L b RE : Removal efficiency [%] v f : Superficial gas velocity through foam packing [m/s] vg : Superficial gas velocity [m/s] vl : Superficial liquid velocity [m/s] 3 X f : Biofilm density [kg/m ] Y : Yield coefficient [1] Greeks

f : Biofilm thickness [m]

l : Liquid thickness [m]

b : Bed porosity [1]

f : Foam porosity [1]

L : Liquid viscosity [kg/m/s]

max : Monod’s maximum velocity [kgH2S/kgbiomass/s]

3 L : Liquid density [kg/m ] : Fraction of PU foam support covered by biofilm [1] Subscripts i : Inlet o : Outlet

II.VI. REFERENCES

1. Aizpuru, A., Khammar, N., Malhautier, L. and J. Fanlo. (2003) “Biofiltration for the treatment of complex mixtures of VOC. Influence of the packing material.” Acta Biotechnologica. 23(2-3) 211-226. 2. Govind R. and S. Narayan (2005). “Selection of bioreactor media for odor control.” Chapter 4 in “Biotechnology for Odor and Air Pollution Control.” Edited by Z. Shareefdeen and A. Singh. Springer Verlag, Germany. 65-100. 3. Gabriel, D. and M. Deshusses. (2003) “Performance of a full-scale biotrickling filter treating H2S at a gas contact time of 1.6 to 2.2 seconds.” Environmental Progress. 22(2) 111-118 4. Li, C. and W. Moe. (2003) “Sequencing batch biofilter operation for treatment of methyl ethyl ketone (MEK) contaminated air.” Environmental Technology. 24(5) 531-544. 5. Kim, H., Xie, Q., Kim, Y. and S. Chung. (2002) “Biofiltration of ammonia gas with sponge cubes coated with mixtures of activated carbon and zeolite.” Environmental Technology. 23(8) 839-847. 6. Van Groenestijn, J. and J. Liu. (2002) “Removal of alpha-pinene from gases using biofilters containing fungi.” Atmospheric Environment. 36(35) 5501-5508. 7. Moe W. and R. Irvine. (2001) “Polyurethane foam based biofilter media for toluene removal.” Water Science and Technology. 43(11) 35-42. 8. Park, S., Nam, S. and E. Chol. (2001) “Removal of odor emitted from composting facilities using a porous ceramic biofilter.” Water Science and Technology. 44(9) 301-308. 9. Vancha A., Govindaraju S., Parsa K., Jasti M., Gonzalez-Garcia M. and R. Ballestero. (2004) “Use of polyethyleneimine polymer in cell culture as attachment factor and lipofection enhancer.” BMC Biotechnology. 4. Art. 23. 10. Clapper, D. and W. Hu. (1996) “Cell culture support containing a cell adhesion factor and a positively-charged molecule.” US Patent 5,512,474. 11. Drumheller, P. (1999) “Materials and methods for the immobilization of bioactive species onto polymeric substrates.” US Patent 5,914,182. 12. Dean, R., Silver, F., Berg, R., Phillips, P. and P. Runstadler. (1989) “Weighted collagen microsponge for immobilizing bioactive material.” US Patent 4,861,714. 13. Swann, W. (1985) “Vermiculite as a carrier support for immobilized biological materials.” US Patent 4,504,582.

14. Goncalves, J. and R. Govind. “Simulation of Biotrickling Filters using Novel Foams for Treating Odors and Volatile Compounds.” Proceedings of the 2005 AIChE Fall Annual Meeting. Cincinnati, Ohio (2006). 15. Hirai, M., Kamamoto, M., Yani, M. and M. Shoda. (2001) “Comparison of the biological H2S removal characteristics among four inorganic packing materials.” Journal of Bioscience and Bioengineering. 91(4) 396-402. 16. Wani, A., Lau, A. and R. Branion. (1999) “Biofiltration control of pulping odors - hydrogen sulfide: performance, makrokinetics and coexistence effects of organo- sulfur species.” Journal of Chemical Technology and Biotechnology. 74(1) 9-16. 17. Duan, H., Koe, L., Yan, R. and X. Chen. (2006) “Biological treatment of H2S using pellet activated carbon as a carrier of microorganisms in a biofilter.” Water Research. 40(14) 2629-2636. 18. Chen, J.M., Jiang, L.Y. and H.L. Sha. (2006) “Removal efficiency of high- concentration H2S in a pilot-scale biotrickling filter.” Environmental Technology, 27(7) 759-766. 19. Hartikainen, T., Martikainen, P., Olkkonen, M. and J. Ruuskanen. (2002) “Peat biofilters in long-term experiments for removing odorous sulphur compounds.” Water, Air and Soil Pollution. 133(1-4) 335-348. 20. Oyarzun P., Arancibia F., Canales C. and G. Aroca. (2003) “Biofiltration of high concentration of hydrogen sulphide using Thiobacillus thioparus.” Process Biochemistry. 39(2) 165-170. 21. Chung, Y., Huang, C. and C. Tseng. (1996) “Operation optimization of Thiobacillus thioparus CH11 biofilter for hydrogen sulfide removal.” Journal of Biotechnology. 52(1) 31-38. 22. Jin, Y., Veiga, M., & Kennes, C. (2005). “Autotrophic deodorization of hydrogen sulfide in a biotrickling filter.” Journal of Chemical Technology and Biotechnology. 80(9) 998-1004 23. Rittmann, B. and McCarthy, B. (1980) “Model of steady-state biofilm kinetics.” Biotechnology and Bioengineering. 22(11) 2343-2357. 24. Robbins, L. (1991) “Improved pressure drop prediction with a new correlation.” Chemical Engineering Progress. May 1991 87-91. 25. Cho, K., Hirai, M. and M. Shoda. (2001) “Degradation characteristics of hydrogen-sulfide, methanethiol, dimethyl sulfide and dimethyl disulfide by Thiobacillus-thioparus DW44 isolated from peat biofilter.” Journal of Fermentation and Bioengineering. 71(6) 384-389. 26. Parvatiyar, M., Govind, R. and Bishop, D. (1996) “Biodegradation of toluene in a membrane biofilter”. Journal of Membrane Sciences. 119(1) 17-24. 27. Li, H., Mihelcic, J., Crittenden, J., and Anderson, K. (2003). “Field measurements and modeling of two-stage biofilter that treats odorous sulfur air emissions.” Journal of Environmental Engineering-ASCE, 129(8) 684-692. 28. Rittman, B. (2004) “Biofilms in the water industry.” In “Microbial Biofilms.” Edited by Ghannoum, M. and O’Toole, G. ASM Press. 359-378.

CHAPTER III

QUALITATIVE STUDY OF BIOFILM GROWTH AND METABOLIC ACTIVITY IN THIOSULFATE OXIDIZING BACTERIA USING IMPEDIMETRY

SUMMARY

The time response of the impedance of mixed strains of Halothiobacillus spp., Burkholderia spp., and Rhizobium spp. growing in culture wells containing 10 active gold microelectrodes (250 μm diameter) under AC currents with an approximate intensity of 1 µA were experimentally measured in this study. Based on a proposed equivalent RC circuit that describes the electrode-culture well, the time response of the imaginary component of the observed impedance was anticipated to be dependent on the formation of a biofilm atop the gold microelectrode, with the shape of such time response indicating the preferential biofilm growth direction (perpendicular to or along the gold microelectrode surface). Polyethyleneimine (PEI), poly-d-Lysine (PDL) and Collagen from calf skin (Col) were used to coat the electrode surface so as to monitor their effect on the biofilm growth direction. It was determined that PEI tends to favor the spreading of the biofilm more than PDL and Col when used independently, yet when combined with Col, PEI and PDL both exhibit a tendency to spread the biofilm rather than promote its accumulation perpendicularly to the support surface. Also, PEI, PDL and Col favor the spreading of the biofilm when the electrode is pretreated with L-Cysteine (Cys), mostly due to the stabilization of the gold surface and its easier readability. The total observed capacitance was determined to be controlled by the capacitance in a pseudo surface most likely composed of the electrode, its double layer and biofilm, whereas the total observed resistance is controlled by the bulk of the media. Finally, the bacterial activity, proportional to the resistance decrease in the cultured well, seemed to be higher when PEI was the coating used, when compared to PDL or Col.

III.I. INTRODUCTION

Impedance microbiology has long been regarded as a reliable technique for the rapid detection of bacterial activity in food, e.g. dairy and meat products [1] and more recently for the determination of the occurrence of microorganisms in blood as an alternative for bacteremia studies [2, 3]. The principle of functioning of the method relies in the fact that bacteria metabolizes organic compounds encountered in the media they grow in, increasing the concentration of ionic, more conducting metabolites that can be measured by the application of low intense AC currents, which can be sensed by small metallic electrodes, made mainly of platinum and gold. AC currents are used since, first, homogeneous, sinusoidal AC electric fields translates in negligible electrophoretic mobility of the charged species [4] which eases the interpretation of the electrical properties of the cultures by reducing one dimension of analyses. In turn, only the change of the electrical properties with time is monitored. Second, the elements that form the culture media such as the electrodes, nutrient media and suspended cells, especially microorganisms, can be represented as ideal resistors or capacitors depending upon their intrinsic electrical properties [5]. This way, by drawing an equivalent RC circuit, the impact of such elements and therefore changes on the media or bacteria, or both on the observed impedance data can be studied provided that the biological system can be fitted to the proposed RC model.

Most of the information currently available on the literature regarding impedance microbiology relates the metabolic activity of bacterial cultures from the observation of an onwards change with time of the resistance and capacitance of the system [1, 2]. From the observed values, some authors have reported that specific strains of bacteria under some definite growing conditions confirm activity or not, and that such activity exhibits the classical growth phases shown by microorganisms; i.e., lag phase, exponential growth, stationary phase, decay, etc. These results are mostly obtained in suspended cultures and agar plates; therefore, they lack information as to the structure and spatial distribution of the bacterial consortia, for which microscopy tests have to be carried out to address this subject. Others have partially dealt with this question by quantifying the biofilm thickness grown on metallic coupons, relating the change in the electrical properties of the supporting metallic solids with the bacterial attachment [6]. This approach, however, assumes that bacteria grow perpendicularly with respect to the solid support, when, in

reality, the growth can happen in three directions; when a single bacterium divides, the second element can be located upon the plane of the support parallel to the mother bacterium, or on top of it. Such behavior leads to the formation of three dimensional structures as diverse as patches partially covering the support, clusters, fibrils, uniform layers densely packed or mushroom like biofilms, depending upon physiological properties of the microorganism and the physicochemical properties of the environment [7].

The lack of information on the biofilm structure using impedance tests is not a problem inherent to limitations from the electrical technique itself; in fact, in several studies carried out in tissue cell growth with this method, it was reported that the use of polymeric cofactors coating the electrodes can facilitate the spread over the electrode of the reproducing cells, based on the transient change of the observed capacitance and its interpretation with respect to a model of electricity flow [8]. By changing the initial concentration of cells in the culture and by using electrodes with different relative sizes, such change in capacitance can be attributed to collocation of new cells on the plane of the mother cells, or the taxonomic movement of the latter. Moreover, since the metabolite accumulation in the culture and the change in the culture resistance is a function of the cells concentration and maintenance, both properties, resistance and capacitance, can be combined to determine whether the cells are being produced and which direction they move predominantly towards. This approach is particularly useful for the study of cultures where biofilms form, since in some engineering applications it is preferred that bacteria spread on the support media rather than amorphously accumulate; for instance, in the early colonization of biofilters filled with synthetic packing media where bacteria need be inoculated by trickling the culture broth inside the bed.

As for the literature available to date, no studies have been found on the use of impedance microbiology tests to determine the evolution of the three dimensional distribution of biofilms forming on the electrode surface by combining the transient signals for resistance and capacitance of the system. Such approach is introduced in the present work by studying the growth and metabolic activity of cultures of mixed thiosulfate oxidizing bacteria over gold microelectrodes in an impedance testing device. Although gold is leached out by thiosulfate [9], this reaction generally happens at alkaline conditions (pH higher than 8-9) and in presence of

ammonia and copper, conditions different from those under which the experiments in this study were carried out (pH lower than 6-7 and no ammonia or copper in the media). The effect of adding proteins and cationic polymers on the biofilm spreading and activity will also be studied using the herein proposed approach. The motivation of adding these cofactors obeys reported results showing increased cell attachment and spreading onto supports previously coated with similar substances on in vitro and suspended tissue cell culture studies [10, 11] results that can be extrapolated usefully to other applications like biofilters, as previously mentioned.

III.II. MATHEMATICAL DESCRIPTION OF THE BIOFILM GROWTH KINETICS AND THEIR ELECTRIC PROPERTIES MEASURED BY IMPEDIMETRY

In impedance microbiology, systems consisting of bacteria growing in substrate media, either in a batch culture or a continuous flow scheme, have been modeled as RC circuits whose particular configuration of resistors and capacitors are left to the discretion of the researcher, based upon factors such as the physical setup; electrode dimensions and materials, distance between electrodes, flow pattern, and the properties of the medium and microorganisms, suspension mixing, etc. [5, 12].

The outline of the RC circuit that best describes the experimental system used in this study lies in the ground of having three distinctive spatial domains whose electrical properties are thought of as being controlling variables in the overall impedance measurements. Such domains are the small round gold electrode, the biofilm that forms due to precipitation and bacterial binary fission in three dimensions, and the media, which accounts for the rest of the culture in the test well (Figures III.1a and III.1b). The latter may be controlled itself by the culture contained within a few hundred microns above the upper limit of the biofilm, rather than by all the remaining content of the well. When the counter electrode is much bigger than the test electrode, the contribution to the overall impedance measurements from both the bacteria and the electrode itself around the big electrode is neglected [13]. A simple sketch of the aforementioned RC circuit is depicted in Figure III. 2. For simplicity and due to the expected high concentration of bacteria on the surface of the well (107-109 cells) the double layer and biofilm equivalent capacitances and resistances are drawn in series with respect to one another, unlike the media

above the biofilm where suspended biomass does not necessarily cover all the space parallel to the surface of the electrode.

insulated area insulated 250 µm counterelectrode

bacteria media

biofilm and double layer

electrode

Figure III.1b. Representation of the three domains of the electrical circuit depicted in Figure III.2.

In the absence of magnetic fields, the total measured impedance Zt of the RC circuit shown in Figure III.2 can be written as follows:

1 Zt Rt Ze Zb Z m (III.1) Ct

The impedance of the individual components of Equation (III.1) can be broken down into their real and imaginary resistance and reactance components. The calculation of the equivalent overall impedance of elements arranged in series or parallel follows the same rules as for circuits formed of resistors only. Since the media is assumed to consist of capacitors and resistors in parallel, as shown in Figure III.2, its total impedance is the inverse of the summation of the individual inverted impedances of the resistors and capacitors. It can be proven that Equation (III.1) can be written as follows:

2 Rm 1 1 ωR mCm Z t Re Rb 2 2 2 2 2 2 j (III.2) 1 ω RmCm ωC e ωC b 1 ω RmCm

The real and imaginary components of Equation (III.2) describe mathematically the observed total resistance and reactance of the culture in the testing well.

Re Ce Rb Cb

Figure III.2. Equivalent RC circuit for the electrode-biofilm-culture media system representing the three domains in the culture wells atop the gold microelectrodes.

Since the total impedance and its components can be measured at several different frequencies, these magnitudes can be compared with the expressions in Equation (III.2) to determine the individual values of the electrical properties of the electrode, biofilm and media by fitting the observed total values of the impedance assuming that each of the resistances and capacitances for the electrode, biofilm and media are not inherently functions of the current frequency, particularly the low frequency range used in this study (less than 105 Hz) [5]. Equation (III.2), however, does not explicitly depend on the dimensions of the spatial domain where the electrode,

biofilm and media reside; thus, giving a physical meaning of such magnitudes is a difficult task, even more on those cases when the electrode is covered with the polymeric coatings tested. This way, instead of determining electrical properties at the electrode and biofilm separately, it is assumed that there exists a surface consisting of the electrode and its coating whenever present, the ionic double layer distributed according to the actual properties of the culture tested, and a layer of biomass atop the latter. Beyond this pseudo surface, there is the media that may or may not contain accumulated bacteria with a similar structure as that within the pseudo surface, in addition to suspended biomass. Due to the symmetry and intensity of the electric field in the test well, the measured electrical properties are believed to be controlled by a thin layer of both biofilm and surrounding media.

The resulting pseudo surface resistance and reactance can be calculated as:

Rp Re Rb (III.3) 1 1 1 (III.4) C p Ce Cb

Since only three different frequencies could be simultaneously monitored during this study, the resulting system is comprised of 6 nonlinear equations (total resistance and reactance at three different frequencies) and 4 unknowns (resistance and capacitance of the pseudo surface and the media), which can be solved using an appropriate algorithm that minimizes the error of the calculated values by fitting an over specified system as previously suggested [5]. Some impedance testing equipment allow for the determination of such electrical properties at several different frequencies, which would provide with a better fitting and a more physically representative set of values.

Mathematically, from the schematic shown on Figure III.3 that illustrates the biofilm as a tridimensional stack of bacteria:

1 1 1 1 1 1 Cb ...... N s,1,1 N s,1,2 N s,1,k N s,2,1 N s,2,2 N s,2,k

Cbac Cbac Cbac Cbac Cbac Cbac

1 1 1 ...... (III.5) N s,j,1 N s,j,2 N s,j,k

Cbac Cbac Cbac

electric flow bacterium Ns

k j electrode

Figure III.3. 3D representation of the bacterial stack accumulating during the biofilm formation on the electrode surface. “j” and “k” represent the number of cells on the two dimensions of the plane of the electrode and in a parallel arrangement with respect to the electric flow, whereas “Ns” represents the cells located perpendicularly to the electrode surface, and in series with respect to the electric flow.

Where N s, j,k represents the number of bacteria in series in the j-th and k-th columns in the two dimensional directions of the biofilm plane parallel to the electrode. Equation (III.5) can be rewritten as:

j k 1 Cb (III.6) l 1 i 1 N s,l,i

Cbac

For simplification, it will be assumed that bacteria form a rectangular parallelepiped biofilm for an area of the order of magnitude of the small gold electrode (~10-8 m2). Thus, the capacitance of the biofilm stack simplifies to:

jk Cb Cbac (III.7) N s

The binary fission of the biofilm on top of the coated or uncoated electrode can happen in two directions; upwards or laterally. In the former, the capacitance of the biofilm measured in series with the media is expected to decrease due to the incremental number of individual capacitors represented by each bacterium, whose capacitance Cbac is assumed to be constant and uniform. On the other hand, when bacteria grow laterally, the capacitance of the biofilm should increase following the rules of addition of RC circuits. Thus, the transient trend of the calculated capacitance of the biofilm, or in this case the pseudo surface, is an indication of the direction of biofilm growth. Due to the high ionic strength and conductivity of the culture media and the production of ionic species, the capacitance and resistance of the gold electrode and the Nernst double layer are assumed not to change with time significantly compared to the capacitance and resistance of the biofilm and surrounding media. Therefore:

dC p (t) dCb (t) d j(t)k(t) d Ab (t) Cbac Cbac (III.8) dt dt dt N s (t) dt b (t)

Where Ab is the biofilm area on the electrode surface, and b represents the biofilm thickness. When Equation (III.8) is positive, the biofilm is spreading along the electrode surface rather than accumulating on top of itself, and vice versa. Equation (III.8) provides therefore with a tool to determine the kinetics of the biofilm geometric evolution. Moreover, the influence of several operation parameters such as type and number of bacterial species, media initial composition and pH, starvation period, etc. on the biofilm spatial evolution can be qualitatively monitored, too.

