University of Nevada, Reno
Nanobiocatalytic Degradation of Acid Orange 7
A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN Materials Science and Engineering
by Jason Hastings
Dr. Dev Chidambaram / Thesis Advisor December 2010
THE GRADUATE SCHOOL
We recommend that the thesis prepared under our supervision by
JASON THOMAS HASTINGS
entitled
Nanobiocatalytic Degradation Of Acid Orange 7
be accepted in partial fulfillment of the requirements for the degree of
MASTER OF SCIENCE
Dev Chidambaram, Ph.D., Advisor
Manoranjan Misra, Ph.D., Committee Member
Amy Childress, Ph.D., Graduate School Representative
Marsha H. Read, Ph. D., Associate Dean, Graduate School
December, 2010
i
Abstract
The catalytic properties of various metal nanoparticles have led to their use in environmental remediation applications. However, these remediation strategies are limited by their ability to deliver catalytic nanoparticles and a suitable electron donor to large treatment zones. Clostridium pasteurianum BC1 cells, loaded with bio‐Pd nanoparticles, were used to effectively catalyze the reductive degradation and removal of Acid Orange 7
(AO7), a model azo compound. Hydrogen produced fermentatively by the C. pasteurianum
BC1 acted as the electron donor for the process. Pd‐free bacterial cultures or control
experiments conducted with heat‐killed cells showed limited reduction of AO7.
Experiments also showed that the in situ biological production of H2 by C. pasteurianum BC1
was essential for the degradation of AO7, which suggests a novel process where the in situ microbial production of hydrogen is directly coupled to the catalytic bio‐Pd mediated reduction of AO7. The differences in initial degradation rate for experiments conducted using catalyst concentrations of 1ppm Pd and 5ppm Pd and an azo dye concentration of
100ppm AO7 was 0.39hr‐1 and 1.94hr‐1 respectively, demonstrating the importance of
higher concentrations of active Pd(0). The degradation of AO7 was quick as demonstrated
by complete reductive degradation of 50ppm AO7 in 2 hours in experiments conducted
using a catalyst concentration of 5ppm Pd. Dye degradation products were analyzed via Gas
Chromatograph‐Mass Spectrometer (GCMS), High Performance Liquid Chromatography
(HPLC), UltraViolet‐Visible spectrophotometer (UV‐Vis) and Matrix‐Assisted Laser
Desorption/Ionization (MALDI) spectrometry. The presence of 1‐amino 2‐naphthol, one of
the hypothesized degradation products, was confirmed using mass spectrometry.
ii
Acknowledgements
First, I would like to express my overwhelming gratitude to my advisor Dr. Dev
Chidambaram for his leadership and unwavering positive attitude to the completion of this thesis. I am extremely thankful to have such a knowledgeable and available advisor and role model during this process. I am very thankful for Dr. Dev accepting me as a student before he was planning to accept any students. I would like to thank Dr. Amy Childress and
Dr. Manoranjan Misra for having accepted to serve on my thesis examination committee and also for their valuable time and patience.
I would also like to acknowledge financial support from the Office of the Vice
President for Research and the Chemical and Materials Engineering Department at UNR, which was provided to Dr. Chidambaram in the form of his start‐up package. Further, I acknowledge the UNR Graduate School for providing me with an ACCESS graduate student fellowship for the Fall 2010 semester.
I would like to thank Narasimharao Kondamudi for his continual advice and services when it came to characterizations of my products and Dr. Manoranjan Misra for providing me with access to the equipment in his laboratory. I thank York Smith for allowing me to bother him on a daily basis for chemicals or supplies at start up. I thank Dr. Mojtaba
Ahmadian‐Tehrani for training me on the usage of the SEM. I thank Rebekah Woosley and the Proteomics Center for allowing me to run tests in their facilities. Finally, I would like to thank my fellow graduate students on the fourth floor for their continual humor, advise and availability to converse with.
Now I get to the good stuff. I thank Jesse Ruppert and Dharshini Balasubramaniyan for continual support in and out of the laboratory. I am greatly indebted to Dr. Liz Charan iii
for her expertise in microbiology and microbial lab techniques. I would like to thank my family for their remarkable support in my decision to continue in higher education. I would like to blame my love, Lisa Loe, for delaying my thesis writing. The support from all of my friends and family makes this process fairly painless. I also would like to thank whoever decided it was ok to make an acknowledgements section because I like that I can write whatever I want.
iv
Table of Contents
Section Page Number Abstract i Acknowledgements ii Table of Contents iv List of Table vi List of Figures vii 1. Introduction 1
1.1 Environmental Contamination 1 1.2 Azo Compounds 1 1.3 Treatment Methods for Azo Dyes 6 2. Material and Methods 14
2.1 Microbial Culture and Growth Medium 14 2.2 Microbial Synthesis of Pd Nanoparticles 15 2.3 Degradation of Acid Orange 7 16 2.4 Analytical Techniques 17 2.4.1 UV‐visible Spectroscopy 17 2.4.2 Gas Chromatography Mass Spectrometry 18 2.4.3 High Performance Liquid Chromatography 19 2.4.4 Matrix‐Assisted Laser Desorption/Ionization 20 3. Results and Discussion 22 3.1 Proof of Catalyst Formation 22 3.2 Rate of Reaction 28 3.3 Biocatalytic Reduction of Azo Dyes Using Palladium 29 3.4 Degradation Products 32 3.4.1 UV‐Vis 35 3.4.2 Gas Chromatogram 37 3.4.3 HPLC 38 3.4.4 MALDI 40 v
3.5 Effectiveness of Nanobiocatalytic reduction of Azo Dyes 42 4. Conclusions and Future Directions 49
4.1 Implication of Research 50 4.2 Future Directions 51 References 53
vi
List of Tables
Table Name Page Number 1.1 Classes of dyes and their structures 2 1.2 Comparison of microbial remediation of dyes 8 2.1 Composition of the growth medium for C. pasteurianum BC1 14 2.2 Matrix of palladium‐AO7 experiments 16 3.1 Degradation of 1ppm, 3ppm and 5ppm Pd at 50ppm and 30 100ppm AO7 3.2 Viability tests performed on C. pasuerianum with 3ppm Pd 31 3.3 Table comparing rates and amounts of dye degradation using 44 a multitude of bacteria
vii
List of Figures
Figure Name Page Number 1.1a Chemical dyes being released into the environment in China 6 1.1b China’s yellow river turned red with additions of dye 6 2.1 Anaerobic glove chamber 15 2.2 UV‐1800 spectrophotometer from Shimadzu 17 2.3 Shimadzu GC‐MS QP 2010 19 2.4 Shimadzu LCsolution‐20‐AB 20 3.1 Increasing concentrations of Pd 22 3.2 SEM image of Pd(0) on C.pastuerianum 23 3.3 Proof of experiment for degradation of AO7 with 3ppm Pd and 24 50ppm AO7 3.4 Varying concentrations of AO7 in bacterial growth 25 3.5a Degradation of AO7 (50 and 100ppm) at 1 ppm Pd 27 3.5b Degradation of AO7 (50 and 100ppm) at 3 ppm Pd 27 3.5c Degradation of AO7 (50 and 100ppm) at 5 ppm Pd 27 3.6 The exponential degradation of AO7 and a model generated 29 using a 1st order reaction rate 3.7 Hypothesized breakdown of AO7 32 3.8 Electrochemical pathways for the degradation of AO734 3.9 UV‐Vis spectrum of cultures containing 5ppm Pd and 50ppm 35 AO7 3.10 Comparison of concentrated degradation products with stock 36 dye solution on UV‐Vis 3.11 Mass Spectrometer showing 2‐naphthol as possible 37 degradation product 3.12 The chromatogram of the azo dye degradation products37 3.13 HPLC of the degradation products as compared to spent media 38 and sulfanilic acid standard 3.14 Magnified HPLC of dye degradation products compared to 39 spent media and sulfanilic acid 3.15a MALDI of the matrix background on which the samples were 40 prepared 3.15b MALDI of dye degradation products 40 3.16 MALDI of the spent media of C. pastuerianum BC1 after 19hrs 42 of growth
1
Chapter 1‐Introduction
Water supplies (both surface and sub‐surface) at thousands of sites across our country remain contaminated at this time [1]. The Environmental Management (EM) division of the
United States Department of Energy (US DOE) reports that only 17 of the 50 states in
America do not have remediation sites [2]. Out of the 33 states that have remediation sites, there are 14 host states that are currently under remediation. Within the 14 states with active sites, there are a combined 81 sites that account for a total budget of more than $5.27
Billion [2]. The estimates above do not include the sites or the costs for remediation of radioactive contaminants. Contaminants can be broadly classified into organic, inorganic and radioactive. Organic contamination is the main topic of this study. Organic contamination results from the production, usage and recycling of chemicals with organic structures. Three major classes of organic contaminants are chlorinated organics, pesticides and azo compounds. Azo compounds form the contaminant of interest in this study.
Azo Compounds:
Azo compounds, the chemicals of interest in this study, are widely used in the textile
industry, and account for a little more than half of the world’s production of colorants [3].
Azo compounds may be classified into seven distinct categories: Acid, Basic, Direct,
Disperse, Reactive, Sulfur and Vat dyes as shown in Table 1.1.
2
Table 1.1: Classes of azo dyes with characteristic information and their structures.
Fixation Dye Class (%)/Fibers Structure Apllied
OH 80‐90/Wool Acid and Nylon N N
1-Phenylazo-naphthalen-2-ol H3C N+
S N N 97‐98/Acrylic Basic and some Polyesters
N
CH3 3-Methyl-2-(1-methyl-2-phenyl-1H- indol-3-ylazo)-thiazol-3-ium
80‐ OH O 92/Polyester, Disperse Acetate and other N N NH synthetics N-[4-(2-Hydroxy-5-methyl-phenylazo)-phenyl]-acetamide SSHO3
CH3 60‐70/Cotton N Sulfur and other H3CO N cellulosics N HO N
C6H5 Sulphur Orange 2
3
Table 1.1: (Cont.)
HO N N
H N 2 SO3Na
N 70‐ 95/Cotton, Direct Rayon and O3SNa other cellulosics
NH2
H2N N N
Direct Black 38 O NaO S
O
NH N
N OH H3C O
60‐90/ Cotton, other Reactive cellulosics and Wool
O S O
O O S
NaO O Reactive Orange 16 4
Table 1.1: (Cont.)
O NH O
O HN O
80‐95/Cotton Vat other cellulosics
N O N
O N H H N O
O
Vat Yellow 10
Acid dyes are water‐soluble anionic compounds that generally have a dye fixation of
80‐90% onto wool or nylon. The pollution associated with acid dyes are in the form of color, organic acids and unfixed dyes. Basic dyes are water‐soluble, applied in weakly acidic dye baths and are very bright dyes. Basic dyes have an extremely high fixation rate compared to other dyes at 97‐98% on acrylic and polyester materials. Since pollutants from 5
basic dyes are so low, there is very little information on them. Disperse dyes are water soluble and have a fixation of 80‐92% on polyesters, acetates and other synthetics.
Pollution resulting from the use of disperse class of azo dyes include organic acids, carriers, leveling agents, phosphates, defoamers, lubricants, dispersants, delustrants and diluents.
Sulfur dyes are organic compounds containing sulfur or sodium sulfide. Sulfur dyes have the lowest fixation rate of approximately 60% on cottons and other cellulosics. Sulfur dye pollution includes alkali, oxidizing agent, reducing agent and unfixed dye. Direct dyes are water‐soluble, anionic compounds and can be applied directly to cellulosics without mordants. Direct dyes have a fixation rate of 70‐95% on cotton, rayon and other cellulosics.
The pollution associated with directs dyes are color, salt, unfixed dye, cationic fixing agents, surfactant, defoamer, leveling and retarding agents, finish and diluents. Reactive dyes are water‐soluble, anionic compounds and are the largest dye class. Reactive dyes have a fixation of 60‐90% on cotton, wool and other cellulosics. The pollution associated with reactive dyes are salt, alkali, unfixed dye, surfactants, defoamer, diluents and finishing agents. Vat is the oldest dye and is more chemically complex than the others and is also water‐soluble. Vat is also fixed on cotton and other cellulosics with a fixation of 80‐95%.
Typical pollution associated with Vat dyes are alkali, oxidizing agents and reducing agents
[4, 5].
The colorant industry manufactures more than 700,000 tones of synthetic textile azo dyes each year, and approximately 15% of the dyes produced are lost in wastewater during the dyeing operation [6, 7]. The three azo compounds namely Acid Orange 7 (AO7), Methyl
Orange (MO) and Acid Red 14 (AR14), represent 50%‐70% of dye produced around the world [8]. AO7 was used in this paper for its simple, yet common structure found in most acid dyes. AO7 is used extensively as a model compound for degradation. The formation of 6
AO7 comes from the combination of sulfanilic acid and 2‐naphthol. Environmental effects from the release of textile dyes can be seen from the following pictures in Figure 1.1.
(a) (b) Figure 1.1: (a) shows chemical dyes being released into the environment in China [9]. (b) shows China’s yellow river turned red with additions of dye [10].
Textile dyes have serious health effects. Mutagenic effects of textile dyes have been demonstrated in Salmonella typhimurium, mouse lymphoma cells and Bacillus subtilis [11,
12]. Azo dyes have long been recognized as a human bladder carcinogen [13].
Treatment Methods for Azo Dyes
Treatment of azo compounds uses both electrochemical pathways; reductive and oxidative. Oxidative reduction has shown promising results with and without catalysts.
Photoreduction of azo compounds have been conducted using various substrates; such as, titania [14] and zeolite [15]. Fixed substrate reactions are ideal for continuous flow reactors because the catalyst is not in suspension; however, since they are not in suspension, the available contact sites with the flow of the dye are low [14, 15].
Photocatalytic degradation of azo compounds using only titanium dioxide in suspension has been shown by Gettai et al. [16]. Having the titanium dioxide in suspension is expected to increase the surface contact and allow for a more rapid degradation of the dye. Titanium 7
dioxide nanotubes have also been used as a solid substrate for the degradation of azo compounds [17‐20]. Recent studies conducted using doped titanium dioxide nano‐tubes
[21] showed the addition of the dopants to result in a decrease in the energy required to degrade the dye. The methods above generally require expensive catalysts and use harmful chemicals in the production of the catalyst. For an extensive review on these types of degradation processes please refer to the review article by Konstantiou and Albanis [22].
Electrocoagulation has also been used in combination with photo‐oxidation for the removal of azo dyes [23]. The process is based on the principle of addition of electrical charge to a solution to change the charge distribution on dissolved substances and thereby coagulate and precipitate them.
Oxidation through chemical means is another option that is being explored.
Chemical processes have some advantages over catalytic processes; such as, fast reaction kinetics and the lack of the need for a process step for the recovery of the catalyst. Chemical degradation of azo compounds can also be accomplished with H2O2 and a iron(III)
complexed to tetra amido macrocyclic ligands catalyst as seen from the degradation of AO7
reported by Chahbane et al. [24]. Punj and John used enzymes to degrade azo compounds
[25]. Tauber et al. used Laccase, an enzyme secreted by white rot fungus to degrade azo
compounds [26]. Since Laccase is secreted by a living organism, the environmental effects
were hypothesized to be minimal. The neutrality in the environment enables the effluent
stream, containing azo compounds and Laccase, to flow into the next process step with little
to no preparation. Various studies have demonstrated the use of members of the genera
Hirschioporus, Inonotus, Phlebia, Coriolus [27], Enterobacter, Pseudomonas, Morganella [28],
Bacillus, Acinetobacter, Legionella, and Staphylococcus [29] in the degradation of azo
compounds. A comprehensive list of microbes demonstrated to be useful in the degradation 8
of azo compounds and the results obtained in those studies is given in Table 1.2 One study showed a 99% decolorization of artificial textile effluent in 7 days [30]. Activated sludge has been used in the decolorization of azo dyes; a process that is based on adsorption and not degradation [31]. The advantages for using microbes for remediation are two‐fold: low cost and fewer post processing steps. The often reported shortcoming of microbial remediation is the slow rate of reaction as shown in Table 1.2. Studies, including this thesis research, are currently being conducted to overcome this shortcoming via the use of nano‐ particles to increase the reaction kinetics.