The ratio Ab (t) / b (t) is difficult to deconvolute, and its magnitude is mostly a qualitative indication of how the biofilm grows along its three degrees of freedom. Equation (III.8),

however, does not have any mathematical or physical restriction towards the sign of the slope, for which the possibility of observing decreasing capacitance is open. Others have stated that the capacitance has to increase due to increment in the concentration of highly conductive and smaller ions near the electrode surface due to organic breakdown by the bacteria [1]. This statement, nevertheless, does not explain the observed decreasing capacitance trends reported elsewhere [14].

The change in the total resistance of the test culture Rt is a function of several variables, among which the production of ionic metabolites and deposition of non conductive debris (membranes from dead bacteria, unbreakable organics and other suspended solids) are thought to be the controlling factors. The rate of production of conductive metabolites in the well can be described as proposed by Luedeking-Piret [15]: dP(t) dX (t) X (t) bac (III.9) dt bac dt

Equation (III.9) establishes that the metabolites are produced as a result of bacteria maintenance and multiplication. When the rate of resistance decline becomes a constant, either bacteria have reached the stationary growth phase, or they are decaying at an exponential rate proportional to the ratio / . Since the conductance is directly proportional to the metabolite production, the rate of change of the resistance will be inversely proportional to the latter. Determining the individual values of the parameters in Equation (III.9) requires numerous tests controlling a not less extensive set of variables. Instead, by inspecting the transient resistance curves, a qualitative indication of whether the cells are growing or decaying can be accomplished, which may lead to a better interpretation of the transient capacitive curves. For instance, if a resistance curve has an increasing rate of change, decay or inactivation due to flocculation is occurring in the system. If the slope of the capacitive curve is decreasing itself during the same period, then rather than being an indication of a biofilm forming perpendicularly to the electrode surface, such behavior is due to bacterial death and detachment, or agglomeration by flocculating agents.

An additional difficulty arises in impedance microbiology when deposition of organic, non conductive material or debris is present, which makes complicated to interpret the sign of the exponential growth factor and the activity coefficients at the beginning of the experiments when settling of such species is still taking place. In particular, when agar plates are used, no deposition of debris or diffusion of material is important due to the gelatinous character of the media, and only a decrease in the resistance is observed beyond the lag phase for bacterial acclimation when bacteria is active and growing [1].

III.III. MATERIALS AND METHODS

III.III.I. Impedance monitoring on gold electrode arrays

The growth of thiosulfate oxidizing bacteria on golden electrodes and the effect of coating the latter with PEI, PDL, Col, Cys and a combination of these on the attachment and growth of such bacteria was monitored using the impedance method. The experimental setup has been reported elsewhere and was provided by Applied Biophysics Inc. (1600R model, NY). In brief, experiments were carried out using sets of two arrays of 8 polycarbonate/polystyrene squared wells, with 10 gold microelectrodes of 250 µm in diameter on their surface, and a large counter electrode whose area is at least 300 times bigger than the area of the microelectrode, located inside the well on one side of its surface as depicted in Figure III.1a. The total surface area inside the well available for attachment and spreading of bacteria is 0.8 cm2. The electrodes are connected to a system with the appropriate electronics featuring a collection of relays and power sources that allow for the supply of an AC signal whose frequency can be varied as per the researcher needs. The system supplies an approximately constant current of 1 µA, which translates into electrical potential across the electrodes of 100-103 mV, for working frequencies 2 4 on the range of 10 -10 Hz. Continuous monitoring of the impedance ( Zt ), capacitance ( Ct ) and in resistance ( Rt ) of the media within the wells can be achieved, and data stored automatically upon starting each run. Other technical features can be found in the literature and are available from the manufacturer.

Thiosulfate oxidizing bacteria (symbol: B) were obtained after inoculation of secondary sludge from Cincinnati’s Mill Creek Waste Water Treatment Plant (MCWWTP) in equal volumes of a nutrient solution, in six different beakers, each containing between 500 and 600 mL of the nutrients and sludge liquor. The nutrient solution composition was [g compound per 1000 g of deionized water]: Na2HPO4, 1.2; KH2PO4, 1.8; MgSO47H2O, 0.1; (NH4)2SO4, 0.1; CaCL2, 0.03;

FeCl3, 0.02; MnSO4, 0.02 and agar, 1.5. Each beaker was fed with 1-5 grams of sodium thiosulfate at least once a week, and all beakers received the same amount of substrate at the same frequency. The beakers were kept loosely covered in a shaker at 150 rpm. One of the beakers was stirred on a magnetic plate and generous amounts of air were continuously bubbled for comparison purposes between the acclimation procedures. The former was identified as B1, whereas the latter holds the symbol B2. Once every 10-15 days the biomass on each container was let still for 20 minutes, around 30-50% of its liquid content poured (most of the supernatant) and equal amounts of fresh nutrients added. This process lasted around 30-40 days before the start of the impedance tests, and was continued afterwards for other 30-40 days.

The electrolyte, which is identified herein as E, consisted of the nutrient solution as described above at a 1:1 dilution with deionized water. The coating of the well surfaces was accomplished by treating them with 50 µL of aqueous solutions of PEI (15, 30 and 45 mg/L) (Supelco, PA), PDL (50 and 100 mg/L) (Sigma, MO), Col (3 g/L on 0.1% v/v aqueous acetic acid) (Sigma, MO) and Cys (0.01 M) (Applied Biophysics, NY). Henceforth, the coatings will be referred to as their abbreviation and concentration in one word; for instance, PEI15 means PEI coated foam at 15 mg/L concentration. The coating was let inside the wells for 30 minutes and then poured. Wells were let dry on a forced stream of room air. When coating the wells with a combination of collagen and one of the cationic polymers, the wells were treated with Col or Cys first. More than 50 tests were performed using different well coatings at several concentrations and/or combinations, filed with 200 µL of media at different ratios of sludge to electrolyte, as shown on Table III.1. Experiments where bacteria were present were performed in triplicate, and without bacteria at least in duplicate. Due to the dynamic character of the bacteria growing in the beakers, each time the tests were performed both pH and the ratio of Total Solids (TS) and Volatile Solids (VS) of the sludge was determined by standard dry weight measurements of 5 mL samples at 120-130ºC for 24 hours (TS) and 550 ºC for at least 5 hours (VS).

Three different frequencies (400 Hz, 4000 Hz and 40000 Hz) were evaluated simultaneously during our transient experiments when testing the impedance behavior on each well before going to the next one. Whenever indicated, some arrays were placed inside an incubator shaker at different angular speeds, 150 rpm unless specified, and the behavior of the transient response of electrical properties monitored in order to qualitatively determine whether the solids were firmly attached onto the electrode surfaces.

III.III.II. Analytical and quantification

An Electric Cell-substrate Impedance Sensing (ECISTM) apparatus, model R1600 and several 8W10E electrode arrays were employed for the impedance testing on the biofilm attachment and spreading experiments (Applied Biophysics, Inc., NY). Confocal Light Fluorescent Microscopy (CLFM) was used to visualize the biofilm grown on the electrodes at different experimental conditions dying the cultures with propidium iodide. The net electrophoretic mobility and zeta potential of the biomass was determined with a Laser Doppler Velocimeter (BIC, Inc., NY) by diluting the bacterial sludge in the electrolytic nutrient solution and/or deionized water at different pH values, fitting the data with the classic Smoluchowski model. The mixed bacterial culture used to colonize the 8W10E electrodes during the impedance tests were identified by means of Denaturing Gradient Gel Electrophoresis (DGGE) (Microbial Insights, Inc., TN).

III.IV. RESULTS AND DISCUSSION

III.IV.I. Bacteria characterization

DGGE analyses on samples taken from the beakers were the collected WWTP sludge had been exposed to thiosulfate as described in the materials and methods section, was performed after 40 days of the beginning of the acclimation process. The results indicated the presence of a mixed culture of 3 distinct gram-negative genera: Halothiobacillus spp., a chemolithoautotrophic aerobic genus that oxidizes sulfur reduced species and has been observed to grow at a pH range of 3.5-8.5 [16]; Burkholderia spp., a mixotrophic genus that has rarely been reported in the

oxidation of sulfur compounds, which occur at a pH range of 7.0-8.4 [17, 18], and Rhizobium spp., which, just like the previous strain, has scarcely been regarded as a sulfur oxidizing microorganism, either on its own [19] or symbiotically with other species located in plants [20]. All similarity indexes for the detected strains were above 90%.

III.IV.II. Transient behavior of the total (observed), media (calculated) and pseudo surface (calculated) resistances.

All results shown for the transient trend on the observed resistances were obtained at the lowest working frequency of 400 Hz, at which more sensible results between experimental conditions can be distinguished compared to those obtained at 4000 Hz or 40000 Hz. Due to the amount of experimental values and curves in the following Figures, experimental error bars have been omitted. However, the maximum standard deviation obtained in all data sets shown in Table III.2. The maximum standard deviation calculated for the total observed resistance for all tests was below 12% in average, which is a fairly good value for systems including living cells like bacteria. Also, even though some errors reach values up to 90%, these represent only a few data points, particularly during the early minutes of the test where settling of cells is expected, and they are only a fraction of the average standard deviation for the rest of the data points in the curves.

Figures III.4a through III.4c show the observed, normalized transient behavior of the total resistance and the calculated medium and pseudo surface resistances for the wells seeded with the thiosulfate oxidizing bacteria on the gold electrodes and electrodes coated with PEI solutions at the indicated concentrations. Values for the media and pseudo resistances were calculated as indicated in the introduction section. For comparison purposes, most results shown herein include the transient response of the electrodes with the electrolyte alone, with the electrolyte and coating, and with the electrolyte, coating and bacteria, for the conditions shown in Table III.1.

total/volatile solids Estimated Mother Mother sludge Coatings used when Figures mother sludge %VS/TS maximum sludge dilution on ECIS well a b applicable [mgTS/ml]/[mgVS/ml] number of cells pH B:E:H2O-pH III.4a-III.4c; 43.27-7.58 18 8.33·1009 3.92 1:1:1-NA PEI15, PEI30 III.8a-III.8c III.5a-III.5c; 55.14-10.71 19 1.25·1010 3.83 1:1:1-NA PDL50, PDL100 III.9a-III.9c III.6, III.10c 70.63-22.71 32 2.85·1010 1.39 1:1:1(B1)-NA Col

III.6, III.10c 42.84-7.83 18 8.67·1009 4.20 1:1:1(B2)-NA Col

III.7, III.11 34.43-9.53 28 1.09·1010 3.83 Col-PEI45, Col-PDL100

CysTest 18.39-9.12 50 1.04·1010 2.65 1:1:1-NA Cys, Cys-PEI45, Cys-PDL100, Cys-Col a based on an average bacterium dry weight of 50 fg and assuming measured volatile solids concentration represents only viable bacteria. b when electrolyte is not used, its equivalent proportion is replaced with water; NA: not available. c B1 and B2 represent two types of sludge used on the same test, as shown in the related Figures.

Table III.2. Initial values for the observed total resistance ( Rt ) and capacitance (Ct ) for all data sets collected. Resistance values are measured at 400 Hz, whereas capacitance values are measured at 40000 Hz. Values in parenthesis represent the maximum calculated percentage error for data sets corresponding to the same experimental conditions and curves.

Figure Conditions Resistance Capacitance Figure Conditions Resistance Capacitance [ohm] [nF] [ohm] [nF]

III.4a-III.4c E+B 681 (6) 66.1 (3) III.6, III.10 E 2544 (4) 52.3 (23) III.8a-III.8c E+B+PEI15 1002 (3) 51.8 (8) E+B1 588 (1) 35.1 (17) E+B+PEI30 988 (5) 52.5 (7) E+B2 720 (1) 63.7 (7) E 4253 (1) 29.3 (20) E+Col 2868 (11) 47.7 (37) E+ PEI15 4870 (13) 17.7 (38) E+B1+Col 654 (1) 28.3 (11) E+ PEI30 4695 (6) 16.4 (80) E+B2+Col 691 (1) 43.6 (6) III.3-III.4 E+B 695 (7) 61.6 (7) III.7, III.11 E+B+Col 1114 (4) 27.9 (3) E+B+PEI15 905 (6) 46.1 (8) E+B+Col+PDL100 1065 (1) 33.4 (39) E+B+PEI45 952 (90) 44.7 (6) E+B+Col+PEI45 1319 (1) 28.0 (21) E 2857 (3) 68.5 (9) E+ PEI15 4233 (17) 25.2 (44) E+ PEI45 3630 (11) 24.5 (41) III.5a-III.5c E+B 696 (27) 51.7 (22) CysTest E+B+PEI45 883 (19) 39.9 (22) III.9a-III.9c E+B+PDL50 720 (1) 40.4 (24) E+B+ Cys 582 (22) 80.5 (20) E+B+PDL100 808 (1) 35.1 (12) E+B+Cys+PEI45 592 (13) 86.3 (17) E 2761 (2) 57.8 (20) E+B+Cys+PDL100 580 (10) 89.1 (12) E+PDL50 3044 (1) 33.1 (21) E+B+Cys+Col 562 (11) 82.7 (20) E+PDL100 2983 (1) 30.1 (46) E+Cys 2516 53.3

400 Hz 1.2 E+B E+B+PEI15 1.1 E+B+PEI30 E E+PEI15

R E+PEI30 1.0

0.9

Normalized Normalized

0.8

0.7 0 5 10 15 20 Elapsed time [hr] Figure III.4a. Observed normalized total resistance of the PEI coated and uncoated culture wells, with and without bacterial cells. Experimental conditions are summarized in Table III.1. Resistance measurements were recorded at 400 Hz, since at this frequency the sensitivity between curves is maximum.

400 Hz 1.1

1.0

m 0.9

0.8

E+B E+B+PEI15

Normalized 0.7 E+B+PEI30 E 0.6 E+PEI15 E+PEI30 0.5 0 5 10 15 20 Elapsed time [hr]

Figure III.4b. Calculated normalized media resistance of the PEI coated and uncoated culture wells, with and without bacterial cells. Experimental conditions are summarized in Table III.1. Resistance measurements were calculated from data recorded at 400 Hz, since at this frequency the sensitivity between curves is maximum.

400 Hz 1.06

E+B 1.04 E+B+PEI15 E+B+PEI30

b E

R 1.02 E+PEI15

e E+PEI30

R 1.00

0.98

Normalized

0.96

0.94 0 5 10 15 20 Elapsed time [hr]

Figure III.4c. Calculated normalized pseudo surface resistance of the PEI coated and uncoated culture wells, with and without bacterial cells. Experimental conditions are summarized in Table III.1. Resistance measurements were calculated from data recorded at 400 Hz, since at this frequency the sensitivity between curves is maximum.

Several conclusions are drawn from Figures III.4a to III.4c. First, the resistance behavior of the cells shows a decreasing tendency on those where bacteria is present. Even though this phenomenon is attributed to the breakdown of organics, the species Halothiobacillus, which had been believed to be predominant in the cultures due to the acidic pH of the mother sludge (as shown in Table III.1) are autotrophic and do not synthesize added organic carbons, under limited oxygen availability in liquid cultures [16, 21]. However, the conductivity of diluted solutions of thiosulfate are in the same order of magnitude of the corresponding sulfate salts; thus, the initial hypothesis that Halothiobacillus is the predominant species in the culture may not be true, and indeed, heterotrophic, sulfur and acid tolerant Rhizobium and Burkholderia strains co- metabolized the sulfur containing species and organics present in the wells. The rate of resistance decrease is enhanced by the presence of the PEI coating, with no major difference in the cells coated with PEI solutions at concentrations of 15 and 30 mg/L. Also, an initial increase on the resistance is observed in the wells with bacteria and no coating, which may indicate either a bacterial lag phase and/or non conductive deposition of debris or inactive cells during the settling of the seeded biomass at the beginning of the experiments. Secondly, the observed total

resistance behavior is mostly controlled by the resistance in the medium rather than the resistance at the interface. Indeed, even though both Figures III.4b and III.4c show decreasing tendencies for all curves just like in the case of the total resistance in Figure III.4a, the magnitude of the relative change is less than 6% for the pseudo surface panel, as opposed to the media panel, whose value of 40% more closely resembles the total observed value of around 20%. This result is obtained for almost all tests carried out in the present study. Similar results were obtained from tests where the PEI concentration of the coating solution was increased to 45 mg/L (data not shown).

Figures III.5a to III.5c show the normalized resistance trends of the PDL coated wells and corresponding uncoated ones, as described in Table III.1. Unlike for the PEI case, the resistance of the culture media in the coated cells increases with time. This might be an indication of rapid growth and proliferation of bacteria with low metabolic activity per cell, which is a phenomena already observed in some cultures [22]. Bacteria, like animal cells, exhibit an insulating character; however, the redistribution of the surface charge on their membrane, and the membrane “softness” or capacity to be more easily penetrated by ions makes them slightly more conductive than animal cells. When bacteria grow, the amount of their insulating membrane in the culture well increases as well, and the result is a higher opposition for electricity to flow, which coupled with low activity and production of conductive metabolites, results in the increasing resistance trend as observed for the PDL coated cultured wells. In addition, though less likely to have happened, highly cationic, soluble PDL could have diffused towards the bulk of the media neutralizing the anionic acidic compounds produced by the consumption of the organic ingredients of the electrolyte. The PDL migration towards the bulk of the media is plausible considering that a lower resistance in the pseudo surface for the coated wells was calculated, as shown in Figure III.5b, with relative decrease of up to 20%. Such decrease is much more relevant than for the PEI coated wells. Furthermore, PDL could have initiated the flocculation of bacteria into bigger units which grow yet do not produce metabolites at the same rate when not in intimate contact with the surroundings, particularly for the inner cells in the flock [23]. Lastly, the absorption of ions into the bacteria soft membrane could have played an important role in the irregular resistance trend [1].

400 Hz 1.3 E+B E+B+PDL50 1.2 E+B+PDL100 E E+PDL50 Shaking t 1.1 E+PDL100

1.0 150 rpm

Normalized Normalized 0.9

0.8

0.7 rpm 50 0 10 20 30 40 Elapsed time [hr]

Figure III.5a. Observed normalized total resistance of the PDL coated and uncoated culture wells, with and without bacterial cells. Experimental conditions are summarized in Table III.1. Resistance measurements were recorded at 400 Hz, since at this frequency the sensitivity between curves is maximum.

Figure III.5a shows, too, that some activity was present in the electrolyte itself due to its reduction of resistance down to values of 80% the original one. Yet, for wells coated with PDL the resistance increased more than 20% of the initial values for wells with bacteria in presence of the coating. Similar to PEI, the concentration of PDL in the coating solution had negligible effect on the resistance trend. Table III.2 shows the initial values of each parameter monitor for all tests carried out, indicating that the coatings themselves are not very conductive and add an external resistance to the measurement, either due to their organic character or the formation of an additional double layer including the polymeric cations.

400 Hz 1.6 E+B Shaking E+B+PDL50 1.4 E+B+PDL100 E+PDL50 E+PDL100

R 1.2 150 rpm

50 rpm 1.0

Normalized

0.8

0.6 0 10 20 30 40 Elapsed time [hr]

Figure III.5b. Calculated normalized media resistance of the PDL coated and uncoated culture wells, with and without bacterial cells. Experimental conditions are summarized in Table III.1. Resistance measurements were calculated from data recorded at 400 Hz, since at this frequency the sensitivity between curves is maximum.