Table 1.2: Comparison of microbial remediation of dyes. Reference Bacterial species Dyes Comments s Decolorization of DBMR was Acinetobacter 91.3% in static anoxic Direct brown MR calcoaceticus condition, whereas agitated [32] (DBMR) NCIM 2890 cultures showed less (59.3%) after 48hr Acinobacter sp., Acid Red 88,Reactive The mixed culture of bacteria Citrobacter Black 5, Direct Red removed 88‐100% dyes [33] freundii, klebsiella 81, Disperse Orange (100ppm) in 10hr oxytoca 3 Aeromonas caviae, More than 90% decolorization Proteus mirabilis, Acid Orange 7 of the dye was achieved in [34] Rhodococcus sp. 16hr Treatment times required by most efficient strain, AS96 (Shewanella putrefaciens), Aeromonas, Reactive Black 5, were as short as 4hr for Pseudomonas, Direct Red 81, Acid complete decolorization of Bacillus, [35, 36] Red 88, Disperse 100ppm of AR‐88 and DR‐81 Shewanella and Orange 3 dyes under static conditions, Massillia spp. and 6 and 8hr, respectively, for complete decolorization of RB‐ 5 and DO‐3 High decolorization efficiency (95‐98%) achieved within 6 hr Acid Blue 25, for 100mM Acid Blue 25, 4 hr Bacillus cereus Malachite Green, for 55mM Malachite Green, [37] DC11 Basic Blue and 2 hr for 750mM Basic Blue X‐GRRL under anaerobic conditions 9
Table 1.2: (cont.) Disperse Blue 79, The dyes were completely Bacillus fusiformis [38] Acid Orange 10 mineralized within 48hr The complete decolorization Bacillus sp. Congo Red was achieved in 24‐27hr for a [39] concentration of 100‐300ppm Under the near‐optimal conditions, 99% of the Bacillus subtilis HM Fast Red [40] decolorization was achieved in 6hr Bacillus The dye was decolorized up to Acid Red 119 [41] thurengiensis 70% in 24hr The dye (25ppm) was Bacillus velezensis Direct Red 28 completely decolorized within [42] AB 10hr About 95% dye (200ppm) was Citrobacter sp. CK3 Reactive Red 180 [43] removed in 36hr The bacterium removed 53‐ 63% of the dye in 24hr in Entrerococcus Direct Black 38 minimal medium while 71‐ [44] gallinarum 85% of decolorization was observed in Luria broth media After acclimation, time for Escherichia coli Reactive Red 22 50% color removal lowered [45] NO3 from 5.7 to 4.3hr The complete decolorization was achieved at the end of 9 Escherichia coli, Congo Red, Direct days of incubation in case of E. [46] Pseudomonas sp. Black 38 coli while Pseudomonas sp. Decolorized in 5 days The dye was decolorized up to Escherichia coli YB Acid Red 27 [47] 75% in 2hr Reactive Brilliant Red X, Acid Black 10B, Acid Scarlet GR, The decolorization of the dyes Halomonas sp. [48‐50] Acid Red B, Acid Red was up to 90% in 24hr G, Reactive Brilliant Red K Remazol Black, Maxilon Blue, The bacterium was capable of Sulfonyl Scarlet decolorizing the dyes in wide Halomonas sp. BNLE, Sulfonyl range of NaCl concentrations [51] Green BLE, Remazol after 4 days of incubation Black N, Entrazol period Blue IBC
10
Table 1.2: (cont.) The first four dyes decolorized Amaranth, fast R, by the bacterium by 100% Ponceau S, Congo R, Kerstersia sp. VKY1 while the remaining three [52] Orange II, Acid O 12, decolorized by 84, 73 and Acid R 151 44%, respectively, in 24hr Monoazo dyes RY107 and RR Reactive Yellow 107, 198 were decolorized in 72 Klabisiella sp. VN‐ reactive Red 198, and 96hr; the diazo dyes (RB5 [53] 31 reactive Black 5, and triazodye DB71) Direct Blue 71 decolorized in 120 and 168hr Lactobacillus casei The complete decolorization of Methyl Orange [54] TISTR 1500 the dye was achieved in 2.5hr Paenibacillus The bacterial consortium polymyxa, Reactive Violet 5R showed complete [55] Micrococcus luteus decolorization in 36hr Proteus vulgaris, Bacterial consortium Micrococcus Scarlet R [56] decolorized 90% in 3hr glutamicus The 59‐99% color removal after 206 days static Reactive azo dyes, Pseudomonas incubation, at the dye Direct azo dyes and [57] luteola concentration of 100ppm, leather dyes monoazo dyes showing fastest rate of decolorization P. aeruginosa showed Methyl Orange, Y87, Pseudomonas decolorization efficiency over B86, R91, B19, R90, aeruginosa, 98% after 48hr while 76% B69, B31, B36, Y15, [58] P. oleovarons, decolorization was achieved R34, B15, Y79 AND P. putida by P. oleovarons after 54hr. P. B54 utida showed lower efficiency The dye GHE4B was Pseudomonas Direct Blue 6, Green completely decolorized in 12hr [59] desmolyticum HE4B, Red HE7B while DB 6 and RHE7B were decolorized in 16hr The E. coli improved the ability of Pseudomonas sp. to Pseudomonas decolorize the dye by luteola, Escherichia Reactive Red 22 [60] producing decolorization‐ coli stimulating extracellular metabolites
11
Table 1.2: (cont.) Complete biodegradation of azo dye up to 200ppm was achieved in 49hr under Pseudomonas Acid Violet 7 shaking while the [61] putida mt‐2 biodegradation time was reduced to 37hr under static conditions The strain was capable of degrading dye in a wide range Pseudomonas sp. Reactive Red 2 of concentrations (up to [62] SUK1 5000ppm) and almost 80% dye was removed in 114hr The dye up to 700ppm Rhodopseudomonas Reactive Black 5 concentration was completely [63] palustris decolorized in 40hr After 4hr incubation, more than 90% of the color was removed under anaerobic Shewanella Fast Acid Red GR conditions while 12.8 and [64] decolorationis S12 33.7% decolorizing rates were observed under aerobic and microaerophilic conditions Shewanella Fast Acid Red GR, The 90% decolorization of the decolorationis sp. reactive Brilliant dyes were achieved within [65] nov. S 12T Blue 12hr Anaerobic cultures of Shewanella strain J18 143 rapidly removed color from Remazol Black B, Shewanella J18 143 the azo dye Remazol Black B in [66] Acid Orange 7 the growth medium to produce an absorbance at 597nm of less than 1 in under 40min The bacterium was capable of Sphingomonas decolorizing bromoamine acid Anthraqiunone dyes [67] herbicidovorans dye (1000ppm) more than 98% within 24hr
Nanoparticles have been used to enhance the degradation of azo dyes both chemically and catalytically. Nanomaterials, such as zero valent iron (ZVI), have been used to rapidly degrade the organic dyes [68‐71]. The rate of degradation of dyes with ZVI is much higher than that of microbial degradation. However, it should be noted that ZVI is used in those studies as a chemical reactant rather than as a catalytic material. The 12
shortcoming of this approach is based on the inherent properties of ZVI; ZVI oxidizes in the presence of air, which makes production, transportation and storage a costly endeavor [72].
Further, since it is used as a chemical reactant and not as a catalyst, the amount of ZVI required is often more than the stoichiometrically required amounts when losses due to unwanted reactions are taken into account.
The use of nano catalysts have also been reported. Nano catalysts have the ability to increase the degradation rate and be recycled for multiple cycles. Examples of nanocatalysts include zinc oxide [73, 74], titanium oxide [75, 76] and palladium.
Microbes, such as, Shewanella oneidensis [77] and Desulfovibrio desulfuricans [78],
have been shown to create palladium nanoparticles in the presence of a palladium salt.
Reactor studies have been conducted using bio‐synthesized palladium [79]. However, these
studies require two steps: one step in which the Pd(0) nanoparticles, hence forth bio‐
nanopalladium, are generated and then a second step in which they are supplemented to a
reactor for the remediation of contaminants.
This thesis explores the use of concomitantly prepared palladium nanocatalysts in
enhancing the microbial degradation of azo dyes. Palladium catalyst was chosen due to its
known catalytic hydrogenation and electron transfer properties. Clostridium pasteurianum
BC1 is capable of forming palladium (0) nanoparticles when challenged with a Pd ion
solution [80]. Microbial metabolism requires oxidation of a carbon source. The oxidation of
the glucose leads to the generation of excess electrons which are transferred to terminal
electron acceptors. Palladium ions act as the terminal electron acceptor and are reduced to
metallic Pd(0). Confirmation of the synthesis of Pd(0) was done previously using SEM, XRD
and XANES. While these nanoparticulate catalysts have been used for the degradation
and/or remediation of inorganics such as hexavalent Cr [80], to our knowledge this is the 13
first instance where this system has been demonstrated for use against organic compounds.
C. pasteurianum BC1 was isolated from a metal contaminated coal mining site [81]. Unlike
other aerobic bacteria, C. pasteurianum BC1 has the ability to produce hydrogen as a byproduct of metabolism, which can then act as an electron donor in the reduction of azo dyes. When the treatment is conducted in a process reactor, the advantages of combining the two steps, formation of nanoparticles and injection of hydrogen source, are faster reaction rate and easier recovery of catalyst.
The specific hypothesis and expected outcomes of this thesis are as follows:
(I) Biologically generated nanopalladium will act as a catalyst in reductive degradation of azo dyes.
Expected outcome: AO7, the model compound, will be degraded via reduction at a
rate faster than controls without Pd.
(II) Biosynthesis of Pd nano particles and the degradation of azo compounds can be
combined in an in‐situ process.
Expected outcome: In‐situ degradation of model azo compound AO7 will lead to its
breakdown products.
(III) Biodegradation of azo compounds will require the presence of living bacterial cells, bio‐nanopalladium and the biogeneration of hydrogen.
Expected outcome: Azo compounds will be significantly degraded only in the
presence of growing culture of C. pasteurianum BC1 and bio‐nanopalladium.
(IV) In the presence of bio‐nanopalladium, microbial viability is increased.
Expected outcome: the toxicity of the AO7 will have limited effect on microbes in the
presence of bio‐nanopalladium.
14
Chapter 2‐Materials and Methods
Ultrapure water (18.2MΩ) was used throughout this study. All reagents were of
analytical grade or better. All gases used were of ultrahigh purity grade.
Microbial Culture and Growth Medium
Clostridium pasteurianum BC1 (ATCC No. 53464) was grown in a culture medium
composed of the ingredients listed Table 2.1 and was prepared as per the protocol given
below.
Table 2.1: Composition of the growth medium for C. pasteurianum BC1.
Ingredients Amount (g)
Glucose 5
Ammonium Chloride 0.5
Glycerol Phosphate 0.3
Magnesium Sulfate 0.2
Calcium Chloride 0.5
Peptone 0.1
Yeast Extract 0.1
FeSO4.7H2O 0.00278
Ultrapure water 1000
The chemical ingredients listed above, with the exception of iron sulfate, were
added to one liter of ultrapure water which was pre‐reduced by boiling it for 15 minutes
while purging the solution with nitrogen. The media was then cooled to room temperature
using a water bath during which, the nitrogen purging was maintained. The flask was then 15
transferred to an anaerobic chamber shown in Figure 2.1. 6.25mL of a 1.6mM FeSO4.7H2O solution was added to the media solution once inside the anaerobic chamber.
Figure 2.1: anaerobic glove chamber.
The pH of the medium was adjusted to 6.8 using 1M NaOH. 40ml and 22ml aliquots of the medium were dispensed into several 60ml and 30ml serum bottles, respectively. The bottles were sealed with butyl rubber stoppers and aluminum crimps, autoclaved at 121oC for 20min and cooled prior to use.
All liquid culture experiments were performed in triplicates. Culture viability and growth relative to a pure culture, which was growth in parallel, was tested by comparing the time required for a 2mL subculture to reach O.D.600nm=0.6 in fresh media.
Microbial synthesis of Pd nanoparticles
Cultures were grown in an incubator shaker (125rpm, 28±1˚C) to the mid to late log
phase of growth (O.D.600nm = 0.6), which typically required 19 hours. Unless otherwise
stated, all cultures were then spiked with aqueous Pd(2+), using a 90ppm or 900ppm 16
+ Na2PdCl4 stock solution, to achieve the necessary initial Pd(2 ) concentration. Formation of bio‐nanopalladium was confirmed using Scanning Electron Microscope (SEM) and X‐Ray
Diffraction (XRD) [82].
Degradation of Acid Orange 7 (AO7)
A spectrophotometric method was used to determine the concentration of AO7 remaining in the serum bottles. The cultures were left undisturbed after the addition of Pd for approximately 10min, although an earlier study showed the conversion of ionic palladium (Pd2+) to Pd(0) nanoparticles occur in less than a minute after the addition of
Pd(2+) [80]. The orange‐brown color of the palladium salt turns black as the nanoparticles
form and visually the culture appears grey to black, depending on the concentration of the
Pd. In this study, AO7 was added 10min after the addition of Pd.
The concentration of the AO7 used varied from 50ppm to 100ppm. The concentration of AO7 in the serum bottle was monitored spectrophotometrically at 484nm.
The experiments and controls performed are illustrated in Table 2.2.
Table 2.2: Matrix of palladium‐AO7 experiments. The “” represents the presence of that constituent in the experiment. The “X” in the bacteria column denotes that the use of a heat‐ killed culture as a control. C. Pasteurianum BC1 (Alive () or heat‐ Palladium Acid Orange 7 killed(X)) X X
The check marks in Table 2.2 indicate the inclusion of the constituents in that set of
experiments. For example, in the experiment described in the first row in the table, a living
bacterial culture and AO7 were the only addition to a bottle of media. The “X” indicates the 17
use of heat‐killed cultures. All heat‐killed cultures were initially grown to mid to late phase
(typically 19 hours) and then autoclaved. Row two of Table 2.2 explains that AO7 was added to a bottle of media containing a heat‐killed culture. In the case with a check mark in all three columns, the bacterial culture was allowed to grow for 19 hours, then palladium salt was added and finally the AO7 was added. Experiments and controls described in Table
2.2, were conducted to unequivocally prove the necessity of using living C. pasteurianum
BC1 in conjunction with palladium for the catalytic reduction of azo compounds.
Analytical Techniques
The degradation products were analyzed by three different techniques: Gas
Chromatography Mass Spectrometer (GCMS), High Performance Liquid Chromatography
(HPLC) and Matrix‐Assisted Laser Desorption/Ionization (MALDI). Hexane was used to extract any non‐polar compounds out of the spent media with degradation products.
Ultraviolet‐Visible spectroscopy
All UV‐Vis experiments were conducted on the UV‐1800 spectrophotometer from
Shimadzu (Figure 2.2).
Figure 2.2: UV‐1800 spectrophotometer from Shimadzu. 18
There are two existing light sources within a UV‐VIS spectrophotometer: one for the
UV (deuterium lamp) and one for the visible light (halogen lamp) spectrum. While the spectrophotometer is running, the wavelengths will change, warranting the use of multiple bulbs. The sample chamber is equipped with multiple slots to allow for continuous measurements of several samples at a particular wavelength. The instrument is blanked after any change in wavelength. Quartz cuvettes were used to prevent any interference and allow measurements at lower wavelengths. The Beer‐Lambert law states that there is a logarithmic dependence among transmittance of the light through a substance, the product of the absorption coefficient of the substance and the distance the light travels through the substance, as shown in equation 2.1.
= (2.1)
Where a is the absorbance coefficient, b is the light path length and c is the concentration. Equation 2.1 implies that the absorbance is linear with the concentration.
Beer’s law must be obeyed; otherwise a non‐linear relation will develop between concentration and absorbance.
A standard curve for the color of the azo compound was generated prior to any experiments. A concentration of 100ppm was found to be the upper limit for obeyance of
Bragg’s law. A full spectrum, from 190nm to 900nm, was run on every sample; this allowed for visual confirmation of the degradation of the dye and the formation of any degradation products.
Gas Chromatography Mass Spectrometry
The samples were analyzed using gas chromatography mass spectrometer GC‐MS;
Shimadzu GC‐MS QP 2010 (Figure 2.3). Samples were filtered using a 45µm syringe filter before being placed in GC vials. RTX‐5MS capillary column (29.5m length, 0.25µm thickness 19
and 0.25mm diameter) was used for the analysis. Ultra pure helium gas was used as the carrier gas. The temperature gradient was set from 90oC to 240oC during the course of
95min (1.57 oC/min), which showed good resolution between 2‐naphthol and dibutyl
phthalate. 2‐naphthol is a known substance in the National Institute of Standards and
Technologies (NIST) database, which closely resembles one of the hypothesized
degradation products. Dibutyl phthalate is the autooxidation response to one of the
hypothesized degradation products. All MS peaks were compared to NIST databases.
Figure 2.3: Shimadzu GC‐MS QP 2010.
High Performance Liquid Chromatography
The reduction of azo dyes was monitored using a High Performance Liquid
Chromatography (HPLC), Shimadzu LCsolution‐20‐AB (Figure 2.4) with ultraviolet detector.
Degradation products and spent media were separated using a Biorad® silica 5µm column
with the deuterium lamp set at 254nm. The oven was set at 40oC. The eluent stream used 20
was a (25/75) mixture of acetonitrile/ammonium acetate, where the ammonium acetate solution had a concentration of 0.025mol l‐1, with a flow of 1mL/min. Samples were filtered using a 5µm syringe filter (VWR). The filtrate was then aliquoted into HPLC vials and ready for use. Samples were injected and diluted to 1mL using the eluent solution.
Figure 2.4: Shimadzu LCsolution‐20‐AB.