400 Hz 1.10 Shaking 1.05

b 1.00

R 0.95

0.90 E+B E+B+PDL50 0.85 E+B+PDL100

Normalized E+PDL50 150 rpm E+PDL100

0.80 50 rpm

0.75 0 10 20 30 40 Elapsed time [hr]

Figure III.5c. Calculated normalized pseudo surface resistance of the PDL coated and uncoated culture wells, with and without bacterial cells. Experimental conditions are summarized in Table III.1. Resistance measurements were calculated from data recorded at 400 Hz, since at this frequency the sensitivity between curves is maximum.

The impact of collagen as coating cofactor did not exhibit a definite trend, as shown in Figure III.6. For the highly acidic bacteria B1 (mother sludge pH of 1.39, VS relative concentration of 32%) the resistance decrease has a lower slope during the initial stage of the seeding, and reaches a plateau faster than for the less acidic bacteria B2 (mother sludge pH of 4.20, VS relative concentration of 18%) where a faster decrease of resistance is observed initially. This is because for highly acidic bacteria such as B1, an increment on the metabolite concentration from bacterial activity does not change the conductivity of the surroundings noticeably, even at higher cell concentrations in the well. The presence of high contents of agar and other organic carbons in the electrolyte and sludge, which themselves contain proteins, may have mitigated the effect of collagen, too, especially for the B2 case where both curves for the coated and uncoated wells reach very similar normalized resistance values after 6-8 hours and onwards; indeed, collagen has been reported to act as an attachment enhancer for tissue cells since the latter, as well as bacteria, contain integrins on their surface that bind to the proteins in collagen, and perhaps agar, by mechanisms different than electrostatic attraction, but biological compatibility [8, 24].

400 Hz 1.10 E Shaking 1.05 E+B1 E+B2 1.00 E+Col

t E+B1+Col

R E+B2+Col 0.95

0.90

Normalized 0.85

0.80

0.75 0 10 20 30 40 Elapsed time [hr]

Figure III.6. Observed normalized total resistance of the Col coated and uncoated culture wells, with and without bacterial cells. B1: bacteria from mother sludge with pH of 1.39; B2: bacteria from mother sludge with pH of 4.20. Resistance measurements were recorded at 400 Hz, since at this frequency the sensitivity between curves is maximum.

Figure III.7 shows the effect of coating the electrodes with a combination of Col and a cationic polymer, PEI or PDL at the concentrations specified. It has been reported that combining a protein source such as Col with a cationic polymer results in better cell attachment onto suspended biocarriers than when either substance is used separately for higher animal cells [11]. According to the latter, the cationic polymer neutralizes the typically negative charge of the cell surface, which deactivates the repulsive forces among them and facilitates their accumulation in the initial coagulation mechanism, whilst the protein source promotes the subsequent accumulation on bigger flocks and their spreading onto surfaces by providing the material for the rapid build-up of extracellular polymer matrices (EPM) needed to colonize a surface [25, 26]. Even though these EPM can be produced by the outer membrane layers of the bacteria, their presence as an added source in the environment speeds up their growth. A steeper and faster decrease in the observed resistance is obtained when collagen is combined with PEI, indicating higher bacterial activity. PDL seemed not to have any effect when combined with collagen on the transient resistance response of the cultured wells. When compared with the results shown in Figure III.5a, it appears that PDL inhibits the activity of the bacteria or neutralizes the metabolites produced by the organic breakdown.

400 Hz 1.10

1.05

t R 1.00 Shaking 0.95

Normalized E+B+Col 0.90 E+B+Col+PDL100 E+B+Col+PEI45

0.85 0 10 20 30 40 Elapsed time [hr]

Figure III.7. Observed normalized total resistance of Col, PEI and PDL coated culture wells with bacterial cells. Experimental conditions are summarized in Table III.1. Resistance measurements were recorded at 400 Hz, since at this frequency the sensitivity between curves is maximum.

III.IV.III. Transient behavior of the total (observed), media (calculated) and pseudo surface (calculated) capacitances.

Results for the observed total and calculated media and pseudo surface capacitances are summarized in the section herein. These values were obtained at the highest working frequency of 40000 Hz since the sensitivity between experimental conditions was maximum. Like the resistance panels, the subsequent Figures omit the error bars for better visualization of the curves. The maximum calculated standard error is included in the Figure titles and in Table III.2 for all tests carried out for reference. Once more, even though some error maxima reach values up to 80%, the average of all maxima for the tests was below 20%, and the former was obtained only during a few measurements at the early stages of the incubation.

The transient observed total capacitance and calculated medium and pseudo surface capacitances of the wells coated with a solution of PEI are indicated in Figures III.8a to III.8c. Figure III.8a shows a dramatic difference in the capacitance trend for the bacterial seeded wells in presence of the coating, as opposed to the wells with no treatment. Based on the arguments described by Equation (III.8), it is concluded that PEI promotes the spreading of the bacterial biofilm along the electrode surface. The total capacitance of the well is controlled by the capacitance of the pseudo surface, as expected, when inspecting the shapes of Figures III.8a and III.8c. This behavior is consistent in all tests carried out, and it can be concluded therefore that, providing the equivalent RC circuit depicted in Figure III.2 represents the physical system in the well, by just looking at the transient trend of the total capacitance, the relative direction of the bacterial growth can be estimated, and therefore the feasibility of the cells to spread rather than accumulate for the conditions of the experiment.

40000 Hz 1.4

1.2

C 1.0

0.8 E+B E+B+PEI15 Normalized Normalized E+B+PEI30 E 0.6 E+PEI15 E+PEI30

0.4 0 5 10 15 20 Elapsed time [hr]

Figure III.8a. Observed normalized total capacitance of PEI coated and uncoated culture wells, with and without bacterial cells. Other experimental conditions are summarized in Table III.1. Resistance measurements were recorded at 40000 Hz, since at this frequency the sensitivity between curves is maximum.

40000 Hz 2.0 E+B 1.8 E+B+PEI15 E+B+PEI30 1.6 E E+PEI15

C E+PEI30 1.4

1.2

Normalized 1.0

0.8

0.6 0 5 10 15 20 Elapsed time [hr]

Figure III.8b. Calculated normalized media capacitance of PEI coated and uncoated culture wells, with and without bacterial cells. Other experimental conditions are summarized in Table III.1. Capacitance measurements were calculated from data obtained at 40000 Hz, since at this frequency the sensitivity between curves is maximum.

40000Hz 1.4

1.2

C 1.0

0.8 E+B E+B+PEI15 Normalized E+B+PEI30 E 0.6 E+PEI15 E+PEI30

0.4 0 5 10 15 20 Elapsed time [hr]

Figure III.8c. Calculated normalized pseudo surface capacitance of PEI coated and uncoated culture wells, with and without bacterial cells. Other experimental conditions are summarized in Table III.1. Capacitance measurements were calculated from data obtained at 40000 Hz, since at this frequency the sensitivity between curves is maximum.

Figures III.9a to III.9c show the capacitance response for the wells coated with PDL solutions at the conditions specified. Unlike for PEI, the capacitance trend tends to decrease for all cases where bacteria is present, though for the coated cells the slope is just slightly less negative, specially for an incubation period higher than 10 hours. This might indicate some tendency to spread the biofilm by presence o the PDL coating, although the predominant direction for bacterial growth is parallel to the electrode surface. A negligible effect of the coating in the overall capacitance trend is also observed for collagen, compared to the untreated wells (Figure III.10). The synergy between the polymeric coatings PEI and PDL and the added protein source collagen on the overall capacitance trend is depicted in Figure III.11. The transient trend indicates a preferential direction perpendicular to the electrode surface for the biofilm growth for the conditions of the experiment. Even though it is estimated that bacteria tend to accumulate atop one another instead of spread, the presence of PEI reduces this tendency by exhibiting a less negative slope of the capacitance curve. This result is congruent with the previous finding that PEI promotes lateral expansion of the biofilm rather than perpendicular one. That the capacitance curve tends to decrease rather than increase like in Figure III.8a for PEI might be a consequence of the higher initial concentration of VS in the mother sludge used for the tests shown in Figure

III.9a (28% compared to 18%) for which more bacteria could have settled and accumulated vertically, specially during the first 10 hours of incubation. That the biofilm tends to grow vertically rather than horizontally was observed by means of CLFM on bacteria incubated in PEI coated and uncoated wells, as shown in Figures III.12a and III.12b, for wells after 24-30 hr of incubation. It can be seen, as reported elsewhere, the formation of fibrils and mushroom like spots that exhibit a longer vertical distance from the electrode surface than horizontal one.

40000 Hz 1.6 E+B Shaking E+B+PDL50 1.4 E+B+PDL100 E E+PDL50 t 1.2 E+PDL100

C 150 rpm 1.0

50 rpm

Normalized 0.8

0.6

0.4 0 10 20 30 40 Elapsed time [hr]

Figure III.9a. Observed normalized total capacitance of PDL coated and uncoated culture wells, with and without bacterial cells. Other experimental conditions are summarized in Table III.1. Resistance measurements were recorded at 40000 Hz, since at this frequency the sensitivity between curves is maximum.

40000 Hz 3.5 E+B Shaking 3.0 E+B+PDL50 E+B+PDL100 E+PDL50 E+PDL100 m 2.5

2.0

1.5

Normalized 150 rpm

1.0 50 rpm

0.5 0 10 20 30 40 Elapsed time [hr]

Figure III.9b. Calculated normalized media capacitance of PDL coated and uncoated culture wells, with and without bacterial cells. Other experimental conditions are summarized in Table III.1. Capacitance measurements were calculated from data obtained at 40000 Hz, since at this frequency the sensitivity between curves is maximum.

40000 Hz 1.6 E+B Shaking 1.4 E+B+PDL50 E+B+PDL100 E+PDL50

p 1.2 E+PDL100

1.0 150 rpm

50 rpm

Normalized 0.8

0.6

0.4 0 10 20 30 40 Elapsed time [hr]

Figure III.9c. Calculated normalized pseudo surface capacitance of PDL coated and uncoated culture wells, with and without bacterial cells. Other experimental conditions are summarized in Table III.1. Capacitance measurements were calculated from data obtained at 40000 Hz, since at this frequency the sensitivity between curves is maximum.

100

40000 Hz 1.4 Shaking

1.2

C 1.0

0.8 E

Normalized E+B1 E+B2 0.6 E+Col E+B1+Col E+B2+Col 0.4 0 10 20 30 40 Elapsed time [hr]

Figure III.10. Observed normalized total capacitance of the Col coated and uncoated culture wells, with and without bacterial cells. B1: bacteria from mother sludge with pH of 1.39; B2: bacteria from mother sludge with pH of 4.20. Capacitance measurements were recorded at 40000 Hz, since at this frequency the sensitivity between curves is maximum.

40000 Hz 1.1

Shaking E+B+Col 1.0 E+B+Col+PDL100 E+B+Col+PEI45

C 0.9

0.8

Normalized

0.7

0.6 0 10 20 30 40 Elapsed time [hr]

Figure III.11. Observed normalized total capacitance of Col, PEI and PDL coated culture wells with bacterial cells. Experimental conditions are summarized in Table III.1. Capacitance measurements were recorded at 40000 Hz, since at this frequency the sensitivity between curves is maximum.

101

Figure III.12a. CLFM micrograph of the mushroom/fibril like biofilm on a PEI coated well after 24 hours of incubation. Picture dimensions: 650 µm side, 120 µm depth. Adjacent panels show the perpendicular profile of the biofilm. Biofilm solid support bottom is located in the outermost edge of the adjacent panels.

Figure III.12b. CLFM micrograph of the mushroom/fibril like biofilm on an uncoated well after 24 hours of incubation. Picture dimensions: 650 µm side, 86 µm depth. Adjacent panels show the perpendicular profile of the biofilm. Biofilm solid support bottom is located in the outermost edge of the adjacent panels.

102

Since bacterial growth and decay follow an exponential trend, it is predictable that the capacitance behavior of the cultured wells changed in the same fashion with respect to time, as indicated:

1t 2t Ct (t) 1e 2e (III.10)

The transient capacitance curves have been fitted with the logarithm of time rather than with the exponential of time, as it could have been expected, yielding a linear regression. This can be mathematically interpreted as an unequal transient evolution of the capacitive behavior of the two elements in series; either the pseudo surface and media pair, or the electrode and biofilm pair. Indeed, it can be proven that, when the capacitors are arranged in series, the resulting overall capacitance, which is the corresponding inverse of the summation of the exponential inverse values of the individual capacitors, fits a logarithmic trend with time when the rate of change of the capacitance of one of the domains is controlling; i.e., using the nomenclature of

Equation (III.10), when 1 2 and 1 2 , being negative and for large times. The resulting fitting is shown in Table III.3. Here, the slope of such linear regression is a quantitative indication of the capacitance change with time, and can be used to estimate the relative direction of biofilm growth and the impact of the different coatings on such direction. Due to settling during the early stages of incubation and evaporation effects after more than 24 hours, only the data corresponding to the period 2-10 hours has been included in the fitting.

From the values shown in Table III.3, it can be concluded that, for the experimental conditions of pH and relative VS concentration indicated in Table III.2, in absence of another coating PEI promotes the spreading of the thiosulfate oxidizing bacteria, whereas PDL induces the bacteria to accumulate as in a vertical stack. When collagen is the coating, however, the spreading is promoted on bacteria with a highly acidic pH in their mother sludge and low VS concentration. Further, when collagen is combined with a cationic polymer, namely PEI and PDL, both arrangements tend to spread the bacteria compared with collagen alone, yet the spreading power of PEI is more intense. It is worth noting, too, that upon treating the electrodes with Cys, which is a thiol containing aminoacid used to stabilize the electrode rather than to promote any binding between the electrode and bacteria, all coatings, i.e. PEI, PDL and Col, tend to spread the biofilm

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compared to the control well when present individually (“CysTest” in Tables 1 to 3). The spreading capability is slightly higher for PDL than PEI in this case, though both cationic polymers exhibit much higher spreading capacity than collagen alone. Due to the stabilization brought about by the thiol aminoacid, this results might be more representative of the real effect of the studied coatings, and an indication that PDL can actually increase the spreading of the biofilm even though results shown in Figure III.9a indicate otherwise.

III.IV.IV. Effect of pH on the transient behavior of the observed electrical properties.

The main premise for the utilization of positively charged polymers coating the electrode surface is the charge cancellation of the bacterial surface and therefore enhanced collisions with the biofilm support. Even though most bacteria have a negative charge at physiological pH in the range 5-7, gram-negative bacteria has been reported to exhibit an isoelectric point at a pH just slightly lower of around 4.0 [27]. Since the pH of most mother sludge used in this study is slightly lower than such value, both proton titration and zeta potential determination were carried out in order to determine the net surface charge of the final bacterial suspension. The former showed inflection points at pH values in the range 3.27-4.29 for 1:1:1 dilutions of E:H2O:B such as those used in the wells. Further examination revealed zeta potential values of -5.2 to -24.3 mV for the same bacterial dilutions at pH range of 2.43-5.83. Although the pH of the diluted bacteria cultures was not measured during the tests, most sludge samples had a pure pH within this range as shown on Table III.1, and after dilution it was expected that the net surface charge had a negative value. However, pH, and equally importantly but much more difficult to measure ionic strength, have to be considered when comparing the effect of different coatings on the transient electrical properties. As previously indicated, comparing the effect of coating the electrodes with the studied polymers is only meaningful when comparing tests using the same mother sludge and VS concentration.

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Table III.3. Calculated values of the linear regression of the type Ct (t) a ln(t) b for all the tests carried out. Results obtained on 10 electrode wells.

Coated Uncoated Bacteria Abiotic Bacteria Abiotic Figure Conditions a b a b III.4a-III.4c E+B -0.0879 0.8139 III.8a-III.8c E+B+PEI15 0.014 1.2454 E+B+PEI30 0.014 1.2454 E -0.0075 0.9052 E+ PEI15 0.0050 0.9894 E+ PEI30 -0.0057 0.9891 III.5a-III.5c E+B -0.0581 0.9665 III.9a-III.9c E+B+PDL50 -0.1004 0.9887 E+B+PDL100 -0.0996 1.0545 E -0.0066 0.8456 E+PDL50 0.0403 0.9697 E+PDL100 0.0465 0.9983 III.6, III.10 E 0.0185 0.778 E+B1 -0.0148 0.9728 E+B2 -0.0556 0.8213 E+Col 0.0309 0.9866 E+B1+Col 0.0064 1.0108 E+B2+Col -0.056 0.9768 III.7, III.11 E+B+Col -0.0834 0.9657 E+B+Col+PDL100 -0.0782 0.9563 E+B+Col+PEI45 -0.0272 0.9325 CysTest E+B+PEI45 0.0098 0.9173 E+B+ Cys -0.0469 1.0638 E+B+Cys+PEI45 0.6114 1.4339 E+B+Cys+PDL100 0.6338 1.0558 E+B+Cys+Col 0.2168 1.2861 E+Cys 0.0082 0.9162

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III.IV.V. Effect of shaking the culture well on the transient observed electrical properties

Inspection of the curves shown in Figures III.5a to III.8 and III.9a to III.11 show that whenever bacteria was present in the culture well, the transient behavior of both resistance and capacitance did not show and appreciable change in their time trends after shaking the wells at 150 rpm (or 75 rpm where indicated) and it was only appreciable for some wells containing just the electrolyte. Further examination of all collected data during this study confirmed that changes are only observable in the resistance of some cells where bacteria is growing in the absence of coating, or cells with electrolyte only. Experiments were carried out where shaking was applied at the beginning of the incubation, after which the cultures were removed from the shaker and let still (data not shown). No change in the time trends of the capacitance was observed, whereas a deviation of the resistance trend was detected for cells with electrolyte only. Thus, it was concluded that the biomass, in presence or absence of a coating agent, is heavy enough to settle and stay atop the electrolyte, whereas some ions might migrate towards the bulk of the wells after shaking.

III.V. CONCLUSIONS

AC currents in the range 400-40000 Hz were used to determine the transient impedance characteristics of an electrode-culture well system containing biofilms of thiosulfate oxidizing bacteria, in order to determine the effect of coating the electrodes with polymeric and protein cofactors on the impedance properties of the forming biofilms. Based on an equivalent RC circuit and the hypothesis that the elements of the cultured well behave as ideal capacitors and resistors, it was proposed that an increasing observed capacitance indicates a biofilm growing along the surface of the electrode, as opposed to perpendicularly to it when such trend is decreasing. Results showed that when PEI solutions are used to coat the electrodes, the biofilm tends to spread alongside, whereas it grows vertically when the coating is PDL. When the electrodes are stabilized after pretreatment with Cys, PEI, PDL and Col tend to promote the sideward spreading of the biofilm, being a combination of Col with a cationic polymer more predominant than the polymer alone. Finally, no effect of shaking the cultured wells was observed on their capacitance

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properties, suggesting stable biofilms even in absence of a binding coating; however, some mixing of suspended ions was detected through a change on the observed resistance.

III.VI. SYMBOLS a : Slope of logarithmic fitting of total capacitance, Table III.3.