Matrix‐Assisted Laser Desorption/Ionization
The final characterization method used was the Matrix‐Assisted Laser
Desorption/Ionization (MALDI) conducted using 4700 Proteomics Analyzer with
TOF/TOF Optics at the Proteomics Facility at University of Nevada, Reno. The MALDI was used to obtain the Molecular Weight of the products within samples of interest. A matrix containing 10mg/mL α‐cyano‐4hydroxycinnamic acid and 10mM of ammonium phosphate was used as a background for all samples. Samples were mixed with the matrix at a ratio of 1:1. The MALDI was operated at room temperature and in reflection positive 21
mode. The samples were filtered in a manner similar to that conducted for HPLC and GCMS analyses. A matrix is used as the substrate to hold the sample. Every run of the MALDI requires a standard of the matrix to be performed, as the chemicals on the matrix may change depending on the run.
22
Chapter 3‐Results and Discussion
Proof of catalyst formation
Within a few minutes of the addition of Pd(2+) solution to the Clostridium pasteurianum BC1 culture at the end of log phase growth (O.D.600nm = 0.6), a gray precipitate
formed. As shown in Figure 3.1, the color varied, with increasing concentrations of Pd(2+), ranging from 0ppm to 5ppm. The broth in the vial does not turn completely black, as demonstrated by De Windt et al. [77] and Lloyd et al. [83], as the concentration of Pd is
almost two orders of magnitude lower.
Figure 3.1: Microbial cultures of C. pasteurianum BC1 with increasing palladium concentration from left to right. The concentration of palladium is indicated above the respective bottle.
The precipitate identified from analysis using Scanning Electron Microscope (SEM),
was found to be nanoparticulate in nature as seen in Figure 3.2. The mean size‐distribution
of the bio‐nanopalladium particles was 11.8±4.5nm. Sizes varied from less than 1 to 15nm 23
[80]. Further analysis of the precipitate, using XRD, showed it to be Pd(0). This was subsequently confirmed using XANES [82]. Although the reduction is complete in less than a minute after the addition of Pd solution [82], 10 minutes were given to ensure complete conversion of Pd(2+) to Pd(0). In the absence of growing culture of C. pasteurianum BC1,
Pd(2+) will still be reduced to Pd(0), albeit very slowly, by the small amount of hydrogen
present in the headspace. This small amount of hydrogen in the headspace is a consequence
of sealing the serum bottles in an anaerobic chamber containing ~4% hydrogen, which is
used to catalytically remove any trace amounts of oxygen. However, as demonstrated
earlier, the particles size, as well as the catalytic behavior of chemically formed Pd(0), is not
similar to the nanopalladium synthesized by living cells [82].
Figure 3.2: SEM image of Pd(0) on C. pasteurianum BC1. The size of the Pd(0) Ranges from 1‐15nm.
24
Figure 3.3: Proof of experiment for degradation of AO7 with 3ppm Pd and 50ppm AO7. Samples without both living microbial cultures and biogenerated palladium show little to no degradation of the azo dye.
Figure 3.3 shows the rate of degradation of AO7 for the experiments and controls described in Table 2.2. 45mM stock solution of AO7 was used to supplement plain media to obtain a starting concentration of 50ppm AO7. A bright orange‐red color appeared instantly and the color intensity increases with an increasing concentration of AO7, as shown in
Figure 3.4. The orange color, in the absence of a living microbial culture and bio‐ nanopalladium, did not subside even after multiple weeks. The absorbance at 484nm for the dye is a direct result of the orbital energy gap shrinking caused by the azo linkage.
484nm is located in at the edge of the blue range of the UV‐Vis spectrum, but from delocalization of the charge, the complementary color, yellow‐red, is formed. 25
Figure 3.4: Increasing concentrations of AO7 among bacterial growth from left to right. The numbers on the front of the bottles represents the concentration of AO7 in ppm.
Media, with or without Pd, showed limited degradation of AO7 during the time of the experiment. Media without Pd showed an average change in AO7 concentration from
42.4ppm to 40.3ppm. In the presence of Pd, the average concentration decreased from
41.6ppm to 35.6ppm. Complexation of AO7 with minor nutrients in the media could account for the change seen in the media without Pd. Abiotic controls consisting of media and Pd demonstrated that a small amount of Pd is reduced by headspace hydrogen as described earlier, forming a shiny metallic film at the interface of the media and the bottom of the glass bottle. Although a poor catalyst, this reduced Pd will degrade some amount of azo dyes using the headspace hydrogen and could explain the slightly higher removal of
AO7 observed in Pd‐containing control. The initial values in Figure 3.3 do not begin at
50ppm AO7 because the time required for sample preparation was neglected. Sample preparation took approximately 15min. In the 15min during which concentrations were not recorded, the concentration in each bottle decreased. 26
Figure 3.3 also shows the degradation of AO7 with heat‐killed faction as a function of time. The dye was added after the media had been autoclaved and purged of hydrogen.
Specifically, samples were purged to eliminate the biogenically generated hydrogen as an electron donor to exclude the effect of hydrogen in the degradation process. Centrifugation of cultures, performed in all experiments containing microbial cells (live or heat‐killed), always leads to some loss of concentration of AO7, as monitored using a spectrophotometer due to biosorption of the dye onto the cells. This biosorption could be attributed to the peptidoglycan layers around the Gram‐positive C. pasteurianum [84]. Knapp and Newbym showed adsorption in cells as a possible reason for decrease in dye concentration [85].
Figure 3.3 shows that there is no significant reduction in the concentration of AO7 in heat‐ killed controls with and without Pd. Confirmation of hypotheses (I) and (III) are revealed through figure 3.3, which illustrates (i) that bio‐nanopalladium acts as a catalyst for the degradation of azo dyes and (ii) living microbes and bio‐nanopalladium are needed for degradation.
Living microbial cultures without the addition of Pd show an initial decline in AO7
concentration and the curve can be compared to two‐zero order curves. Figure 3.5a shows
the degradation of 50 and 100ppm AO7 with 1ppm Pd(0).
The initial downward curve represents dye being absorbed by the cells. When the
cell surface binding sites are fully saturated, the concentration does not show a dramatic
decrease, which is similar to a second zero‐order curve. The steady decrease in the almost
horizontal curve, in Figure 3.5 b and c, is a direct result from either centrifugation removing
AO7 from the solution and pelletizing it with biomass, or from cell replication creating more
binding sites for the adsorption of the dye. Cell replication is a more likely scenario, as the
1ppm Pd curves does not show a gradual decline in concentration. 27
Figure 3.5: Degradation of AO7 (50 and 100ppm) at 1ppm (a), 3ppm (b) and 5ppm (c) Pd. In a, b and c, all 100ppm AO7 curves show a slower percent degradation, but a higher overall degradation. Palladium concentrations of 3 and 5ppm show different time required for total dye degradation at different dye concentrations. 28
Another study suggests that the mechanism behind reduction in concentration is enzymatic rather than adsorptive, which is indicative of Clostridium spp. possessing azoreductace activity [86]. The rapid decrease in concentration, before time zero, could then be attributed to high enzymatic concentration before inoculation, followed by slow regeneration of enzymes as metabolic growth occurs.
Figures 3.5 b and c show exponential decrease in dye concentration indicative of first order rate kinetics. Mendez‐Paz et al. also found a first order degradation kinetics using a microbial culture [87]. Longer degradation times are required for the higher dye concentration in 3 and 5ppm Pd samples for complete dye degradation. Additional time is required for complete degradation of AO7 at a concentration of 100ppm.
Rate of Reaction
For the purposes of this study, a simple first order model, as shown by equation 3.1, will be used.
− = (3.1)
The degradation curves resemble a first order curve, which is the reason behind choosing
first order kinetics. The initial rate of reaction is determined through the following
equation:
ln = (3.2) t
Time (t) in the above equation represents the initial time interval between the first two measurements. The initial time for 1ppm, 3ppm and 5ppm Pd(0) is 0.5hr, 1hr and 2hr, respectively. 29
Figure 3.6: The graphs above show the exponential degradation of AO7 and a model generated using a 1st order reaction rate at 50ppm (left) and 100ppm (right) with 3ppm Pd.
First order rate models compared to dye degradation are shown in Figure 3.6. The model, when compared to degradation of 50 and 100ppm AO7 with 3ppm Pd mimicked the
AO7 degradation curve. The curve similarities for both 50 and 100ppm AO7 are 99.9% and
98.3%, respectively. The similarities in the curves further confirm the first order rate of reaction.
Biocatalytic reduction of azo dyes using palladium
NanoPd(0), in the presence of living cultures of C. pasteurianum BC1, leads to maximum degradation of the azo dye. NanoPd acts as a catalyst for the reductive hydrogenation of the double bonded nitrogen group in azo compounds. Thus, hydrogen becomes the electron donor necessary for the reduction reaction to take place. This hydrogen is produced by C. pasteurianum BC1 as a metabolic byproduct.
The percent degradation was calculated as follows:
% degradation = 1 ‐ AO7 amount measured x 100 (3.3) Total amount of AO7 at time zero
30
Greater than 90% degradation was observed within two hours for both 3ppm and 5ppm Pd with 50ppm AO7. Within two hours 39.76±0.23ppm of AO7 was degraded in the presence of 5ppm Pd yielding an initial rate of reaction of 2.84hr‐1. The azo dye degradation rate for
5ppm is 126% higher than 3ppm with only a 66% increase in the amount of Pd. The reason
behind the faster rate at higher Pd concentration could be the presence of more catalytic
sites for degradation of AO7. Also, Pd has been shown as an excellent species for the
adsorption of hydrogen [88] and could further explain the increase in AO7 degradation rate
seen with increasing concentration of palladium. Hypothesis (I) is confirmed by figures 3.5 a, b and c, which show faster azo reduction rates in the presence of bio‐nanopalladium compared to samples without bio‐nanopalladium. Our results show 5ppm Pd concentration
to be a much better choice than 1ppm or 3ppm Pd concentrations, as it has a much higher
reaction rate and a much more expedient total degradation.
Table 3.1: Degradation rates for AO7 concentrations at 50 and 100ppm with palladium at 1, 3 and 5ppm. N/A illustrated the inability of 1ppm Pd to fully degrade the given concentrations of AO7. The initial rate of reaction for each concentration was calculated 2hr, 1hr and 0.5hr for 1ppm, 3ppm and 5ppm, respectively. AO7 Concentration 1ppm 3ppm 5ppm (ppm) % degradation @ 50 71.98±0.36 91.01±1.24 99.04±0.23 2hr Initial rate of 50 0.63 1.86 2.84 reaction(1/hr) Time required 50 for 100% N/A 8 2 degradation (hr) % degradation @ 100 30.54±5.02 74.21±1.51 84.54±2.28 2hr Initial rate of 100 0.39 0.89 1.94 reaction(1/hr) Time required 100 for 100% N/A 52 6 degradation (hr)
31
As seen in Table 3.1, the percent degradation of AO7 in the presence of 5ppm Pd at two hours is significantly higher when compared to 1 and 3ppm Pd. Although Table 3.1 shows a lower percent degradation for the experiments containing 100ppm concentration of azo dye, it should be noted that in the two hour time period, a higher total amount of azo dye is degraded when compared to the experiments conducted using 50ppm AO7.
Specifically at a given concentration of 5ppm Pd, degradation studies conducted using two different concentrations of azo dye, namely 50ppm and 100ppm AO7 demonstrate total
AO7 degradation in two hours of 39.76±0.23ppm and 84.54±2.28ppm, respectfully. These results show that high concentrations of azo dyes could be quickly degraded using the biocatalytic process at optimized concentration of catalyst.
Table 3.2: Viability tests performed on C. pasteurianum with 3ppm Pd and increasing concentrations of AO7. Samples were allowed to grow for 120 hrs before pressure and O.D. readings were taken. Concentration AO7 (ppm) Pressure (psi) O.D. at 600nm 50 33 1 100 28 0.9 200 0 0.05 500 0 0.05
Table 3.2 shows the pressure and optical density (O.D.) of C. pasteurianum BC1 samples from the viability studies conducted using increasing concentrations of AO7. C.
Pasteurianum BC1 was allowed to grow for 19 hours to late log phase before the introduction of 3ppm Pd into four serum bottles. 10 minutes were given to allow complete reduction of Pd(2+) to Pd(0). Then, AO7 was added in increasing concentration as follows:
50ppm, 100ppm, 200ppm and 500ppm. After addition of AO7, samples were incubated for an additional hour. Each bottle containing bacterial growth and AO7, four in all, were used as inoculums for four fresh bottles. Another 120hrs of growth were permitted for the four bottles before the pressure and O.D. were recorded. 32
As shown in Table 3.2, 200ppm and 500ppm concentrations of AO7 resulted in no change in pressure or optical density and leads to the hypothesis that nearly all, if not all, of the bacterial cells have been killed by the AO7, which is the reason for the lack of pressure and inability to grow as measured by O.D. Similar experiment was conducted without the addition of Pd, which resulted in the absence of growth even after an entire week for all concentrations of AO7. Comparing the two tests, with Pd and without Pd, one can see that the addition of Pd helps mitigate the ill effects of the azo compound to the microorganism, which confirms hypothesis (IV). Xu et al. show the existence of peptides, called metallothioneins, in microbes that attach themselves to toxins surrounding the host cell in an attempt to keep them from damaging vital parts of the cell [89]. As the microbes absorb more of the AO7, the cell will inadvertently be damaged, as this microbial peptide is meant to function at low toxin levels. The Pd(0) on the cell surface is hypothesized to help the microbe by lowering the concentration of the azo dye to which the microbe is exposed.
Degradation products
Figure 3.7: Hypothesized breakdown of AO7. 33
The breakdown of 4‐(2‐Hydroxy‐1‐naphthylazo)benzenesulfonic acid sodium salt
(AO7) is a simple reduction reaction that breaks the nitrogen double bond connecting the 1‐
amino 2‐naphthol to sulfanilic acid, which are the two hypothesized degradation products.
This is shown in Figure 3.7.
There are two proposed methods for the reductive degradation of AO7: biological
and catalytic. Biologically mediated reduction of the dye is hypothesized to have a minimal
effect on the overall quantitative reduction, as the dye is toxic to the microbes and in the
absence of the catalyst was found to lead to cell death. In biological reduction, the azo bond
cleavage proceeds through two stages involving the transfer of four electrons. In each stage
two electrons are transferred to the dye. The hydrogenation reaction is likely mediated by a
redox mediator (i. e. NADH, FMNH2, FADH2). The redox mediators donate the associated
hydrogen and the reduction of the dye is complete [90]. Electron density around the azo
bond controls the extent to which the azo dyes are reduced. As the electron density is
decreased, the azo group is more easily reduced and the aromatic amine is released.
Charged functional groups in the proximity of the dye or the presence of a second polar
compound will interfere with the reaction. Electron density is negatively affected by
electron‐withdrawing groups. The hydrogenation reaction occurs when an electron‐donor
group is present in the ortho‐position with respect to the azo linkage [91].
The chemical method proposed follows Figure 3.8. Step one in Figure 3.8 starts
after the first protonization has occurred. Microbes produce electrons, which can be used
effectively as one‐electron donors to reduce the dye to its radical anion. Radical anions of
substituted azobenzenes are reported to have pKa values in the range of ca. 13‐18 and to protonate rapidly to form hydrazyl radicals at neutral pH [92, 93]. Radical anions of substituted azobenzenes are reported to have pKa values in the range of ca. 13‐18 and to 34
protonate rapidly to form hydrazyl radicals at neutral pH [92, 93]. Studies have shown the rapid protonization of dye radical anions at a neutral pH [94]. AO7 is reported to have a higher pH when compared to similar dyes. Since AO7 has a higher pH, it is likely that its radical anion also has a higher pKa; therefore, it is more likely to protonate rapidly to give the hydrazyl radical (structure 3 in Figure 3.8). Disproportionation decay produces two compounds: the parent dye (1) and the hydrazine (4). The 2‐hydroxly group on (4) enables the rapid decay to give sulfanilic acid (5) and 1‐amino 2‐naphthol [95].
Figure 3.8: Electrochemical pathways for the degradation of AO7 [94].
Experiments were also conducted with biogenerated palladium nanoparticles and heat‐killed microbes, as mentioned above; however, the head space gas was not purged with nitrogen. The combination of bio‐nanopalladium and hydrogen gas did not
demonstrate efficient degradation of the azo dye. The highly limited degradation is
hypothesized as being due to low solubility of hydrogen gas with water. If an insignificant
amount of hydrogen is dissolved in solution, then most of the degradation will need to occur
at the interface between the head‐space gas and the spent media contains nanoparticles.
The chemical reduction is then surface area limited; therefore, reduction rates are also 35
limited. With combination of bio‐nanopalladium and viable microbial cells, an increased reduction rate is observed. A proximity effect could be the reason behind this increased reduction rate. Living microbial cells will produce localized hydrogen, which is in proximity of the bio‐nanopalladium and azo dye. This synergy ultimately leads to increased reaction kinetics.
Ultra Violet‐Visible Spectrophotometry
Figure 3.9: UV‐Vis spectrum of cultures containing 5ppm Pd and 50ppm AO7 obtained at 0.25hr, 0.75hr, 1.417hr and 3.25hr.
Figure 3.9 shows the degradation trend for a 5ppm Pd and 50ppm AO7 sample.