Ab : biofilm thickness [m] B: Bacteria b : Intercept of logarithmic fitting of total capacitance, Table III.3. C : Capacitance [F] E: Electrolyte j : Number of bacteria in biofilm in one dimension parallel to electrode plane [1] k : Number of bacteria in biofilm in one dimension parallel to electrode plane [1]

Ns : Number of bacteria in biofilm perpendicular to electrode plane [1] P : Metabolite concentration [kg·m-3] R : Electrical resistance [ohm] t : Time [sec] X : Biomass concentration [kg·m-3] Z : Capacitance [ohm] Subscripts bac : Individual bacterium b : Biofilm e : Electrode m : Media p : Pseudo surface : Total Greeks : Metabolite production rate coefficient due to maintenance, Equation (III.9) [sec-1] : Metabolite production rate coefficient due to growth, Equation (III.9) [1]

b : Biofilm thickness [m]

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: AC frequency [Hz] : Capacitance rate linear coefficient, Equation (III.10) [F] : Capacitance rate exponential coefficient, Equation (III.10) [sec-1]

III.VII. REFERENCES

1. Firstenberg-Eden, R., & Eden, G. (1984). Impedance microbiology. Innovation in microbiology series, 3. Letchworth, Hertfordshire, England: Research Studies Press. 2. Chang, T. and A. Huang. (2000) “Rapid differentiation of fermentative from nonfermentative gram-negative bacilli in positive blood cultures by an impedance method.” Journal of Clinical Microbiology. 38(10) 3589-3594. 3. Wu, J., Huang, A., Dai, J. and T. Chang. (1997) “Rapid detection of oxacillin-resistant Staphylococcus aureus in blood cultures by an impedance method.” Journal of Clinical Microbiology. 35(6) 1460-1464. 4. Dukhin, A. and S. Dukhin. (2005) “Aperiodic capillary electrophoresis method using an alternating current electric field for separation of macromolecules.” Electrophoresis. 26(11) 2149-2153. 5. Sengupta, S., Battigelli, D. and H. Chang. (2006). “A micro-scale multi-frequency reactance measurement technique to detect bacterial growth at low bio-particle concentrations.” Lab on a Chip. 6(5) 682-692. 6. Cachet, H., El Moustafid, T., Herbert-Guillou, D., Festy, D., Touzain, S. and B. Tribollet. (2001). “Characterization of deposits by direct observation and by electrochemical methods on a conductive transparent electrode. Application to biofilm and scale deposit under cathodic protection.” Electrochimica Acta. 46(24-25) 3851–3857. 7. Rittman, B. (2004) “Biofilms in the water industry.” In “Microbial Biofilms.” Edited by Ghannoum, M. and O’Toole, G. ASM Press. 359-378. 8. Wegener, J., Keese, C. and I. Giaever. (2000) “Electric cell-substrate impedance sensing (ECIS) as a noninvasive means to monitor the kinetics of cell spreading to artificial surfaces.” Experimental Cell Research. 259(1) 158-166. 9. Aylmore, M. and D. Muir. (2001) “Thiosulfate leaching of gold - A review.” Minerals Engineering. 14(2) 135-174. 10. Vancha, A., Govindaraju, S., Parsa, K., Jasti, M., Gonzalez-Garcia, M. and R. Ballestero. (2004) “Use of polyethyleneimine polymer in cell culture as attachment factor and lipofection enhancer.” BMC Biotechnology. 4(23). 11. Clapper, D. and W. Hu. (1996) US Patent 5,512,474. 12. Yang, L., Ruan, C. and Y. Li. (2003) “Detection of viable Salmonella typhimurium by impedance measurement of electrode capacitance and medium resistance.” Biosensors and Bioelectronics. 19(5) 495-502. 13. Giaever, I. and C. Keese. (1991) “Micromotion of mammalian-cells measured electrically.” Proceedings of the National Academy of Sciences of the United States of America. 88(17) 7896-7900. 14. Noble, P., Dziuba, M., Harrison, D. and W. Albritton. (2006) “Factors influencing capacitance-based monitoring of microbial growth.” Journal of Microbiological Methods. 37(1) 51-64.

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15. Lan, C., Oddone, G., Mills, D. and D. Block. (2006) “Kinetics of Lactococcus lactis growth and metabolite formation under aerobic and anaerobic conditions in the presence or absence of hemin.” Biotechnology and Bioengineering. 95(6) 1070-1080. 16. Sievert, S., Heidorn, T. and J. Kuever. (2000) “Halothiobacillus kellyi sp nov., a mesophilic, obligately chemolithoautotrophic, sulfur-oxidizing bacterium isolated from a shallow-water hydrothermal vent in the Aegean Sea, and emended description of the genus Halothiobacillus.” International Journal of Systematic and Evolutionary Microbiology. 50(3) 1229-1237. 17. Jung, S., Jang, K., Sihn, E., Park, S. and C. Park. (2005) “Characteristics of sulfur oxidation by a newly isolated Burkholderia spp.” Journal of Microbiology and Biotechnology. 15(4) 716-721. 18. Wittke, R., Ludwig, W., Peiffer, S. and D. Kleiner. (1997) “Isolation and characterization of Burkholderia norimbergensis sp. nov., a mildly alkaliphilic sulfur oxidizer.” Systematic and Applied Microbiology. 20(4) 549-553. 19. El-Tarabily, K., Soaud, A., Saleh M. and S. Matsumoto. (2006) “Isolation and characterisation of sulfur-oxidising bacteria, including strains of Rhizobium, from calcareous sandy soils and their effects on nutrient uptake and growth of maize (Zea mays L.).” Australian Journal of Agricultural Research. 57(1) 101-111. 20. Anandham, R., Sridar, R., Nalayini, P., Poonguzhali, S., Madhaiyan, M. and T. Sa. (2007). “Potential for plant growth promotion in groundnut (Arachis hypogaea L.) cv. ALR-2 by co-inoculation of sulfur-oxidizing bacteria and Rhizobium.” Microbiological Research. 162(2) 139-153. 21. Ito, T., Sugita, K. and S. Okabe. (2004) “Isolation, characterization, and in situ detection of a novel chemolithoautotrophic sulfur-oxidizing bacterium in wastewater biofilms growing under microaerophilic conditions.” Applied and Environmental Microbiology. 70(5) 3122-3129. 22. Bernard, L., Courties, C., Duperray, C., Schafer, H., Muyzer, G. and P. Lebaron. (2001). “A new approach to determine the genetic diversity of viable and active bacteria in aquatic ecosystems.” Cytometry. 43(4) 314-321. 23. Hantula, J. and D. Bamford. (1991) “The efficiency of the protein-dependent flocculation of flavobacterium sp is sensitive to the composition of growth-medium.” Applied Microbiology and Biotechnology. 36(1) 100-104. 24. Rich, R., Deivanayagam, C., Owens, R., Carson, M., Hook, A., Moore, D., Yang, V., Narayana, S. and M. Hook. (1999) “Trench-shaped binding sites promote multiple classes of interactions between collagen and the adherence receptors, alpha(1)beta(1) integrin and Staphylococcus aureus Cna MSCRAMM.” Journal of Biological Chemistry. 274(35) 24906-24913. 25. Julian Wimpenny. (2000) “An overview of Biofilms as functional communities.” In “Community Structure and Cooperation in Biofilms”, pp 1-24. 59th Symposium of the Society for General Microbiology. Edited by Allison, DG, Gilbert, P, Lappin-Scott, HM and Wilson, M. 26. Busscher, HJ., van der Mei., HC. (2000) “Initial microbial adhesion events: mechanisms and implications.” In “Community Structure and Cooperation in Biofilms”, pp 25-36. 59th Symposium of the Society for General Microbiology. Edited by Allison, DG, Gilbert, P, Lappin-Scott, HM and Wilson, M.

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27. Poortinga, A., Bos, R. and Busscher, H.(2001) “Reversibility of bacterial adhesion at an electrode surface.” Langmuir. 17(9) 2851-2856.

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CHAPTER IV

ABIOTIC H2S UPTAKE IN IRON (III) (OXY)(HYDR)OXIDES AT STP CONDITIONS IN TRICKLE BEDS

SUMMARY

This study encompasses the evaluation of the performance of three different, commercially available iron oxide and hydroxide solid materials in packed and trickle beds for the adsorption and abiotic oxidation of H2S in airstreams (50-200 ppmv) at standard temperature and pressure conditions (STP) in two operation modes: in presence of low humidity air (30 ppmv H2O, “dry”) and in excess of water in a closed-loop liquid, trickle bed (“wet”). The materials were mixed iron oxide, open-pore foams (IOPF), iron oxide pellets (IOP) and iron hydroxide grains (IHG). The wet operation removal capacities were evaluated after saturation of the catalyst in dry operation, where the IOPF and IHG showed chemisorption behavior, compared to physisorption for the IOP. It was found that in wet beds the operation time of the IOPF and IOP before breakthrough was achieved increased, respectively, by four and almost one thousand times compared to the dry operation values, with removal efficiencies in excess of 80% and H2S adsorption capacities of 23.31 and 27.10 mgH2S/gcatalyst after 1000 hr for the IOPF and IOP, correspondingly. The enhancement in H2S uptake by water is suggested to be a consequence of both the dissociation of H2S into active sulfhydryl anions, and the conversion of iron sulfides into iron oxides in presence of oxygen at slightly alkaline pH, producing elemental sulfur which was readily detected in the IOPF and IOP. XRD and 57 Fe Mossbauer spectroscopy revealed crystalline hematite (α-Fe2O3) as the active phase in the IOPF, being amorphous ferrihydrite (5Fe2O3·9H2O) and lepidocrocite (γ-FeOOH) the plausible phases in the IOP, with no detectable phase in the IHG material. The nature of the iron (III) bearing catalyst influenced its dry adsorption performance, whereas the water presence and trickle bed hydrodynamics played an additional role in the wet adsorption mode.

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IV.1. INTRODUCTION

Most of the scientific efforts concerning the heterogeneous catalytic desulfurization of gas streams are aimed at sweetening coal gas and other fuel derivatives at high temperatures and pressures, due to the commercial value and nature of the processes carried out at these conditions. Conversely, the equivalent process for sulfur containing gases at low or ambient temperature and pressure is generally less attractive in terms of economic potential, and therefore research and development; this way, a wider variety of simpler, cheaper processes like adsorption on activated carbon (AC) or other porous materials, alkaline chemical scrubbing and biooxidation using microorganisms are applied as alternatives to the heterogeneous catalysis method.

The adsorption of H2S on AC, though, has been observed to produce sulfur oxides or elemental sulfur as byproducts, suggesting chemisorption phenomena occurring that consume the active phases, produce new species leaving the active phases unaltered, or both, just like in heterogeneous catalytic processes. Moreover, experiments carried out at room temperature and pressure have shown that depending on operating conditions such as pH and metal oxide speciation and content, the adsorption of H2S in AC and similar organic derived adsorbents may produce sulfur oxides or elemental sulfur at different ratios, and that the moisture content of the adsorbing material has a definite impact in the amount of incoming H2S that can be removed before observing the performance breakthrough, either in batch reactors or beds in continuous operations. These observations have encouraged the study of the surface physical chemistry properties of such AC in order to identify the active phases and to better understand the role of pH, moist and temperature on the activity and selectivity of the catalytic process.

In general, iron, zinc, copper, magnesium and calcium are some of the typical oxides that have been reported to exhibit activity towards H2S removal in AC beds or in supported solid packing [1]. In particular, it was reported that iron increased the catalytic activity of

AC and activated alumina during dynamic H2S adsorption experiments in moist air applications [1]. Two mechanisms are proposed that explain the catalytic oxidation of

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H2S in AC and on metal oxide containing materials in presence of water; the first one involves the dissociation of adsorbed H2S onto a water layer, producing sulfhydryl ions that later reacts with adsorbed oxygen. The second one involves the direct reaction of the metallic oxide with the H2S. The mechanisms can be described for each approach as follows in Equations (IV.1a) to (IV.1d) and (IV.2a) to (IV.2d), respectively:

H2S → H2Sads (IV.1a) - + H2Sads → HS ads + H (IV.1b) - - HS ads + Oads → Sads + OH (IV.1c) + - H + OH → H2O (IV.1d)

Also:

Fe3+ + HS- → 2Fe2+ + S + H+ (IV.2a) 2+ + 3+ - 4Fe + O2 + 2H → 4Fe + 2OH (IV.2b)

It is the second set of equations that makes the use of iron oxide phases particularly interesting since there is potential for recovery of the active ferric oxide by means of a reverse reaction in excess of oxygen and moist. These reactions, however, happen at slightly neutral to alkaline conditions; otherwise, inert byproducts such as iron disulfide (pyrite, marcasite) are formed. In addition, when these reactions happen at warm temperatures the iron sulfide indirectly produced in reaction (IV.2a) can form ferrous sulfate, which will react in excess of oxygen to produce sulfuric acid, lowering the pH and increasing the selectivity of the reaction producing inert pyrite or marcasite. Some ambiguity would be expected as to whether the species offering the ferric cation in reaction (IV.2a) is an oxide or a hydroxide.

The catalytic properties of these mixed-oxide or iron oxide rich AC can be incorporated to better packing materials that overcome some operating problems of the original AC, or improve their adsorbent capacity. A recent, typical example is the external impregnation of iron oxides or the selective replacement of aluminum or sodium for iron species in the

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core of highly porous zeolites. Moreover, ingredients can be incorporated that buffer the acidification of the adsorbent and inhibit the parallel reactions that produce inert iron disulfides, as well as hydrophilic materials that spread better the water in the catalytic surface. Also, wider pores can be made available that make water and the fouled gas more accessible to unreacted catalyst surface, which decreases the pore clogging due to water, for instance, in trickling beds packed with these materials. Furthermore, wider pores would decrease the chances of pore clogging due to solid sulfur accumulation.

This said, the present study aims at amplifying the research put forward in AC and iron rich zeolite, packed beds by evaluating three commercially available materials containing iron in oxide supports, not only from a surface chemistry perspective, but also from mass transfer rate limitations. The materials to be employed exhibit different surface area and unit sizes and will be tested in batch and dynamic experiments where H2S will be removed from airstreams at low humidity and in presence of excess water, covering the effect of catalyst characteristics such as surface area, oxide composition, support nature, iron oxidation and crystallinity, water pH and bed hydrodynamics on the catalytic activity of iron containing packing material towards the removal of H2S from airstreams at room temperature and pressure. Desulfurization of gases at these conditions can prove very useful in low energy applications such as environmental control of odorous gases from wastewater treatment plants and landfills, which can be easily accomplished in trickle beds where water is recirculated in a closed loop for minimization of water consumption.

IV.II. MATERIALS AND METHODS

IV.II.I. Adsorbent/catalysts characterization

Three different materials were tested as catalysts in this study, silicate supported, mixed iron/silica/alumina oxide, open-pore foams (IOPF), iron/silica oxide pellets (IOP) and iron hydroxide grains (IHG). The IOPF was tested in batch and continuous experiments, whereas IOP and IHG were tested in continuous mode, only. All materials are commercially available; however, their properties were unknown when received since

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either they represent proprietary packing elements (IOPF, IOP) or their properties were not handed on by the manufacturer (IHG). All three materials were used “as received” without further treatment. The geometric, physical and chemical properties of each catalyst prior to their treatment in H2S are summarized in Table IV.1.

IV.II.II. Batch experiments

A closed recirculating system was assembled to study the transient adsorption of the H2S in consideration onto IOPF units. Foam pieces were crushed into approximately eight units of 2.2-3.6 cm large and single ones were introduced, one at a time in each experiment, in a glass aspirator of 2000 mL. The air contained in the aspirator was recirculated through an electrical pump and its flow was maintained at 1 L/m by using a flowmeter equipped with a valve. A 5% v/v mixture of H2S in nitrogen (Matheson, IL) was injected in the system by using a 50 mL sealed syringe through a compressed tee fitting with septum located at the suction of the pump. The system temperature was monitored and observed to remain between 22 and 26 °C. The calculated initial relative humidity of the air ranged between 55-65% at room temperature. No water was supplied in the system, or its humidity controlled, either.

IV.II.III. Continuous experiments

A schematic of the experimental set-up where the continuous tests were carried out is shown in Figure IV.1. Compressed air (room temperature and pressure, 0.1% relative humidity, 30 ppmV H2O) from the lab air piping system entered a transparent PVC packed bed reactor (90 cm length, 10 cm diameter) filled up to a height of 60 cm (bed porosity of 53%) with IOPF units of around 2.20 to 3.60 cm in side length, with macroporosity of 69%, or with IOP units with a particle size of 0.20-0.35 cm up to a bed height of 9 cm (bed porosity of 39%). The Empty Bed Residence Time (EBRT) of the air stream in the PVC system was maintained at 60 sec for the IOPF bed, and at 9 sec for the IOP bed. IHG particles were tested in a polycarbonate column with 1.2 cm in diameter, packed with particles having a size of up to 0.10-0.20 cm, operating at an EBRT between

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1.7 and 5.1 sec. For all tests, the same H2S source as that of the batch experiments was used, being diluted with the incoming lab air at concentrations up to 280 ppmv. For the IOPF and IOP beds, experiments were carried out on a “dry” basis (air containing the initial lab supply relative humidity of 0.1%) and a “wet” basis, in which water was trickled down the bed from the column top, counter currently with respect to the air stream at a flux of 0.061 GPM/ft2 (0.0025 L/min/cm2) in a closed loop; make up water was fed as appropriate every 5-7 days to compensate for the evaporation into the dry air.

Sensor and Data Acquisition Mass Flow Controller

Biofilter

Recycling pump Hydrogen Sulfide

Compressed Air

Nutrient Tank

Figure IV.1. Schematic of the adsorption used in the present study for the removal of H2S polluted airstreams in beds packed with IOPF and IOP materials.

Since no water could be trickled down for the IHG bed, runs were done with as received, wet IHG (IHGw, 14% w/w water content) and dry IHG (IHGd, dried overnight at 105°C).

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Table IV.1. Selected physicochemical and operation properties of the raw and spent (dry and wet) catalysts used in this study for the H2S adsorption over airstreams.

IOPF raw, IOPF cont., IOPF cont., IOP cont., IOP cont., Property/Sample IHGd IHGw batch dry wet dry wet Sample amount [g] 6.18b 1160 1160 497 497 1 and 5 1 and 5 12.99-27.55c Initial surface iron content determined with EDS [%w/w] 1.7-4.6 1.7-4.6 1.7-4.6 3.4-3.6 3.4-3.6 57.5-67.3 57.5-67.3

Bed height [cm] - 60.96 60.96 9.21 9.21 0.6-3.0 0.6-3.0 3 Bed volume [cm ] - 4864 4864 735 735 0.80-3.98 0.80-3.98 Bed porosity [%] - 47 47 39 39 35 35 Average unit sample size [cm/pellet, cm/grain, cm/foam piece] 2.2-3.6 2.2-3.6 2.2-3.6 0.20-0.35 0.20-0.35 0.10-0.20 0.10-0.20

Average solid macropore size [µm] 525 525 525 - - - - Average solid pore size (adsorption-desorption) d 23-29 - - N/A N/A N/A N/A using BJH/N2 adsorption [nm] Average solid surface area (adsorption-desorption) 2 d 5.13-6.31 - - 6.51 - 187.59 - using BJH/N2 adsorption [m /g]

Sample macroporosity [%] 69 69 69 - - - - Intrinsic solid moisture content [w/w %] 12a 12a - 8e - 0 14e Gas phase EBRT [sec] 300 60 60 9 9 1.7-5.1 2.5-5.1 2 Water flow rate during wet experiments [GPM/ft ] - - 0.61 - 0.61 - - b Inlet airstream H2S concentration [µmol/L] 8.93-23.00 4.47-12.50 0.45-4.24 4.47 0.89-5.58 8.93 8.93 5.58-13.40c b g Inlet airstream H2S concentration [ppmv] 200-515 100-280 10-95 100 20-125 200 200 125-300c f Inlet airstream H2O concentration [µmol/L] 1.34 1.34 1.34 1.34 1.34 1.34 1.34 a: after calcination at 550°C for 4 hours. b: saturation experiments. c: one pass experiments. d: particle size smaller than 150 µm. e: after drying at 125 °C on air overnight. f: calculated at 0.1% relative humidity and room temperature and pressure. g: media had been used for three months for dry bed experiments. N/A: Not available.