Attached to each curve in the figure are photos corresponding to the color of the AO7 at different stages in the breakdown. The peak for the AO7 can be observed at 484nm. As time progresses, the AO7 peak disappears along with a sub peaks at 304nm. 36
Figure 3.10: Comparison of concentrated degradation products with stock dye solution on UV‐Vis. The blue curve shows peaks at 330nm and 286nm, which are consistent with those for 2‐naphthol (per NIST). The 484nm peak in the red line shows the presence of azo dye prior to degradation.
UV‐Vis spectrum of the breakdown of AO7 shows an increase in two peaks 330nm and 286nm as shown in Figure 3.10. These two peaks agree with values of those expected for 2‐naphthol as obtained from the NIST database [96]. The spectrum showing the degradation products was obtained from analyzing a culture containing approximately
100ppm Pd and 1500ppm AO7. With a Pd concentration of 100ppm, large quantities of dye can be reduced rapidly. AO7 degradation using 100ppm Pd is not shown in this paper; however, no standard information was found for 1‐amino 2‐naphthol, but UV‐Vis spectrums are expected to be similar. Reduction of AO7 into the 1‐amino 2‐naphthol and sulfanilic acid products, has been reported [97, 98].
Gas Chromatography
GC‐MS of the degradation products indicated 2‐Naphthol was an 83% match to the
NIST website as shown in Figure 3.11. Figure 3.12 shows the chromatogram obtained from 37
the GC‐MS. The first peak shown in the chromatogram shows the presence of 2‐naphthol at
27min. The second peak shows the presence of an autooxidation product, dibutyl phthalate at 55min. When analyzed with the mass spectrometer attached in line with the GC, the first peak is found to resemble 2‐naphthol with an accuracy of 83% based on the values in the
NIST database [96]. Unfortunately, 1‐amino 2‐naphthol is not in the NIST database, and therefore we could not identify its presence in our samples.
Figure 3.11: Mass Spectrum showing 2‐naphthol as possible degradation product. The top graph shows the actual values attained from the mass spectrometer on the azo dye degradation products. The bottom graph is the NIST standard for 2‐naphthol
Figure 3.12: Chromatogram of the azo dye degradation products as observed using GC‐MS.
The second peak is attributed to dibutyl phthalate with 95% accuracy based on NIST database, as shown in figure 3.13 [99]. Dibutyl phthalate can also be seen in the UV‐Vis as a small peak at 275nm, as shown by Figure 3.9 and in agreement with NIST database [99]. 38
Dibutyl phthalate is not expected to be a compound resulting from the reductive degradation of AO7. However, the autooxidation of 1‐amino 2‐naphthol, a dye degradation product, leads to the formation of dibutyl phthalate [100].
Figure 3.13: HPLC of the degradation products as compared to spent media and sulfanilic acid standard. The black curve represents degradation products, pink represents spent media and blue represents sulfanilic acid.
High‐performance liquid chromatography
HPLC samples were analyzed using the procedure from Liu et al. [97]. Figure 3.13 gives the breakdown products formed by the degradation of AO7 when compared to standards of media and sulfanilic acid. Neither media nor sulfanilic acid are observed at this wavelength, while degradation products are formed that are sensitive to this wavelength.
Sulfanilic acid may be broken down further by the microbial cells, as 254nm is the absorbance peak for sulfanilic acid, which should have been seen. 1‐amino 2‐naphthol could not be used as a standard as it undergoes autooxidation, but based on the NIST webbook for the adsorption peaks for 2‐naphthol, it is feasible that if 1‐amino 2‐naphthol 39
were to behaves as 2‐naphthol, then 1‐amino 2‐naphthol would be present in the products.
The autooxidation of 1‐amino 2‐naphthol is slow enough to obtain peaks for 1‐amino 2‐ naphthol. Multiple degradation peaks are visible and this is in disagreement with Liu et al.
[97]. Analysis by Liu et al. shows only one peak at 2min, whereas this study shows multiple
peaks over the range of 1‐8min. The large peak observed by Liu et al. is also present in this
HPLC, but at an earlier time. The lack of subsequent peaks in the spectrum obtained by Liu
et al. can be attributed to the further breakdown of the hypothesized breakdown products
by Zero Valent Iron (ZVI).
1min
Figure 3.14: Magnified HPLC of dye degradation products compared to spent media and sulfanilic acid. The high number of peaks in the dye degradation products proves the presence of degradation products. The black curve represents degradation products, pink represents spent media and blue represents sulfanilic acid.
Figure 3.14 shows a magnified HPLC spectrum of the AO7 degradation products as
compared to spent media and sulfanilic acid. The spent media shows a small peak at 1min,
which is similar to the AO7 degradation peak at the same time, as shown in figure 3.14. The
two samples have the same peak, suggesting the constituent being observed is formed 40
during cellular reproduction. The AO7 degradation curve shows absorbance at a wide range of time, unlike spent media and sulfanilic acid. These extra peaks are indicative of the production of degradation products.
Matrix‐Assisted Laser Desorption/Ionization
Figure 3.15a: MALDI spectrum of the matrix background on which the samples were prepared. The circled area shows the absence of 1‐amino 2‐naphthol.
Figure 3.15b: MALDI spectrum of dye degradation products. The circled area shows an increase in the intensity seen at a molecular weight of 160, which is attributed to 1‐amino 2‐ naphthol.
MALDI analysis provides an accurate representation of the molecular weight of
products formed through degradation. Figure 3.15a shows the MALDI matrix, or
background. Figure 3.15b shows the MALDI spectrum of degradation products formed from 41
AO7. Comparing Figure 3.15a with figure 3.15b shows a peak at molecular weight of 160 to be present only in the spectrum of the degradation products. This peak at molecular weight of 160 is in agreement with the molecular weight expected from 1‐amino 2‐naphthol. This result provides proof to the existence of one of the main degradation products, 1‐amino 2‐ naphthol. 1‐amino 2‐naphthol does autooxidize over time and can be seen visually by the formation of a brownish orange color. At the time of MALDI analysis, the sample showed nearly zero formation of the oxidative products. MALDI samples were conducted immediately after removal from anaerobic conditions, which can explain the lack of autooxidation products. Figure 3.15b shows decreases in peaks found in Figure 3.15a.
These decreases in peak size results from the dilution of the matrix with the sample.
Sulfanilic acid, the second main hypothesized degradation product, would be expected at the molecular weight of 212 peak. Once again, comparing the sample to the
matrix shows a clear increase in the intensity of the peak at 212; this peak is generated
either through cellular reproduction or dye degradation. Spent media of C. pasteurianum
BC1 was also analyzed using the MALDI to verify that the peaks observed were not due to
metabolites formed during cell growth and the resulting spectrum is shown in figure 3.16.
Figure 3.16 shows a large peak at molecular weight of 212, which contradicts the presence
of sulfanilic acid in the solution to be entirely from the degradation of the dye. When
compared to the matrix, the spent media shows an increase in the intensity indicated at
molecular weight of 212, implying that a molecule with a molecular weight of 212 is
generated during cellular reproduction. Since Figure 3.16 does not contain a peak at
molecular weight of 160, 1‐amino 2‐naphthol can still be considered a degradation product
and the peak at molecular weight of 160 observed in figure 3.15b be assigned to 1‐amino 2‐
naphthol. 42
Figure 3.16: MALDI of the spent media of C. pasteurianum BC1 after 19hrs of growth. The increase in the peak at molecular weight of 212 shows formation of molecule with molecular weight similar to sulfanilic acid.
The presence of breakdown products shown through the analytical methods confirms hypothesis (II) in chapter 1.
Effectiveness of nanobiocatalytic reduction of azo dyes
Most microbes, are unable to degrade azo compounds, as bacteria have an innate
ability to absorb the dye instead of degrading them. Adsorption is a timely process as
shown by Pagga and Taeger [31]. After 24 hours of contact with the AO7, the activated
sludge had only adsorbed 10% of the initial starting concentration of 215ppm; however,
90% decolorization was obtained after 14 days [31]. In this study, 90% degradation occurred in less than two hours at both 3ppm and 5ppm. Kocuria rosea MTCC 1532, another decolorizing bacterium showed improved results over activated sludge, albeit at lower dye concentration [101]. For a starting concentration of methyl orange at 10ppm, it took 48 hours for 100% decolorization; in comparison, C. pasteurianum BC1, in the presence of bio‐ nanopalladium, needed only three hours to degrade five times the starting concentration.
More complete degradation than that observed by Pagga and Taeger has been achieved via photo degradation. Degradation of methyl orange was shown to be 90.8% degraded after 40min using vacuum ultraviolet light and a starting concentration of 10ppm. 43
Oxygen was used to increase the degradation of methyl orange to 94.1% in 20min [102].