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Sampling ports were connected to solenoid valves wired to an electronic board that reads an algorithm written in LabView® 7.1 that allows for controlled sampling. The software stores the data and shows them continuously at customized intervals. These and other experimental conditions are partially summarized in Table IV.1.

IV.II.IV. Analytical and quantification

The BET surface area for all the solids was determined by means of nitrogen adsorption at 77 K on 0-150 µm adsorber particles. Scanning Electron Microscopy (SEM) was used to observe the different phases of the solid adsorber, whereas their elemental surface composition was measured with Energy Dispersive X-ray Spectroscopy (EDS). In addition, 57Fe Mossbauer Spectroscopy at 77 K and FT-Raman Spectroscopy (FT- Raman) were employed to analyze the physicochemical state of the iron and sulfur species in the materials. X-ray Diffraction (XRD) with a copper anode (wavelength:

1.5406 Å) was used to analyze the crystal structure of the materials. H2S concentrations were measured in real time by means of an inline electrochemical sensor (iTrans,

Industrial Scientific Corp., PA) able to read concentrations of up to 500 ppmv at an inlet flow of 0.8-1.0 STPL/hr. The IOPF macropore size and PPI were determined by an imaging analysis software (ImagePro 4.0, MediaCybernetics, MD) using a light microscope before the continuous adsorption experiments using this packing material.

IV.III. RESULTS

IV.III.I. Preliminary catalyst characterization

Preliminary characterization of the composition of the surface materials as shown in Tables IV.1 and IV.2 reveals that the IOPF might be composed of iron oxides and hydroxides supported in alumina and silica or alumina-silicate frames, just like in previous works where such metal oxides where impregnated or embedded in zeolite like supports treating H2S in dry and moist airstreams [2, 3] and sulfide rich waters [4] with promising results.

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Table IV.2. Elemental composition and performance of the “as received” and spent materials tested before and after treatment with H2S in “dry” and “wet” conditions as shown in Table IV.1.

Property/Sample IOPF IOP IHG As Spent Spent As Spent Spent As Spent Spent

received “dry” “wet” received “dry” “wet” received “dry” “wet” O: 61.2 - 19.2 37.0 - 43.8 30.0 - - Fe: 4.8 - 1.1 3.9 - 3.4 70.0 - - Al: 11.1 - 0 0.8 - 4.2 - - - Average, carbon free, surface Si: 11.8 - 1.3 6.4 - 47.6 - - - sample speciation determined Na: 11.1 - 0 25.4 - 0 - - - with EDS [% w/w] Cl: 0 - 0 26.5 - 0 - - - S: 0 - 77.7 0 - 1.0 - - -

a a Adsorption capacity [gcatalyst/gH2S] - 127.9 42.9 - 23466 22.9 8.18 11.44 (36.9)c 8.13b 9.12b

Maximum observed instantaneous - - 8.67 - - 46.43 - - - 3 elimination capacity [gH2S/m bed/hr]

Operation time [hr] - 266 1104 - 1.3 1072 - 42a 42a (625)c 95b 95b

Breakthrough at shut down - 75 0 - 85 71 - 60a 40a [% initial concentration] 59b 92b a: 1 gram FeOOH sample. b: 5 gram FeOOH sample. c: values at which removal efficiency was above 90%.

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The surface elemental characterization was determined in a carbon-free basis; this is, the concentrations were normalized assuming no carbon is included in the catalysts. Even though some carbon is expected to exist in the materials due to the binding polymers used to form the foam backbones in the IOPF, from adsorbed CO2 from air dissolved in the intrinsic water of the materials, and from the alkaline buffers, it is likely that a considerable contribution to the carbon concentration is accountable to the carbon tapes used to hold the samples on the apparatus when being struck by the electron beams.

On the IOP material, however, the aluminum concentration is very low, which leads to think the iron oxides and hydroxides are supported just in silica rather than in zeolite-like frames. Of particular interest is the fact that on this material almost 50% of the concentration is made up of sodium and chloride, which might be an indication of an attempt to modify a precursor alumina-silicate with sodium, or the use of sodium chloride in the preparation of the proprietary support with other purposes. The BET surface areas of the as received IOPF and IOP are in agreement with similar iron oxide catalysts supported on alumina and alumina-silicates reported elsewhere [3, 5]. Also, the surface area of the IHG is in agreement with that reported on amorphous iron hydroxide [6].

Even though silica is supposed to exhibit a high surface area, and that the incorporation of alumina increases even further such values, the inclusion of iron species either at the surface or in the core of the silica or alumina-silica supports reduces the latter microporosity considerably [2]. These results can be explained in terms of the crystal dimensions of the metal oxides. Natural variants of minerals made only of these pure oxides, dry or hydrated, have unit cell volumes averaging 263, 662 and 2256 Å3 for iron, aluminum and silicon oxides and hydroxides, respectively. Thus, bigger alumina backbones increase the pore size of the catalyst when in the frame; however, if smaller iron oxide crystals are finely widespread inside the pores, then this can be filled up with the consequent reduction in pore size.

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IV.III.II. Dry continuous operation of packed beds

Table IV.2 summarizes the operation conditions and design characteristics of the continuous tests where the removal of H2S from airstreams in beds packed with IOPF, IOP, IHGd and IHGw were tested. Since the materials were meant to be assessed as received in order to determine their suitability in real packed bed unit processes, such materials exhibited a wide range in their properties, principally, packing and pore size, EBRT, and elemental surface composition. In particular, the use of water trickled down the bed was not possible with the IHGd and IHGw grains due to their little bed void space and the likeliness of clogging, high pressure drop and fluidization of the bed had water been supplied this way. Instead, the IHGw was tested only using its intrinsic water content of 14% w/w. Due to the high content of iron in the IHGd/w adsorbent, the latter were both evaluated using 1 g and 5 g of the material in two separate tests. Results for the dry and wet continuous tests are shown in Figures IV.2a to IV.2f for the IOPF, IOP, IHGd and IHGw adsorbents.

The dry adsorption capacity of the IOPF bed is clearly superior than that of IOP in terms of operation time before breakthrough was achieved (250 hr in IOPF compared to 1.2 hr in IOP) for inlet H2S loads in the same order of magnitude. More interestingly, the adsorption of H2S in the IOPF was irreversible and no pollutant was detected during the course of the operation when desorption tests were carried out twice, once after 122 hr during one hour, and another after the reactor shut down for another hour. Desorption of

H2S previously adsorbed on the IOP, conversely, yielded high detectable concentrations in the outlet gas. The maximum elimination capacity (EC) for these two dry systems reveals a difference of two folds for the IOPF compared to the IOP, as indicated in Table

IV.2. The dry adsorption of H2S in IHGd, both for 1 g and 5 g samples, occurred irreversibly, and 15 times higher EC were attained in this adsorbents compared to IOPF even though the removal efficiency decreased at similar levels in both materials after around the same elapsed time (~40-50% removal efficiency after 60 hr).

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0.12 100 0.04 100 A 0.10 B 80 80 0.03 0.08

S/kgbed/hr]

S/kgbed/hr] 2 60 2 60 0.06 0.02 40 40 0.04

S load [gH S load

2 2 0.01

Inlet H S load efficiency [%]Removal

20 efficiency [%]Removal 0.02 2 Inlet H2S 20 Removal efficiency Removal efficiency

Inlet H

Inlet H 0.00 0 0.00 0 0 50 100 150 200 250 0 200 400 600 800 1000 Elapsed time [hr] Elapsed time [hr]

0.12 100 0.12 100 C D 0.10 0.10 80 80

0.08 0.08

S/kgbed/hr] S/kgbed/hr]

2 60 2 60 0.06 Inlet H2S load 0.06 Removal efficiency 40 40 0.04 0.04

S load [gH S load [gH S load

2 2

20 efficiency [%]Removal 20 efficiency [%]Removal 0.02 0.02 Inlet H2S load Removal efficiency

Inlet H Inlet H 0.00 0 0.00 0 0.0 0.2 0.4 0.6 0.8 1.0 1.2 0 200 400 600 800 1000 Elapsed time [hr] Elapsed time [hr]

3.0 100 5.6 100 E F dry bed 5.2 2.5 80 80

4.8

S/kgbed/hr] S/kgbed/hr]

2 2.0 60 2 60 4.4

1.5 40 humid bed 40 Removal efficiency humid bed 4.0 Inlet H2S load

S load [gH S load [gH

2 Removal efficiency dry bed 2 Removal efficiency humid bed

1.0 Inlet H S 20 Removal efficiency [%] 20 Removal efficiency [%] 2 3.6 Removal efficiency dry bed

Inlet H Inlet H 0.5 0 3.2 0 0 10 20 30 40 50 60 0 10 20 30 40 50 60 Elapsed time [hr] Elapsed time [hr]

Interestingly, the iron content in the IHGd is around 15 times higher than in the IOPF, which would lead to think that the absolute iron content is responsible for the adsorbent performance in terms of EC. Data for the equilibrium isotherms obtained from the continuous operation is shown in Figures IV.3a to IV.3d, where monolayer like coverage is observed in the IOPF and IHGd and IHGw (5 g samples), in agreement with the irreversibility of adsorption, and a type II isotherm for the silica supported iron oxide

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catalyst. Figure IV.3b shows a hysteresis loop atypical from real physisorption systems with condensation of mesopores, due mainly to the irreversibility of the adsorption after the chemical reaction between some of the H2S with the iron in the IOP. Indeed, after two consecutive adsorption/desorption runs, approximately 47% of the adsorbed H2S was desorbed before the outlet concentration of the dry IOP bed reaches zero (data not shown).

5 0.04 B 4 A adsorption 0.03 desorption

3 0.02

S/gcatalyst]

2 2 2 IOPF

[mgH [mgH 0.01

S concentration in solid in S concentration

S concentration in solid

2 1

0 0.00 0 20 40 60 80 100 120 140 0 20 40 60 80 -6 -6 H2S partial pressure [10 Pa/Pa] H2S partial pressure [10 Pa/Pa]

120 120

100 C 100 D

80 IHGd [1 g] 80 IHGw [1 g] IHGd [5 g] 60 60 IHGw [5 g]

S/gcatalyst] S/gcatalyst]

2 2 40 40

[mgH [mgH

S concentration in solid in S concentration solid in S concentration 2 20 2 20

H H

0 0 0 20 40 60 80 100 120 140 0 20 40 60 80 100 120 140 -6 -6 H2S partial pressure [10 Pa/Pa] H2S partial pressure [10 Pa/Pa]

Figure IV.3. Adsorption and desorption isotherms (where applicable) at STP conditions for H2S on different iron containing materials. A: IOPF; B: IOP; C: IHG (1 g sample); D: IHG (5 g sample). Desorption isotherms were possible only in IOP packing. Other materials did not exhibit desorption characteristics. Operation conditions and isotherm fittings in Tables IV.1 to IV.3.

Thus, the H2S uptake capacity of the IOP is higher than if just physisorption occurred. Finally, the shape of the isotherms for the 1 g IHGd and IHGw is a consequence of the little amount of sample in the reactor, for which gas bypassing was a problem and little adsorption attained. This sample, however, exhibited irreversible H2S adsorption, just like

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in 5 g samples. For future reference, the isotherms for the IOPF, IGHw and IGHd were fitted to Langmuir, a new exponential function purely empirical, and Freundlich isotherm curves, respectively, being these the ones that best fitted the corresponding materials. Results for this fitting are summarized in Table IV.3. Attempts to fit of the data obtained in the IOP to a BET isotherm type did not yield positive results, and underpredicted the adsorbate in the solid phase.

Table IV.3. Isotherm data fitting for the iron containing materials in equilibrium with H2S at STP conditions. Data obtained from continuous dry bed experiments.

Isotherm type Equation Sample Parameters Correlation Langmuir 2 Csites ka / d R Csiteska / d Cg IV.3 Cs IOPF 4.6961 0.0723 0.98 1 ka / d Cg Empiric IV.4 Cs exp FOP 0.00017 0.0240 0.99 Cg

k f n IHGd Freundlich 56.9537 0.2671 0.97 n IV.5 C k C (5g sample) s f g IHGw 54.7390 0.2380 0.99 (5g sample)

Inspection of the isotherms shown in Figure IV.3 reveals that the IHG exhibits a constant increased H2S concentration in the solid catalyst of around 26 times that of the IOPF, being the surface area of the former 36 times higher than the latter. This is, the concentration of active sites in the IOPF is uniformly about 40% higher than that in the IHG, assuming the actives sites of both catalysts have the same activity and mechanism of removal towards the H2S. Since the iron content in the surface of the IHG is just 15% that of the IOPF, but its removal 2600% higher, it is concluded that the elemental iron concentration in the surface of the catalyst is not the solely variable affecting the performance of the different solids, nor is it only the surface area; indeed, even though the IOP contains a similar iron concentration than that of the IOPF, its H2S concentration

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in the solid catalyst is just 0.8-1.6% that of the IOPF, having both almost the same surface area.

IV.III.III. Wet continuous operation of packed beds

The wet operation of both the IOPF and IOP beds shows an enhanced performance and longer operation times at which the H2S concentration can be maintained well below 20% of the incoming values, with extended periods increasing 20 and 4000 times for the IOPF and IOP, respectively. The use of water augments significantly the EC of the beds, as reported elsewhere for systems using iron containing AC and zeolites operating at room temperature and pressure [7-9]. The water seems to activate the catalyst by either promoting the dissociation of H2S in the active phase where sulfhydryl ions react with adsorbed oxygen and produce elemental sulfur and water, as indicated in Equations (IV.1a) to (IV.1d), or by promoting the reversible reaction of iron sulfides in presence of oxygen at alkaline pH to recover the original hydroxides while producing elemental sulfur, as in Equations (IV.2a) and (IV.2b).

The wet IOP EC exceeded that of the IOPF by a factor of nearly 10%; however, the latter had already chemisorbed almost 25 grams of H2S for around 10 days, compared to a negligible adsorption capacity of the dry IOP. The material that better increased their performance by the water presence was the IOP. The IGHw exhibited little difference with respect to the IGHd, with the exception of the 1 g sample after 30 hr of operation where the removal of H2S seemed to recover. The intricate hydrodynamics of the 1 g sample bed might have played an important role in the performance recovery, since the 5 g sample exhibited a continuous tendency to decrease its EC, both for dry and wet materials.

Analysis of the elemental surface composition using EDS of randomly chosen packing units after breakthrough during the wet operation indicated a higher production of sulfur in the IOPF than in the IOP. As for other sulfur species and moieties produced, even though the electrochemical cell used to detect the H2S concentration in gas phase does

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not differentiate between H2S and SO2 and accounts for both as the same, the observed zero pollutant concentration in the outlet gas phase in wet operation indicates that all H2S was converted into elemental sulfur or soluble sulfur species. This way, the IOPF was more selective into converting the H2S to elemental sulfur than the IOP. Indeed, the pH of the recirculated water decreased from 9.74 to 8.80 in the IOPF and from 9.30 to 7.13 in the IOP beds, being the sharper decrease in the latter caused by a higher accumulation of soluble sulfur species, or less alkaline buffer ingredients in it. The transformation of

H2S to elemental sulfur or soluble sulfur species is congruent with experiments carried out in iron oxide catalysts at room temperature and pressure reported elsewhere, where

SO2 was hardly detected [8, 10]. Additionally, the better selectivity of IOPF towards elemental sulfur and better buffering capacity improves the H2S adsorption. Indeed, the equilibrium solubility of H2S in water increases with pH in a power of 15 for pH greater than 7.00 at room temperature [11]. Finally, the alkaline ingredients from the IOPF keep the alkaline character of the catalyst which favors the production of iron sulfides that can be converted back to iron oxides while producing sulfur as described before.

Even though the equilibrium solubility of H2S in water is as high as 124 g/L at pH of 8.50 [11] the alkaline adsorption of the gas in the IOPF or IOP beds was not responsible for the pollutant removal. Previous experiments carried out in beds packed with polyurethane foam cubes, with similar hydrodynamic and pollutant loads and at controlled and uncontrolled water pH, demonstrated that the removal efficiency would decrease rapidly to 60% in the pH~ 7-9 water system after just 4 hr, and it would start at just around 50% at pH~7 and plummet to around 20% after 12 hr. It is important to point out that the water is continuously recirculated, which minimizes its consumption, and small make-up volumes were needed only once on a weekly basis.

IV.III.IV. Final catalyst characterization

In order to better identify the active species and products of the adsorbents and produced sulfides, XRD analysis were performed in the as received and spent samples after

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treatment in the wet beds with H2S. Results for the XRD are shown in Figure IV.4 and Table IV.4.

Intensity [a.u.]

5 10 15 20 25 30 35 40 45 50 55 60 2 degrees

Figure IV.4. XRD spectra of the raw and spent IOPF and IOP materials after treatment with H2S in wet continuous operation. A: raw IOPF; B: spent IOPF; C: raw IOP; D: spent IOP. Short dashed lines represent the location of the three most intense peaks of sulfur standards, whereas the long dashed lines represent those of hematite (α-Fe2O3).

The crystallinity of the IOPF is evident, whereas the IOP exhibits an amorphous structure. The XRD spectra of the IHGd and IHGw are not included since only a baseline type of curve was observed, indicating high dispersion and almost inexistent order. Several iron oxides and hydroxides were matched against existing libraries for the IOPF and IOP by corresponding at least two peaks at the broad specified precision. Only hematite was clearly detected in the IOPF, whereas several possible mineral systems consisting of Fe/Al/Si oxides in the IOPF, and Fe/Si oxides in the IOP were also tallied.

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As for the products, elemental sulfur was clearly detected as well, indicating the oxidation of H2S into this solid species as described above. The spent spectra coincided within the specified precision (θ ± 0.05°) with iron sulfides and sulfates as shown in Table IV.4; however, either these iron sulfur species exhibited low peak intensity in the spectra, or their peaks were overlapped with those of sulfur and/or silica (iron sulfates). Sulfur was also easily detected by comparing the FT-Raman spectra of the spent IOPF with a standard, with matching peaks at wavelengths of [cm-1]: 46.46, 78.24, 149.35, 215.76, 243.45, 435.99 and 469.75.

Since crystallographic experiments were insufficient to determine the iron species in the solid catalysts, 57Fe Mossbauer spectra at 77 K was applied on the solids to aid on this task, as shown in Figures IV.5a and IV.5b. The asymmetric character of the 57Fe Mossbauer data for the IOPF and the little resolution for the IHG demonstrates that there exists several iron containing components in the former, whereas in the latter hardy any crystallinity is present. The location of the 57Fe Mossbauer peaks for several known components are also shown in the Figures, aiming at comparing qualitatively the experimental ones with those of the known reported values. Unless stated, the spectra for the known samples was determined at temperatures similar to that of the experimental data; however, the availability of this information is scarce and other low temperature spectra needed be used instead. This temperature difference could cause a significant divergence in the location of the peaks, since the spectra is widely known to be dependent, among other variables, in the sample temperature during the data acquisition [12]. Sophisticated identification of species using 57Fe Mossbauer spectra requires the quantification of properties such as the isomer shift, quadrupole splitting and other energetic parameters specific to the technique itself [12]. Whenever obtained spectra indicates a combination of species such as in the IOPF in Figure IV.5a, the quantification of the abovementioned parameters require assuming the presence of some expected structures in order to deconvolute the final spectra. Since scarce information on the as received materials is available, instead of determining such parameters and comparing their magnitude with known species, the qualitative assessment of the position of the

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Iron Sample Iron silicates Sulfur Iron sulfides Iron sulfates Silica Others (hydr)oxides

IOPF “as Fe (Fe2SiO4)0.456(Fe3O4)0.454 SiO2 received” (H, P) (O, BC) (H, P) Fe2O3 Fe2SiO4 (R, RC) (O, P) FeOOH Fe5.36Si0.64O8 (O, EC) (O)

IOPF Fe (Fe2SiO4)0.456(Fe3O4)0.454 S Fe0.95S1.05 (H, P) FeH(SO4)2·4H2O (O, P) SiO2 spent (H, P) (O, BC) (O, FC) Fe1.05S0.95 (H, P) FeSO4·H2O (M, EC) (H, P) Fe2O3 Fe2SiO4 (O, P) S (O) Fe3S4 (C) SiO2 (R, RC) FeSiO3 (O, P) S (M) Fe7S8 (H, P) (T, P) FeO S8O FeS (H, P) (C, FC) (O, P) FeS (O) FeS2 (O, P) FeS2 (O, P) ISP “as FeSiO3 (O, P) NaCl (C, FC) received” +2 +3 ISP spent Fe2O3 Fe2SiO4 (O, P) S FeS (H) Fe 1.33Fe 0.67(SO4)2(OH)0.67·H2O SiO2 Al2(SiO4)O (O, P) (R, RC) FeSiO3 (O, P) (O, FC) FeS2 (O, P) (M) (T, P) α-Al2S3 (H) Fe(OH)3 FeSO4·7H2O (M, P) SiO2 γ-Al2S3 (R, RC) (O, BC) Fe3(SO4)4·14H2O (A, P) (H, P) Al2(SO4)(OH)4·5H2O (M, P) FeH(SO4)2·4H2O (O, P) SiO2 Al13Fe4 (R, RC) FeSO4·H2O (M, EC) (O, EC)

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1.00

0.95 Ferr

Lep

1.00

0.95 Ferr

Pyr 1.00 B

Relative absorbance [1] absorbance Relative

0.95 Hem

1.00

0.95 -10 -8 -6 -4 -2 0 2 4 6 8 10 velocity [mm/sec]

Figure IV.5a. 57Fe Mossbauer spectra at 77 K of the packing materials before and after treatment with H2S in the wet mode continuous operation. A: raw IOPF; B: spent IOPF; C: raw IOP; D: spent IOP. Standards: Hem: hematite @ 6 K [13]; Pyr: pyrite @ 6 K [13]; Lep: lepidocrocite @ 140 K [14]; Ferr: ferrihydrite @ 70 K [14] and 75 K [15]. peaks of the spectra for some known samples [13-15] is used to approximate the detection of the iron species as an alternative. Inspection of the spectra in Figure IV.5a indicates the plausible presence of hematite in the as received IOPF material, and of amorphous lepidocrocite (γ-FeOOH) and/or a poorly ordered ferrihydrite (5Fe2O3·9H2O) in the as received IOP. Furthermore, both the reported orange color of the as received

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IOP, and the dark red of the IOPF [16] are in agreement with the matching iron species as indicated in Figure IV.5a. However, even though the orange lepidocrocite is believed to be present in the IOP in higher quantities than the reddish ferrihydrite due to the qualitative inspection of the surface color of the catalyst, the former is thermodynamically and kinetically more difficult to obtain than the reddish-brown ferrihydrite. Furthermore, since both minerals exhibit a Neel temperature (temperature at which the ferromagnetic behavior changes) in the range of the temperatures used to obtain the 57Fe Mossbauer spectral data [16] the location of the peaks shown in Figure IV.5a should be thought of as qualitative references instead of definite standards.

1.00

G 0.95

0.90 1.00

0.95 F

0.90

Relative absorbance [1] absorbance Relative 1.00

0.90 E 0.80

0.70 -10 -8 -6 -4 -2 0 2 4 6 8 10 velocity [mm/sec]

Figure IV.5b. 57Fe Mossbauer spectra at 77 K of the packing materials before and after treatment with H2S in the wet mode continuous operation. E: raw IHG; F: spent IHGd; G: spent IHGw.

Spent samples, on the other hand, show the increase in the intensity of a doublet in the mid section of the spectra for both IOPF and IOP, which is congruent with the formation of pyrite and other iron (II) sulfate species [13]. In addition, ferrihydrite has also shown

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magnetic splitting at similar velocities and temperatures [15] as indicated in Figure IV.5a, typical of iron (III) in octahedral coordination [15] which suggests hydration of the hematite crystals. However, due to the unlikely conversion of hematite to ferrihydrite from a thermodynamic standpoint, and the highly oxidizing potential and alkaline pH of the water saturated with oxygen in the trickle bed, both the presence of ferrihydrite and pyrite, as well as other reduced forms of iron (II) sulfides and sulfates (see Table IV.4) as products of the H2S oxidation in the IOPF and IOP are questionable.

Even though the assessment of 57Fe Mossbauer and XRD analyses are not indicates that since both IOPF and IOP bear oxidized iron (III), this variable solely is not responsible for the performance of the catalysts towards the dry adsorption of H2S, but instead, that the crystallinity and solid structure of the iron might have played the most important role in such performance, provided that no differences in the mass transfer resistances in both catalysts were present during the dry experiments. Thus, assuming that the adsorption of

H2S in the solid bearing materials is surface controlled instead of pore controlled, as previously suggested [2], or that mass transfer resistances had the same quantitative impact in both IOPF and IOP dry adsorption of H2S, then the trigonal crystal, anhydrous iron (III) oxide encountered in hematite is more active towards the adsorption and oxidation of H2S than the poorly crystallized, hydrated lepidocrocite (orthorhombic system) and ferrihydrite (trigonal system). When an excess of water is present in the system during wet operation, nonetheless, the crystallinity and intrinsic water content of the catalysts seem no to produce any difference in performance. Thus, free water available for adsorption and dissociation of species could potentially be seen as a limiting reactant in the dry operation of the beds, and the kinetic reaction can be thought of as exhibiting a sigmoidal shape with the concentration of crystal iron species being neglected when that of water is predominant (pseudo zero order).

Finally, an additional qualitative approach towards a deeper characterization of the raw and spent catalysts is possible by inspection of their SEM micrographs, as shown in Figures IV.6. The micrographs show the loss of several heterogeneous phases from the IOPF and IOP in the as received state to more homogeneous superficial structures, with

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the peculiarity that most of the supported phases in the IOP seem to have been washed away in the wet operation as none of the heterogeneous phases previously distinguishable are there present in the spent catalyst. In addition, the spherical polymeric binders in the IOPF (top left, bright circles) were removed and a bulky formation of sulfur solids accumulates in the surface of the material after wet treatment with H2S (top right.)

Figure IV.6. SEM micrographs of the raw and spent (after wet operation) IOPF and IOP solids. Top left: IOPF raw; top right: IOPF spent (bar: 100 μm). Center left: IOPF raw; center right: IOPF spent (bar: 10 μm). Bottom left: IOP raw; bottom right: IOP spent (bar: 200 μm).

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IV.III.V. Kinetics of the H2S adsorption on IOPF

The adsorption kinetics of the H2S in IOPF was measured using the batch protocol described in the materials and methods section. Experiments were carried out in two different modes; in “saturation” mode where the same solid sample was spiked with high concentration up to 500 ppmv of H2S 18 times, as shown in Figure IV.7, and in a “single run” mode where six different samples of different sizes where spiked only once with three different initial concentrations, namely 140, 220 and 310 ppmv.

600

500

400

300

200

H2Sconcentration [ppmv] 100

0 0 5 10 15 20 25 Elapsed time [hr]

Figure IV.7. Adsorption performance of a IOPF sample (6.18 g) saturated with and atmosphere of H2S at STP conditions. Peaks represent spikes of H2S. Continuous experiments carried out in the same sample.

The water content in the recirculating air was initially 100-120 ppmm (55-65% RH) at room temperature and pressure, which yields a molar ratio of water to H2S of around

1:0.75 for an initial H2S concentration of 500 ppmv. Since the material had been stored for several days in an open container, it was assumed to be already in equilibrium with the room moisture before the experiments. The adsorption of H2S onto the IOPF samples in followed a zero order reaction path at the initial stages of the experiment for around 30 minutes, to a first order path for the following 30 minutes when the recirculating gas concentration had decreased to values less than 10% of the initial concentration, both in the first 12 saturation and single run experiments. However, subsequent spikes of similar

134

initial concentrations (200-300 ppmv) resulted in longer time for removal of the H2S, up to twice as much to attain the 10% initial concentration in the remaining 6 saturation experiments, thus indicating the exhaustion of active sites of the adsorption of the gas in the saturated IOPF sample, or the depletion of water or oxygen in the recirculating closed system. Unfortunately, neither the RH nor the oxygen concentration of the recirculating gas was monitored, and their effect in the adsorption kinetics could not be assessed. It is expected that both variables affect the adsorption of H2S in the iron material, as already observed elsewhere [7]. In a similar study [7] coal fly ash was spiked several times at conditions analogous to those carried out in the saturation mode, with a similar rate of adsorption shape and a reduction in the adsorption speed for complete removal of H2S after several spikes. The adsorption rates measured during the zero order path attained values of 76.22 ppmv/min/gFe, which compares less favourably yet in the same order of magnitud than those carried out in the fly coal ash [7] where such values reached the range 186-293 ppmv/min/gFe. However, the surface area and pH of the fly coal ash used in such study was 49% and 23% higher, respectively, than the IOPF samples used in this study.

The proposed adsorption rate follows therefore a sigmoidal shape, as described in Equation IV.6:

dCg k1 Cg (IV.6) dt k2 Cg

The calculated constants are shown in Figures IV.8a and IV.8b (solid circles) where the results for the saturated runs are also included for comparison (empty circles in the inner plots). It can be seen from the results shown on these Figures that the average kinetic constants decrease (or that the adsorption rate increase) with the initial pollutant concentration, even though the rate of adsorption does not change during the first minutes of the operation due to an excess of pollutant in the batch container. That the kinetic constants increase with the pollutant concentration in the reactor for a zero order reaction

135

is an indication of mass transfer limitations in the solid surface of the catalyst or gas phase.

-1

/sec] 1

v 0

-2 /sec]

v -1

[ppm -2

[ppm

k -3 -3 -4 150 200 250 300 350 400 C [ppm ] Maximum velocity of adsorption velocity Maximum o v -4 120 140 160 180 200 220 240 260 280 300 320 Initial concentration Co [ppmv]

Figure IV.8a. Maximum velocity of adsorption k1 for the IOPF material under an atmosphere of H2S at STP conditions. Solid circles are single run experiments, whereas open circels are saturation runs.

1800

]

v 5000 1600 4000

[ppm 1400 ]

k 3000 1200 [ppm 2000

k 1000 1000

800 0 150 200 250 300 350 400 600 Co [ppmv] 400

200

Half saturation constant constant saturation Half 0 120 140 160 180 200 220 240 260 280 300 320 Initial concentration Co [ppmv]

Figure IV.8b. Half saturation constant k 2 for the IOPF material under an atmosphere of H2S at STP conditions. Solid circles are single run experiments, whereas open circels are saturation runs.

When the sample used in saturation tests is considered, however, maxima (minimum rate of adsorption) is observed in the mid of the initial concentration range used. This

136

behavior, unlike being the consequence of some rearrangement of molecules or backwards reaction in the recovery of iron oxides, as indicated in previous sections, is more prone to be the caused of the saturation and consumption of active sites as the runs were carried out; particularly considering the results shown in Figure IV.7 where the adsorption rates decrease progressively whereas the initial concentration of such experiments was increased. The maximum velocity of adsorption k1 and the half saturation constant k 2 correlate with the initial H2S concentration in an exponential fashion, yet they failed to show a relationship with the mass of the catalyst or its surface area in a continuous way; this is, both coefficients did not show continuous increase or decresae of their magnitudes with the amount of catalyst used, which might be a consequence of the intricate geometry of the solids used for each experiment and the heterogeneity of the solid characteristics.

IV.III.V. Mass transfer limitations of the water layer in the wet operation of the IOPF and IOP beds

Assemment of the mass transfer resistance from the liquid layer in the trickle bed is carried out following the criteria suggested by Thomas [17]. In brief, whenever the concentration of the pollutant decreases at least 5% in the liquid layer, mass transfer resistances are to be considered, and in that case they become predominant if:

k k L L 1 (IV.8) dCg 1 1 dCg 1 H

d As Cl d As 0.95Cg

In lieu of the absence of a suitable correlation to determine the value of k L , the following expression is used for its calculation:

Dl k L (IV.9) l

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The liquid layer thickness L and the specific area of the IOPF foam As as well as other transport parameters for the system H2S-water are calculated following the formulas presented by Goncalves and Govind [18].

The effect of the water layer in the overall mass transfer process can be determined assuming a linear rate of removal along the trickle bed, for which the calculation of the left hand side of the Equation (IV.8) yields values of around 26.0 for the IOPF, and of 8.4 for the IOP when the gas concentration is at its maximum, i.e., at the bed inlet. However, as the reaction progresses along the reactor, the ratio between the rate of adsorption of

H2S divided by its absolute local concentration contained in the denominator of Equation (IV.8) may approach any value, even though both numbers in the ratio decrease to zero. Thus, it is not possible to determine without further knowledge of the concentration profile in the bed whether there were mass transfer limitations in the liquid side, as expressed in the criteria of the aforementioned equation. However, even if the liquid layer resulted in difussion limitations of the pollutant in the region of the bed near the exhaustion of the former, this would have happened at concentrations so low that the electrochemical sensor would not have detected them, as it is the case for the observed sustained complete removals.

IV.IV. CONCLUSIONS

The capability of three iron (oxy)(hydr)oxide materials towards the removal of H2S in airstreams was evaluated in both packed beds operating with scarcely moist air (30 ppmv

H2O, “dry”) and under an excess of water (“wet”) in a trickle bed column at room temperature and pressure conditions, for EBRT ranging in 1.7-60 sec and inlet H2S concentrations up to 280 ppmv. A macroporous material containing crystalline, anhydrous hematite exhibited more activity towards the adsorption and oxidation of H2S than amorphous lepidocrocite and ferrihydrite with the same iron surface concentration and surface area in the dry operation. The wet operation time before breakthrough was attained increased by up to three folds for the weakly active lepidocrocite and ferrihydrite, and at least up to four times for the hematite containing material, thus

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evidencing an enhancer property of the water towards the removal of the H2S. Elemental sulfur was detected in these spent materials, with no gaseous sulfur species, including

SO2, identified during the early operation of the trickle beds, indicating therefore that the removal of the H2S followed an already established oxidation mechanism enhanced by the presence of water and requiring oxygen and a non acidic pH for the recovery of the active iron sites. By evaluating the performance of an amorphous iron hydroxide material with a much higher iron content and surface area than that of the hematite and lepidocrocite/ferryhidrite materials, it was determined that the iron content and surface area themselves are not sufficient to determine the activity of the catalyst towards the H2S removal. Thus, in absence of external mass transfer limitations, the crystallinity and/or intrinsic presence of water in the iron oxide seems to play the most important role in the activity of H2S oxidation when an excess of external water is not provided.

IV.V. SYMBOLS

-1 As : Solid catalyst specific macroscopic contact area [m ]

Cg : Gas pollutant concentration [ppmv]

Cg,o : Gas pollutant concentration [ppmv]

Cl : Liquid pollutant concentration [ppmv]

-1 C s : Solid pollutant concentration [mgH2S·gcatalyst ] -1 Csites : Active sites concentration [mgH2S·gcatalyst ] 2 -1 Dl : Pollutant diffusivity in liquid [m ·sec ] H : Dimensionless Henry’s constant [1] -1 k1 : Maximum adsorption velocity [sec ] k 2 : Half saturation constant [ppmv]

-1 ka / d : adsorption-desorption kinetic constant [ppmv ]

-1 -n k f : Freundlich adsorption constant [mgH2S·gcatalyst ·ppmv ]

2 -1 k L : Overall mass transfer coefficient in the liquid phase [m ·sec ] n : Freundlich exponential constant [1]

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-1 : Empirical adsorption constant, Table IV.3 [mgH2S·gcatalyst ]

: Empirical exponential constant, Table IV.3 [ppmv]

l : Liquid layer thickness [m] : Space hydraulic time [sec]

IV.VI. REFERENCES

1. Bagreev, A., Bashkova, S., Locke, D. and T. Bandosz. (2001) “Sewage sludge- derived materials as efficient adsorbents for removal of hydrogen sulfide.” Environmental Science and Technology. 35(7) 1537-1543. 2. Nguyeh-Thanh, D., Block, K. and T. Bandosz. (2005) “Adsorption of hydrogen sulfide on montmorillonites modified with iron.” Chemosphere. 59(3) 343-353. 3. Truong, L. and N. Abatzoglou. (2005) “A H2S reactive adsorption process for the purification of biogas prior to its use as a bioenergy vector.” Biomass & Bioenergy. 29(2) 142-151. 4. Poulton, S., Krom, M., Van Rijn, J. and R. Raiswell. (2002) “The use of hydrous iron (III) oxides for the removal of hydrogen sulphide in aqueous systems.” Water Research. 36(4) 825-834. 5. Lee, E., Jung, K., Joo, O. and Y. Shul. (2005) “Support effects in catalytic wet oxidation of H2S to sulfur on supported iron oxide catalysts.” Applied Catalysis A: General. 284(1-2) 1-4. 6. Su, C, and Suarez, D. (2000) “Selenate and selenite sorption on iron oxides: an infrared and electrophoretic study.” Soil Science Society of America Journal. 64(1) 101-111. 7. Kastner, J., Das, K. and N. Melear. (2002) “Catalytic oxidation of gaseous reduced sulfur compounds using coal fly ash.” Journal of Hazardous Materials. 95(1-2) 81-90. 8. Ros, A., Montes-Moran, M., Fuente, E., Nevskaia, D. and M. Martin. (2006). “Dried sludges and sludge-based chars for H2S removal at low temperature: Influence of sewage sludge characteristics.” Environmental Science and Technology. 40(1) 302-309. 9. Primavera, A., Trovarelli, A., Andreussi, P. and G. Dolcetti. (1998) “The effect of water in the low-temperature catalytic oxidation of hydrogen sulfide to sulfur over activated carbon.” Applied Catalysis A: General. 173(2) 185-192. 10. Jung, K., Joo, O., Cho, S and S. Han. (2003) “Catalytic wet oxidation of H2S to sulfur on Fe/MgO catalyst.” Applied Catalysis A: General. 240(1-2) 235-241. 11. ASCE. (1989) “Sulfide in wastewater collection and treatment systems, manuals and reports on engineering practice” ASCE Manuals and Reports on Engineering Practice No. 69. 12. Murad, E. and J. Cashion. (2004) “Mossbauer spectroscopy of environmental materials and their industrial utilization.” Kluwer Academic Publishing. 1-411.

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13. Melchior, D., Wildeman, T. and D. Williamson. (1982). “Mossbauer investigation of the transformations of the iron minerals in oil-shale during retorting.” Fuel. 61(6) 516-522. 14. Dyar, D. Mount Holyoke College Mars Mineral Spectroscopy Database. Last accessed on 10/01/2007. http://www.mtholyoke.edu/courses/mdyar/database/index.shtml?group=hydrox 15. Childs, C. and J. Johnson. (1980) “Mossbauer spectra of proto-ferrihydrite at 77 K and 295 K, and a reappraisal of the possible presence of akaganeite in New Zealand soils.” Australian Journal of Soil Research. 18(2) 245-250. 16. Schwertmann, U. and R. Cornell. (1991) “Iron oxides in the laboratory: preparation and characterization.” Weinheim. 1-137. 17. Thomas, J.(1997) “Principles and practice of heterogeneous catalysis.” Weinheim. 1-659. 18. Goncalves J. and R. Govind. (2005) “Simulation of biotrickling filters using novel foams for treating odors and volatile compounds.” Proceedings of the 2005 AIChE National Meeting. Cincinnati, OH.

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CHAPTER V

INDIRECT H2S ABATEMENT USING BIOLOGICAL IRON OXIDATION BY ALICYCLOBACILLI ON IRON (III) FOAM, PACKED BIOREACTORS

SUMMARY

3 Airstreams polluted with H2S at inlet loads ranging from 2.39 to 40.92 gH2S/m bed/hr were treated in a biotrickling reactor packed with iron (III) bearing, open pore foam (IOPF) units, at Empty Bed Residence Times (EBRT) ranging from 20 to 60 sec over a period of 80 days. The media was seeded with sludge from a local water works facility. Removal efficiencies in excess of 80% were consistently observed along the operation of the reactor, with an average of 98%. Based on section performance, being a section one third of the bed length, observed Elimination Capacities (EC) reached up to 88.67 3 3 gH2S/m bed/hr and 72.04 gH2S/m bed/hr at section EBRT of 10 and 7 sec, respectively, 3 and 40.92 gH2S/m bed/hr on an overall bed basis. Denaturing Gel Gradient Electrophoresis (DGGE) was performed on the biomass collected in the packing after 80 days of operation service and their most predominant microorganisms were identified as belonging to the type Alicyclobacillus ssp., a ferrous iron oxidizing genus. The H2S adsorption characteristics of the packing in presence and absence of a recycling water flow stream were also evaluated and compared with the biological H2S removal of the reactor after exhausting the bed packing and before the biological operation started. It was found that the bed packing material has a fairly high H2S adsorption capacity which is enhanced in presence of water due to the release of alkalinity and the recovery of ferric oxide in presence of oxygen, yet it was concluded that such mechanisms were not responsible for the consistent removal of the pollutant along the reactor operation during the biological stage. The observed EC values compared much better than data reported on other packed bed reactors using biological iron oxidization to treat H2S airstreams directly or indirectly. This study constitutes the first one reported on the use of Alicyclobacilli for indirect desulfurization of airstreams. Finally, the system herein

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described offers the advantage of no bacterial acclimation time needed and production of solid sulfur than can be more easily disposed-off than sulfates.

V.I. INTRODUCTION

Air biofiltration is mediated through the direct consumption of the pollutants by the microorganisms as their sole or combined source of food and energy, or indirectly by means of other substances present in the system that react with the pollutants of interest, producing new chemical species that are afterwards utilized by the microorganisms. Biological oxidation of iron in presence of iron oxidizing microorganisms for the removal of H2S from airstreams is a typical example of the indirect abatement of such pollutant. In this case, ferric ions present in the system react with H2S carried in the airstreams, producing elemental sulfur and ferrous ions, which are subsequently oxidized by the microorganisms and converted back into the original ferric species.

Several studies have lately dealt with this process in an effort to determine the best operation and design conditions to maximize the speed and selectivity of the conversion of ferrous into ferric ions. In general, reactor configuration, packing material, nutrients nature and concentration, contact time between the phases, pH and initial ferrous ion concentrations are the variables that researchers pay attention the most [1-4]. These investigations, though, systematically focus on the kinetics of the ferric formation rather than the H2S elimination capacity of the system. As a consequence, only a few reports are available where either high rates of oxidation of ferrous ions are attained at the expense of high H2S removal capacities during short periods of time, or low H2S removal capacities at longer operation periods. Thus, long term services of these reactors can only be accomplished in hybrid, combined systems that involve more elaborated, auxiliary processes.

Recently, it was reported that the natural iron content present in lava rock was responsible for the abatement of H2S from airstreams in columns packed with such material, in presence of Thiobacillus ferrooxidans bacteria [5]. The acidic leaching of

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enduring iron ions from the rock and their conversion onto ferric ones in presence of the iron oxidizing microorganisms yielded a system capable of removing the inlet pollutant. The advantages of using a highly concentrated source of iron material as a support for the biomass as in the aforementioned case are obvious; first, a continuous supply of ferric ions can be achieved that react with longer lasting inlet H2S airstreams, and secondly, depending on the chemical nature of the support, the water being fed in the system can be buffered so that the absorption of the H2S is not lowered as more sulfate is accumulating. This way, when the kinetics of the ferrous ions oxidation is not favored, a portion of the ferric ions needed to react with the incoming fouled gas is supplied by the material.

The abiotic reactions happening in the system formed by iron oxides and H2S at different conditions of pH and room temperature are documented elsewhere [6-9]. Hematite

(Fe2O3) in presence of water reacts with H2S to produce elemental sulfur and iron sulfides as follows:

Fe2O3·3H2O + 3H2S → 2FeS + S + 6H2O (V.1)

Fe2O3·3H2O + 3H2S → Fe2S3 + 6H2O (V.2)

The production of troilite (FeS) is less likely to happen in natural waters with a high oxidation potential; i.e. water saturated with a stream of oxygen such as in superficial rivers or lakes. The iron (III) sulfide is uncommon in nature unless combined with others species, such as silver in argentopyrite (AgFe2S3). In presence of high concentrations of dissolved oxygen, the iron sulfides of the reactions described in Equations (V.1) and (V.2) further react to recover the original iron (III) hydrated phases:

2FeS + 6H2O+ 3O2 → 2Fe2O3·3H2O + 2S (V.3)

2Fe2S3 + 6H2O+ 3O2 → 2Fe2O3·3H2O + 6S (V.4)

The recovery of the hematite will happen at pH values slightly alkaline or higher, where otherwise soluble ferrous species would appear in the system. In a parallel set of

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interactions, the hematite further reacts with elemental sulfur to produce pyrite (FeS2) and greigite (Fe3S4):

4Fe2O3 + 12H2S → 2FeS2 + 2Fe3S4 + 12H2O (V.5)

These reactions are thought to no yield high amounts of the abovementioned iron sulfides, since both pyrite and greigite contain ferrous iron, which will only be significant in waters exhibiting low oxidation potential, such as groundwater, water with high concentrations of biomass or brines. Overall, the inorganic reactions involving hydrated hematite and H2S in presence of oxygen would be confined to the following simplified reaction:

HEMATITE 2H2S + O2 2S + 2H2O (V.6)

On the other hand, when a mixed culture of microorganisms is seeded in a system containing iron (hydr)oxide phases in contact with H2S, two different biological processes occur. First, sulfur oxidizing bacteria degrades the H2S releasing high amounts of bioconverted sulfates that increase the acidity in the media, which favors the leaching and maintenance of ferrous ions, specially at low concentrations of dissolved oxygen. The ferric oxide/hydroxide phases uncovered by the microbial community further react with the H2S at acidic conditions to release more ferrous ions:

2Fe2O3 + H2S + 7H2SO4 → 4FeS2O3 + 8H2O (V.7)

4Fe2O3 + H2S + 7H2SO4 → 8FeSO4 + 8H2O (V.8)

The second biological process occurs when the concentration of ferrous ions is sufficiently high, for which the microbial community growing in the media evolves to a predominant iron oxidizing one. Thus, the H2S removal is mediated indirectly by the production of ferric sulfide by means of the bacteria as follows:

4FeSO4 + 2H2SO4 + O2 + bacteria → 2Fe2(SO4)3 + 2H2O (V.9)

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Fe2(SO4)3 + H2S → 2FeSO4 + S + H2SO4 (V.10)

The activity of both types of microbial species, the sulfur and iron oxidizing ones, is enhanced at acidic pH. Thus, when these microorganisms are seeded in the system, reactions (V.1) and (V.2) are progressively replaced as the bacterial communities grow, and the direct biooxidation and/or indirect H2S reaction expressed in Equation (V.10) is responsible for the pollutant abatement. Since very acidic conditions are not favorable for the presence of ferric ions even for water in contact with the atmosphere, the H2S removal will depend greatly in the speed of conversion of ferrous into ferric ones by the iron oxidizing microorganisms whenever this species is the predominant in the bacterial community.

In order to determine experimentally the feasibility of operating equipment where these processes take place, the performance of a biotrickling filter packed with iron enriched, open pore foam (IOPF) units on the removal of H2S from airstreams at different concentrations and EBRT has been tested in the present study. Open pore foam units were used since it has been consistently reported that biofilters packed with similar porous structures exhibited better performance compared to other systems packed with natural or more compact synthetic media. Though most of these studies were carried out using polyurethane foam (PU) units, such material can not be easily modified to incorporate iron that can be leached out. Thus, foams made of a proprietary ceramic and cement combination were selected for the present study. Furthermore, this choice integrates the intrinsic characteristic of this material of releasing alkaline ingredients that buffer the water in the system.

In addition, the use of iron oxidizing bacteria to remove H2S indirectly by its absorption and following chemical reaction with ferric ions has the inherent advantage of producing elemental sulfur that can be disposed off in an easier, better controlled way. On the contrary, the use of sulfur oxidizing bacteria produces soluble sulfates that eventually become gaseous H2S after disposal in sinks where sulfate reducing bacteria are present. Both approaches, however, exhibit the advantage of low cost since they are carried out at

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room conditions, as well as minimal water consumption. Indeed, non biological adsorption and the biotrickling filter in a bed packed with IOPF can be equally operated with the water phase running in a closed loop, with make up water or nutrients, according to the case, being added to compensate their exhaustion. The production of sulfur, however, is counterproductive in that pores get clogged with the insoluble material, and an appropriate internal void space needs be present in the carrying media to avoid this problem, such as in open pore supports.

V.II. MATERIALS AND METHODS

V.II.I. Adsorption experiments

A schematic of the experimental set-up where the continuous tests were carried out is shown in Figure V.1. Compressed air (room temperature and pressure, 0.1% relative humidity, 30 ppmV H2O) from the lab air piping system entered a transparent PVC packed bed reactor (90 cm length, 10 cm diameter) filled up to a height of 60 cm (bed porosity of 53%) with IOPF units of around 2.20 to 3.60 cm in side length, with porosity of 69%. The Empty Bed Residence Time (EBRT) of the air stream in the system was maintained at 60 sec. A pressurized stream of 5% v/v H2S in nitrogen source (Matheson,

IL) was diluted with the incoming lab air at concentrations up to 280 ppmv. Experiments were carried out on a dry basis (air containing the initial lab supply relative humidity of 0.1%) and a wet basis, in which water was trickled down the bed from the column top, counter currently with respect to the air stream at a flux of 0.061 GPM/ft2 (0.0025 L/min/cm2) in a closed loop; make up water was fed as appropriate every 5-7 days to compensate for the evaporation into the dry air. The reactor was equipped with 4 sampling ports separated 30 cm from each other. Each sampling port is connected to a solenoid valve wired to an electronic board that reads an algorithm written in LabView® 7.1 that allows for controlled sampling. The software stores the data and shows them continuously at customized intervals.

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To determine the effect of the pH buffering capacity from the ceramic foam on the absorption of the H2S, the reactor was packed with inert PU foam cubes of 2.54 cm in side length, 8-12 pores per inch (PPI) and porosity of 98%, up to a height of 60 cm (bed porosity of 53%). Adsorption experiments in the PU packed bed were carried out on a wet basis at the same water flux of the ceramic foam experiments, but in two different modes; with and without pH control. In the former, the water contained in the recirculation tank was fed with a dilute solution of NaOH to adjust the pH to a value of around 7-9, being the latter the early pH of the water after some alkalinity is leached out the IOPF during wet absorption experiments on this material, in absence of H2S.

Sensor and Data Acquisition Mass Flow Controller

Biofilter

Recycling pump Hydrogen Sulfide

Compressed Air

Nutrient Tank

Figure V.1. Schematic of the adsorption and subsequent biotrickling filter used in the present study for the removal of H2S polluted airstreams. The column was operated as an adsorber both on a dry and wet basis first. Afterwards, the media was inoculated with activated sludge from a local water works facility to operate as a biotrickling filter.

V.II.II. Biological experiments

After exhaustion of the adsorptive capacity of the H2S on dry basis, and succeeding use of the spent media for another two months on wet basis adsorption, the media was seeded with a mixture containing 1 L of equal volumes of sludge from a secondary clarifier at the Cincinnati’s Mill Creek Waste Water Treatment Plant (WWTP) and a nutrient solution with the following composition [g compound per 1000 g of deionized water]:

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Na2HPO4, 1.2; KH2PO4, 1.8; MgSO47H2O, 0.1; (NH4)2SO4, 0.1; CaCL2, 0.03; MnSO4, 0.02 and agar, 1.5. Make up nutrients were fed in the tank at intervals of around 5-7 days as needed from operation conditions and evaporation rates, and the solid contents of the tank were never replaced from the tank. Thus, the sulfate and suspended or precipitated sulfur in the system were let increase as much as the nature of the reactor allowed it without major disruption of its performance. Inlet concentrations of H2S were varied to up to 235 ppmv and EBRT from 20 to 60 sec, which translates in inlet loads from to 2.39 3 to 40.92 gH2S/m bed/hr. Table V.1 depicts the design and operation properties of the IOPF packing media and reactor used during the adsorption and biological experiments carried out in the present study. Additionally, Figure V.2 shows the profile of the IOPF as taken by an optical image microscope.

Figure V.2. Microscopic picture depicting the IOPF structure and size of the macropores in the IOPF media. Bar represents 1 mm.

V.II.III. Analytical and quantification

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IOPF property Value IOPF property Value IOPF sample amount [g] 1187 Sample macroporosity [%] 69 Bed height [cm] 60.96 Intrinsic solid moisture content [w/w %] 14 Bed volume [cm3] 4864 Zeta potential [mV] (pH) -19.66 (8.63) Bed porosity [%] 47 Gas phase EBRT [sec] 60 Average unit sample size [m] 0.022-0.036 Nutrients/water flow rate [GPM/ft2] 0.61

Average solid macropore size [µm] 525 Inlet airstream H2S concentration [ppmv] 20-280

Average solid pore size (adsorption-desorption) using 23-29 Inlet airstream H2O concentration [ppmv] 30 BJH/N2 adsorption [nm] Average solid surface area (adsorption-desorption) 5.13-6.31 Average iron surface composition measured with EDS 4.6 2 using BJH/N2 adsorption [m /g] [%w/w]

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microscope. The zeta potential of foam particles of size up to 150 µm in aqueous solutions was determined with a Laser Doppler Velocimeter (BIC, Inc., NY) fitting the data with the classic Smoluchowski model. Environmental Scanning Electron Microscopy (ESEM), Energy Dispersive X-ray Spectroscopy (EDS) and Fourier Transformed Raman Spectroscopy (FT-Raman) were used to observe the biomass attached on the foams and to analyze the chemical composition of the foam surface and of some spots of interest, before and after the reaction. Adsorption isotherms of N2 at 77K on 0-150 µm IOPF particles were fitted to the BHJ model to determine their pore size and volume at the mesopore scale. Upon completion of the continuous experiments, Volatile Solids (VS) were determine in the reactor by weighting 3 samples of the collected biomass after drying them at 105°C overnight and 500°C for 4 hours, subsequently. Additionally, Denaturing Gradient Gel Electrophoresis (DGGE) of 16S rRNA biomass fragments was performed on samples containing a 50-50 biomass and sterile nutrients solution mixture. The biomass was retrieved by washing the foam cubes with deionized water, and letting the extracted biomass settle for about 2 hours. The sampling for the VS and DGGE analyses were performed on biomass as extracted from the foam without any further purification method, other than those required by DGGE protocols itself.

V.III. RESULTS AND DISCUSSION

V.III.I. Dry and wet basis adsorption experiments

The dry basis, room standard temperature and pressure (STP) conditions of the H2S adsorption capacity of the bed is depicted in Figure V.3. EBRT was maintained at 60 sec over the experiment, and the concentration of the pollutant at the inlet, outlet and two intermediate points was monitored (intermediate values not shown).

It is clear from the Figure that the dry basis adsorption capacity of the bed had reached a plateau before the experiments were halted after less than 300 hours of operation. After

122 hours, the supply of H2S was stopped to evaluate the desorption behavior of the

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media, resulting in a not detectable outlet H2S concentration for more than 60 minutes. This way, it was firstly assumed that the adsorption of the pollutant in the media was mediated by the formation of chemical bonds between the H2S and the active phases of the material, i.e., iron oxides and hydroxides. Further, the plateau reached an outlet concentration of around 75-80%, indicating that the reverse reaction described in Equations (V.3) and (V.4) are occurring, yet at a slower rate than the chemisorption rate; this way, a steady but low supply of recovered iron oxide is present in the system at any time after long term operation.

300 EBRT 60 sec 250

]

200

150

100

S concentration [ppm concentration S Inlet

2 H 50 Outlet

0 0 50 100 150 200 250 300 Elapsed time [hr]

Figure V.3. Long term operation of the dry basis adsorption bed packed with IOPF media

After the exhaustion of the media in the previous experiments, the reactor was operated on a wet basis by trickling down water in a closed loop. The results obtained are shown in

Figure V.4, which indicates that water had a significant impact in the removal of the H2S in the bed. Throughout 60 days of operation, the removal stayed at consistent values in the range 82-100% with an average of 98%. The pH of the water in the system varied from 9.74 to 8.80 during this period, which represents ideal enhancement conditions for the alkaline absorption of H2S into the water phase. FT-Raman analyses of the media after this operation revealed the presence of elemental sulfur, with peaks within 2% of measured pure sulfur samples with the same technique (spent IOPF peaks at wavelengths

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of [cm-1]: 46.46, 78.24, 149.35, 215.76, 243.45, 435.99 and 469.75). The sulfur had been qualitatively detected by observing the accumulation of yellowish solids on the surface of the IOPF, especially in the units located at the bottom of the bed, indicating that the reverse reaction described in Equations (V.3) and (V.4) had also occurred.

100

EBRT 60 sec

]

v 80

S concentration [ppm concentration S

H 20 Inlet Outlet

0 0 10 20 30 40 50 Elapsed time [d]

Figure V.4. Long term operation of the wet basis adsorption bed packed with IOPF media

In order to determine whether the results obtained during the wet basis adsorption were promoted due to alkaline adsorption or chemical reaction, experiments were carried out in a PU foam bed with comparable foam dimensions and inlet load conditions to the wet basis IOPF bed. Observed collected data are shown in Figures 5 and 6 for both the water pH controlled and uncontrolled systems. The pH of the former was kept in the range 7-9 during around 4 hours of operation, whereas in the latter the bed operated up to 15 hours. These results indicate that even a controlled pH scheme cannot achieve the removal attained in the wet basis IOPF, falling rapidly to around 60% after just 4 hours of operation.

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60 100

] 50

v 80 Inlet Outlet 40 Removal 60 30 EBRT 60 sec 40 20 pH controlled 7-9

S Concentration [ppm Concentration S pH 9.0 [%] efficiency removal S

2 0

20 H 10

0 0 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 Elapsed time [hr]

Figure V.5. Performance of the PU foam packed scrubber removing H2S with deionized water in a closed loop. Water pH was kept in the range 7-9 by adding diluted NaOH.

80 100

]

v 80 60

Inlet 60 Outlet EBRT 60 sec 40 Removal No pH control pH0 7.0 40

S concentration [ppm concentration S

S removal efficiency [%] efficiency removal S

2 20

20 H

0 0 0 2 4 6 8 10 12 14 16 Elapsed time [hr]

Figure V.6. Performance of the PU foam packed scrubber removing H2S with deionized water in a closed loop. Water pH was let decrease naturally through the experiments.

The uncontrolled pH bed did not even reach removal efficiencies higher than 40%, decreasing rapidly to around 25% after just 4 hours, period in which the pH declined from 9.55 to 6.18. Therefore, the goodness of the IOPF does rely neither on its alkaline

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absorption nor its dry adsorption capabilities, but on its capacity to enhance the H2S chemisorption in presence of water, mainly due to the conversion of spent iron oxides and hydroxides at alkaline conditions as depicted in Equations (V.3) and (V.4).

V.III.II. Biological operation

After the media was used during the wet basis adsorption experiments, sludge from a local water works facility was seeded in the bed at the conditions described in the materials and methods section. The liquid in the tank was replaced by the nutrient solution as explained previously. The EBRT of this system was varied between 20 and 60 sec and the inlet H2S concentration fluctuated between 20 and 235 ppmv in alternating modes; increasing and decreasing randomly but keeping the overall removal efficiency at target values of around 90%. Results for the long term operation of this bed are shown in Figure V.7. As seen in the Figure, removal efficiencies were consistently above 80% and had an average of 98%, even from the startup of the reactor. The removal efficiency decreased to a local minimum of around 80% after 42 days of operation when the EBRT had been decreased from 60 to 20 sec, for which the original value was retaken to achieve system stabilization. This steady condition lasted 36 more days until the system was reseeded with acclimatized activated sludge as indicated in the arrow in Figure V.7 due to a second decrease of the removal efficiency to values around 80%.

Figures 8 and 9 show the reactor performance in terms of the inlet H2S loads and total load removal, for the overall bed and each of the monitored sections, respectively. The 3 maximum overall observed EC was 40.92 gH2S/m bed/hr for an EBRT of just 20 sec, and 3 88.67 and 72.04 gH2S/m bed/hr on a section of one third of the bed length at EBRT of 10 and 7 sec, respectively. As shown in Figure V.8, the highest EC are attained at overall

EBRT of 20 sec, which correspond to inlet H2S concentrations in the range 130-150 ppmv. The removal efficiency at this EBRT remains within 95%, compared with values at

EBRT of 30 and 60 sec (inlet H2S concentration up to 235 ppmv) whose removal efficiencies fall outside this limit several times.

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300 100

250 Inlet

]

v Outlet 80 Removal 200 EBRT 60 150 40 100

S concentration [ppm concentration S

H 20 50

S removal efficiency [%] and EBRT [sec] EBRT and [%] efficiency removal S

0 0 H 0 10 20 30 40 50 60 70 80 Elapsed time [d]

Figure V.7. Long term performance of the IOPF biotrickling filter removing H2S at the specified conditions. Iron oxidizing bacteria from the genus Allicyclobacillus was determined to have colonized the IOPF media after 80 days of operation.

Even though such results include values observed when the overall performance of the reactor decreased and reseeding was necessary, and that Figure V.8 does not show a conclusive indication that the maximum EC was achieved in the entire bed, the results obtained at low EBRT indicate that the reactor is likely controlled by the kinetics of the

H2S adsorption, reaction of H2S with ferric ions and bioconversion of ferrous into ferric ions, rather than mass transfer limitations (i.e. axial convective time of the carrying gas compared to the H2S diffusive time into the water and biofilm layers on the IOPF). Also, it is clear from Figure V.8 that most of the pollutant removal is attained in the lowest section of the bed, with a sole contribution averaging 66%, compared with the second and third sections with 22% and 12%, respectively. The pH of the nutrient solution trickled down the bed decreased steadily from 9.02 to 2.36 during the operation of the reactor, as a consequence of the accumulation of soluble products from the H2S conversion.

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bed/hr]

S/m 30

20 EBRT 60 sec EBRT 30 sec

S removal [gH S removal 10 EBRT 20 sec

0 0 10 20 30 40 50

Inlet H S [gH S/m3bed/hr] 2 2

Figure V.8. Overall performance of the IOPF packed biotrickling filter removing H2S at different EBRT

30 4 EBRT 60 sec 25

2 20

bed/hr] 3 15 0 2 4

S/m

S removal per section per removal S

2 10

[gH Lower section 5 Middle section Top section

Abiotic H Abiotic

0 0 5 10 15 20 25 30

Inlet H S load [gH S/m3bed/hr] 2 2

Figure V.9. Relative H2S removal for the wet basis adsorption IOPF column for each of the sections of the bed. Each section equals one third of the total bed length.

157

140 10 EBRT 20-60 sec 120

5 100

80 0

bed/hr]

3 0 5 10

S/m 60

S removal per section per removal S

[gH 40 Lower section 20 Middle section

Biological H Biological Top section 0 0 20 40 60 80 100 120 140

Inlet H S load [gH S/m3bed/hr] 2 2

Figure V.10. Relative H2S removal for the biological adsorption IOPF column for each of the sections of the bed. Each section equals one third of the total bed length.

In order to better visualize the contribution of the biological removal of H2S compared to the removal due to wet chemisorption, the results showed on Figure V.6 were plotted for the three sections monitored as shown in Figure V.10. Comparison of Figures V.9 and V.10 reveals that while the maximum local section EC for the biological system reached 3 88.67 gH2S/m bed/hr as previously indicated, the maximum EC was just 23.09 3 gH2S/m bed/hr for the wet basis adsorption bed. Thus, the long term, high H2S EC results shown on Figures V.8 and V.9 can be attributed to the activity of the microorganism in the IOPF rather than other adsorptive processes.

The performance of the biological IOPF bed is compared to other systems [10-12] where indirect H2S abatement is accomplished and data regarding the H2S removal is available, as shown in Table V.2. As seen in Table V.2, the biological IOPF exhibits a much better performance than other one and two stage systems based on elimination capacity, removal efficiency and duration of operation. Another important consideration is that complete removal efficiencies were attained in the present study from the startup of the reactor, unlike most biofilters packed with synthetic media which need a few days for bacterial colonization when the removal of H2S is directly mediated by sulfur oxidizing

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bacteria. These results show the synergy between the wet adsorption capability of the IOPF during the initial stages of the separation, until the biological process takes over and controls most of the H2S removal in the system.

V.III.III. Biomass characterization

The biomass collected after 80 days of operation in the biofilter was characterized in terms of their most predominant microbial species and VS content. Denaturing Gradient Gel Electrophoresis (DGGE) was performed on a biomass sample, and the only species identified belonged to the genus Alicyclobacillus ssp. Due to the high contents of iron present in the bed (up to 54 g of atomic iron by weight) the activated sludge used to seed the reactor evolved into the enduringness of this particular strain. This bacterial type has been seldom reported to participate in the oxidation of ferrous into ferric ions and iron utilization [13-16] unlike the much better known species Thiobacillus ferrooxidans. Since the starting operation pH (9.02) and oxygen concentration in the trickling nutrient fluids were high enough for ferrous ions to be negligible in solution, it is concluded that during the early stages of the operation a community of sulfur oxidizing bacteria was responsible for the biodegradation of H2S into soluble sulfates, which resulted in a rapid decrease of the liquid pH, until the iron oxidizing strains prevailed due to the leaching of great quantities of ferrous ions in the system. Thus, the H2S mechanism of removal was the reaction between ferric ions and absorbed H2S, as described in Equation (V.10). This identification results are further supported by the observation of quite abundant amounts of yellowish elemental sulfur entrapped in several spots within the bulk of the biomass when extracted from the IOPF. The amount of VS in the extracted biomass was determined to be 92.59 mgVS/IOPFunit, or 6,982.35 gVS/m3bed. Figure V.11 shows ESEM micrograph pictures taken on the collected bacteria after 80 days of operation. Streptobacilli and individual cells were observed, either suspended on the biomass or attached to solid particles.

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Table V.2. Maximum Elimination Capacity (EC) of some systems where explicit indication of H2S removal performance is available, in processes involving the chemical absorption of H2S with ferric ions and following oxidation of ferrous into ferric ions.

Reference Maximum EC observed Comments 3 [gH2S/m bed/hr] [10] 0.058 when removal efficiency reached a Two stage separation; first stage involves chemical scrubbing in a bubble maximum of 75%, before plummeting at 30% column reactor. The second stage is oxidation of ferrous ions with T. after 5 hours of operation. ferrooxidans on glass cubes, packed bed.

[11] 136.60 at beginning of operation, but depleted H2S removal is performed in two stages: the first stage encompasses the to 54.40 after just 60 hours. H2S absorption in a glass beads, upflow packed bioreactor in batch ferric solution, whereas the second stage promotes the bioconversion of ferrous ions after their spending in the first stage by means of T. ferrooxidans.

[12] 11.84 during just 10 hours of test. One stage, countercurrent bioscrubbing of H2S on PVC packing media with immobilized T. ferrooxidans.

This study 40.92 in a reactor operating over 80 days with One stage separation using chemical absorption and iron biooxidation on a average removal efficiency above 98%. biotrickling filter packed bed, with Alicyclobacillus cells supported on IOPF media.

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Figure V.11. SEM micrographs showing Allicyclobacilli collected from the biomass grown on the IOPF bed after 80 days of biological operation. Bars represent, clockwise from top left, 2 µm, 0.5 µm, 0.5 µm and 0.5 µm. Top left picture shows a streptobacilli structure, compared to suspended cells on the other pictures.

V.III.IV. Other operational considerations: Pressure drop, IOPF clogging and effect of carbon source on the accumulation and survival of biomass

The pressure drop measured in the bed was steadily below 34 Pa/m (0.08 inH2O for a bed length of 23 in) along the operation of the reactor, which represents a value much lower than other packed beds where H2S was removed from airstreams by biofilms supported onto different inorganic media with sulfur oxidizing bacteria (60-300 Pa/m [17]) and with iron oxidizing bacteria for indirect abatement of H2S (249 Pa/m [5]). Pressure drop and flooding are major concerns in unit processes where packed beds are used, particularly if biomass is to be grown in the packing film. This is a consequence of the consistent decrease of the void space between the support units due to biomass and solid sulfur accumulation, which translates in higher energy consumption in the fouled air blowers, and a depletion in the removal capacity of the bed since less air is to be driven in the bed to avoid flooding. The good pressure drop behavior of our system compares much better

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than those reported on so called columns packed with iron sponges. Indeed, though higher EC were attained in an iron sponge bed, significant clogging and a decrease of the operation airflow rates down to 50% occurred along the same 80 days of operation of our study on a field test reported in the Literature [18]. The good pressure drop behavior attained in the IOPF system is a result of their properties such as good porosity, ample pore size and rigidity even at very acidic conditions. This, however, did not prevent the media from collecting high amounts of elemental solid sulfur which was visible after biomass was retrieved from the reactor following its termination. This sulfur accumulation is suspected to be the cause of the performance depletion observed after 78 days of operation as shown in Figure V.7. Pagella and de Faveri [10] correlated the performance decrease of their two stage system with an increase of the sulfur suspended in the liquid of the iron biooxidation reactor. It is not clear, however, whether these two effects are directly related or if they are just experimentally observed trends that happen to have a monotonous behavior, but they are affected by other design and operation variables.

Another operation variable directly linked to the biomass accumulation is the carbon source provided to the bacteria. Biological experiments similar to those described in the preceding section were carried out in a bed with IOPF media after exhaustion following 80 days of wet basis adsorption, but whose nutrient solution lacked agar. Results for the performance of this reactor are shown in Figure V.12, which illustrates that the removal efficiency of this system averaged only 63%, at a continuous EBRT of 60 sec and inlet

H2S concentrations lower than 100 ppmv. The maximum EC of this reactor was just 4.68 3 3 gH2S/m bed/hr compared to 40.92 gH2S/m bed/hr for the reactor with agar in its nutrient solution. Bacterial activity was expected in the agar lacking bed due to the observed decrease of pH from 8.06 to 4.13; however, after inspection of the collected IOPF using ESEM pictures, scarce cells were identified (Figures not shown).

The presence of agar is therefore clearly critical for the IOPF colonization considering that Allicyclobacillus, just like most bacteria, exhibit negative surface charge at pH above physiological values, which are actually lower than the initial pH of 9.02 during the

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reactor seeding, and that the charge of the IOPF material is also negative at similar values of pH, as by the measured zeta potential shown in Table V.1. Thus, the electrostatic repulsion between the carrying material and the cells was compensated by the supply of external binding material provided by the agar in the nutrients solution, which formed the spinal for the biofilm fixation. Indeed, it has been reported that biofilm formation is mediated and enhanced by either surface charge cancellation, Extra Polymeric Matrices (EPM) build-up, or both [19].

120

EBRT 60 sec Inlet 100 Outlet

]

S concentration [ppm concentration S

2 H 20

0 0 10 20 30 40 50 60 Elapsed time [d]

Figure V.12. Performance of a IOPF packed biotrickling filter removing H2S for a system whose nutrient solution lacked agar or another organic carbon source.

In order to accumulate, attach to a surface and spread, bacteria have to approach one another, and then either excrete proteins themselves by intrinsic metabolic processes, or process the proteins already available in their surroundings. Even though some bacterial colonization is still possible in iron containing media with negative surface charge as reported elsewhere [20] the presence of agar was the definite factor in the bacterial colonization of the reactor, by being both a source of energy for the Allicyclobacillus activity and the biofilm backbone structure.

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V.IV. CONCLUSIONS

Iron (III) bearing, open-pore foam units (IOPF) were successfully tested as packing media in packed bed reactors for the removal of H2S from airstreams by means of both inorganic chemisorption and indirect oxidation by biological conversion of ferrous into ferric ions, which react with the pollutant carried in the fouled gas. The inorganic adsorption of H2S occurs in presence of low water concentrations in the air phase (relative humidity of 0.1%) and it is greatly enhanced by much higher contents of water on the media surface, which is easily accomplished by trickling down the water from the top of the reactor. Due to the release of alkalinity from the IOPF and the presence of elemental sulfur on their surface, it is concluded that the abatement of H2S occurs due to the reaction of the pollutant with ferric ions, and their conversion in presence of water into the original active phase, leaving elemental sulfur as by-product. No indications of

H2S physisorption were observed. Although the dry basis bed reached a steady breakthrough outlet after less than 250 hr, trickling down water on the IOPF bed promoted a continued removal efficiency averaging 98% over 60 days at the same EBRT and comparable inlet H2S concentrations.

After seeding the media with activated sludge, and due to the high contents of iron present in the media, a colonization of iron oxidizing Alicyclobacilli bacteria occurred which mediated the conversion of ferrous onto ferric ions, which reacted with H2S forming ferrous sulfates. Thus, sustained, high concentrations of ferric ions were responsible for the removal of the pollutant in a biologically mediated, indirect biofiltration. The maximum overall EC of the wet basis inorganic bed and the biological 3 reactor were 8.67 and 40.92 gH2S/m bed/hr, respectively, on a total bed length basis, whereas on a local, one third of bed length basis, these values were 23.09 and 88.27 3 gH2S/m bed/hr, on the same order. This indicates that the contribution of the biological removal reaches at least 2.5 times that of the wet inorganic chemisorption.

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V.V. REFERENCES

1. Kim, T., Kim, C., Chang, Y., Ryu, H. and K. Cho. (2002) “Development of an optimal medium for continuous ferrous iron oxidation by immobilized Acidothiobacillus ferrooxidans cells.” Biotechnology Progress. 18(4) 752-759. 2. Malhotra, S., Tankhiwale, A., Rajvaidya, A. and R Pandey. (2002) “Optimal conditions for bio-oxidation of ferrous ions to ferric ions using Thiobacillus ferrooxidans.” Bioresource Technology. 85 (3) 225-234. 3. Jensen A. and C. Webb. (1995) “Ferrous sulphate oxidation using Thiobacillus ferrooxidans: a Review.” Process Biochemistry. 30(3) 225-236. 4. Mesa, M., Macias, M. and D. Cantero. (2002). “Mathematical model of the oxidation of ferrous iron by a biofilm of Thiobacillus ferrooxidans.” Biotechnology Progress. 18(4) 679-685. 5. Hebi L., Lueking, D., Mihelcic, J. and K. Peterson. (2005) “Biogeochemical analysis of hydrogen sulfide removal by a lava-rock packed biofilter.” Water Environment Research. 77(2) 179-186. 6. Bonnissel-Gissinger, P., Alnot, M., Ehrhardt, J. and P. Behra. (1998) “Surface oxidation of pyrite as a function of pH.” Environmental Science and Technology. 32(19) 2839-2845. 7. Davydov, A., Chuang, K. and A. Sanger. (1998) “Mechanism of H2S oxidation by ferric oxide and hydroxide surfaces.” Journal of Physical Chemistry, B. 102(24) 4745-4752. 8. Baird, T., Campbell, K., Holliman, P., Hoyle, R., Stirling, D. and B. Williams. (1996). “Structural and morphological studies of iron sulfide.” Journal of the Chemical Society: Faraday Transactions. 92(3) 445-450. 9. Afonso, M. and W. Stumm. (1992) “Reductive dissolution of iron(III) (hydr)oxides by hydrogen sulfide.” Langmuir. 8(6) 1671-1675. 10. Pagella, C., and D. de Faveri. (2000) “H2S gas treatment by iron bioprocess.” Chemical Engineering Science. 55 (12) 2185-2194 11. Chung, Y., Ho, K. and C. Tseng. (2006) “Treatment of high H2S concentrations by chemical absorption and biological oxidation process.” Environmental Engineering Science. 23(6) 12. Giro, M., Garcia, O. and M. Zaiat. (2006) “Immobilized cells of Acidithiobacillus ferrooxidans in PVC strands and sulfite removal in a pilot-scale bioreactor.” Biochemical Engineering Journal. 28(2) 201-207. 13. Turova, T., Poltoraus, A., Lebedeva, I., Bulygina, E., Tsaplina, I., Bogdanova, T. and G. Karavaiko. (1995) “Phylogenetic position of Sulfobacillus thermosulfidooxidans - determination based on 5s and 16s ribosomal-RNA sequence-analysis.” Microbiology. 64(3) 306-313. 14. Pepi, M., Agnorelli C. and R. Bargagli. (2005) “Iron demand by thermophilic and mesophilic bacteria isolated from an Antarctic geothermal soil.” Biometals. 18(5) 529-536. 15. Okibe, N. and D. Johnson. (2004) “Biooxidation of pyrite by defined mixed cultures of moderately thermophilic acidophiles in pH-controlled bioreactors: Significance of microbial interactions.” Biotechnology and Bioengineering. 87(5) 574-583.

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16. Rodgers, L., Holden, P. and L. Foster. (2002). “Culture of Acidiphilium cryptum BV1 with halotolerant Alicyclobacillus-like spp.: effects on cell growth and iron oxidation.” Biotechnology Letters. 24(18) 1519-1524. 17. Hirai, M., Kamamoto, M., Yani, M. and M. Shoda. (2001) “Comparison of the biological H2S removal characteristics among four inorganic packing materials.” Journal of Bioscience and Bioengineering. 91(4) 396-402. 18. Zappi, P. (2001) “Ironclad odor control?” Water Environment and Technology. 13(1) 39-44. 19. Starkey, M., Gray, K., Chang, S. and M. Parsek. (2004) “A sticky business: the extracellular polymeric substance matrix of bacterial biofilms.” In Microbial Biofilms. Edited by M. Ghannoum and G. O’Toole. ASM Press. 174-191. 20. Truesdail, S., Lukasik, J., Farrah, S., Shah, D. and R. Dickinson. (1998). “Analysis of bacterial deposition on metal (hydr)oxide-coated sand filter media.” Journal of Colloid and Interface Science. 203(2) 369-378.

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