The advantage of this process is lack of a catalyst; however, it is not easy to achieve these conditions in subsurface or in large areas. Daneshaver et al. reported that 100% degradation could be achieved within 60min using photedegradation and a zinc oxide
nanopowder catalyst [103]. The catalyst and dye concentration needed to achieve this
100% degradation in 60min was 160ppm and 20ppm, respectively. It should be noted that
the concentration of catalyst required is 8 times the concentration of azo dye degraded. The
ratio of azo dye to catalyst is the inverse of what is needed in this work.
Using the spent media of growing microbial culture is an economical approach to
dye degradation; however, it is a time consuming process. Tauber et al. reported a 50% degradation of Acid Orange 52 and Direct Blue 71 after 2 hours with a starting concentration of just 100µmol [26]; whereas, in this study a degradation of 74.21%±1.51 is observed in only 2 hours with a starting concentration of 100ppm and 3ppm Pd. The initial rate of reaction for the previous study, as shown by UV‐Vis, was 0.36±0.2hr‐1 and 1.7±.1hr‐1 for Acid Orange 52 and Direct Blue 71, respectively [26]. The initial rate of reaction shown in this study with same conditions as mentioned above was 0.89hr‐1. The advantage of the
process used by Tauber et al. is that there will be little to no need for removal of the enzyme, as it should be environmentally benign. The disadvantage of the process used by
Tauber et al. is the slower rate of degradation and the complicated method of enzyme extraction.
44
Table 3.3: Table comparing rates and amounts of dye degradation using various bacteria. The table is arranged with the slower degrading microbes at the top and more rapid degradation at the bottom. Bacterial species Dyes Comments References The E. coli improved the ability of Pseudomonas sp. to Pseudomonas decolorize the dye by luteola, Escherichia Reactive Red 22 [60] producing decolorization‐ coli stimulating extracellular metabolites using >1000ppm The complete decolorization of 100ppm was achieved at Escherichia coli, Congo Red, Direct the end of 9 days of incubation [46] pseudomonas sp. Black 38 in case of E. coli while Pseudomonas sp. Decolorized in 5 days The strain was capable of degrading dye in a wide range Pseudomonas sp. Reactive Red 2 of concentrations (up to [62] SUK1 5000ppm) and almost 80% dye was removed in 114hr Remazol Black, Maxilon Blue, The bacterium was capable of Sulfonyl Scarlet decolorizing 5000ppm of the Halomonas sp. BNLE, Sulfonyl dyes in a wide range of NaCl [51] Green BLE, Remazol concentrations after 4 days of Black N, Entrazol incubation period Blue IBC Monoazo dyes RY107 and RR Reactive Yellow 107, 198 were decolorized at Klabisiella sp. VN‐ reactive Red 198, 100ppm in 72 and 96hr; the [53] 31 reactive Black 5, diazo dyes (RB5 and triazodye Direct Blue 71 DB71) decolorized in 120 and 168hr The 59‐99% color removal after 206 days static Reactive azo dyes, Pseudomonas incubation, at the dye Direct azo dyes and [57] luteola concentration of 100ppm, leather dyes monoazo dyes showing fastest rate of decolorization Decolorization of DBMR was Acinetobacter 91.3% in static anoxic Direct brown MR calcoaceticus condition, whereas agitated [32] (DBMR) NCIM 2890 cultures showed less (59.3%) after 48hr
45
Table 3.3: (cont.) P. aeruginosa showed Methyl Orange, Y87, decolorization efficiency over Pseudomonas B86, R91, B19, R90, 98% after 48hr while 76% aeruginosa, B69, B31, B36, Y15, decolorization was achieved [58] P. oleovarons, R34, B15, Y79 AND by P. oleovarons after 54hr. P. P. putida B54 putida showed lower efficiency of 50ppm dye The dyes were completely Disperse Blue 79, Bacillus fusiformis mineralized within 48hr at a [38] Acid Orange 10 concentration of 500ppm The dye up to 700ppm Rhodopseudomonas Reactive Black 5 concentration was completely [63] palustris decolorized in 40hr Complete biodegradation of azo dye up to 200ppm was achieved in 49hr under Pseudomonas Acid Violet 7 shaking while the [61] putida mt‐2 biodegradation time was reduced to 37hr under static conditions The bacterial consortium Paenibacillus showed complete polymyxa, Reactive Violet 5R [55] decolorization of 100ppm in Micrococcus luteus 36hr About 95% dye (200ppm) was Citrobacter sp. CK3 Reactive Red 180 [43] removed in 36hr The bacterium removed 53‐ 63% of the dye (25‐100ppm)in Entrerococcus Direct Black 38 24hr in minimal medium while [44] gallinarum 71‐85% of decolorization was observed in Luria broth media Bacillus The dye was decolorized up to Acid Red 119 [41] thurengiensis 70% of 15‐1000ppm in 24hr Reactive Brilliant Red X, Acid Black 10B, Acid Scarlet GR, The decolorization of the dyes Halomonas sp. [48‐50] Acid Red B, Acid Red was up to 90% in 24hr G, Reactive Brilliant Red K The first four dyes decolorized by the bacterium by 100% Amaranth, fast R, while the remaining three Ponceau S, Congo R, Kerstersia sp. VKY1 decolorized by 84, 73 and [52] Orange II, Acid O 12, 44%, respectively, in 24hr. all Acid R 151 dye concentration was 100ppm
46
Table 3.3: (cont.) The complete decolorization Bacillus sp. Congored was achieved in 24‐27 hr for a [39] concentration of 100‐300ppm The bacterium was capable of Sphingomonas decolorizing bromoamine acid Anthraqiunone dyes [67] herbicidovorans dye (1000ppm) more than 98% within 24hr Aeromonas caviae, More than 90% decolorization Proteus mirabilis, Acid Orange 7 of the dye (200ppm) was [34] Rhodococcus sp. achieved in 16hr Shewanella Fast Acid Red GR, The 90% decolorization of the decolorationis sp. reactive Brilliant dyes were achieved within [65] nov. S 12T Blue 12hr The dye GHE4B was Pseudomonas Direct Blue 6, Green completely decolorized in 12hr [59] desmolyticum HE4B, Red HE7B while DB 6 and RHE7B were decolorized in 16hr, 100ppm After acclimation, time for Escherichia coli Reactive Red 22 50% color removal lowered [45] NO3 from 5.7 to 4.3hr The dye (25ppm) was Bacillus velezensis Direct Red 28 completely decolorized within [42] AB 10hr Acinotobacter sp., Acid Red 88,Reactive The mixed culture of bacteria Citrobacter Black 5, Direct Red removed 88‐100% dyes [33] freundii, Klebsiella 81, Disperse Orange (100ppm) in 10hr oxytoca 3 Under the near‐optimal conditions, 99% of the Bacillus subtilis HM Fast Red [40] decolorization was achieved in 6hr After 4 hr incubation, more than 90% of the color was removed under anaerobic Shewanella Fast Acid Red GR conditions while 12.8 and [64] decolorationis S12 33.7% decolorizing rates were observed under aerobic and microaerophilic conditions Proteus vulgaris, Bacterial consortium Micrococcus Scarlet R decolorized 90% of 50‐ [56] glutamicus 250ppm in 3hr
47
Table 3.3: (cont.) Anaerobic cultures of Shewanella strain J18 143 rapidly removed color from Remazol Black B, Shewanella J18 143 the azo dye Remazol Black B in [66] Acid Orange 7 the growth medium to produce an absorbance at 597nm of less than 1 in under 40min Treatment times required by most efficient strain, AS96 (Shewanella putrefaciens), Aeromonas, Reactive Black 5, were as short as 4hr for Pseudomonas, Direct Red 81, Acid complete decolorization of Bacillus, [35, 36] Red 88, Disperse 100ppm of AR‐88 and DR‐81 Shewanella and Orange 3 dyes under static conditions, Massillia spp. and 6 and 8hr, respectively, for complete decolorization of RB‐ 5 and DO‐3 High decolorization efficiency (95‐98%) achieved within 6 hr for 100mM Acid Blue 25 Acid Blue 25, (anthraquinone dye), 4hr for Bacillus cereus Malachite Green, 55mM Malachite Green [37] DC11 Basic Blue (triphenylmethane dye), and 2 hr for 750mM Basic Blue X‐ GRRL under anaerobic conditions The dye was decolorized up to Escherichia coli YB Acid Red 27 [47] 75% in 2hr Lactobacillus casei The complete decolorization of Methyl Orange [54] TISTR 1500 the dye was achieved in 2.5hr The complete decolorization of Clostridium Current Acid Orange 7 dye (50ppm) was achieved in pasteurianum BC1 Study 2hr with 5ppm Pd catalyst
Table 3.3 shows that out of 34 studies conducted on the degradation of acid azo
dyes, concomitantly synthesized palladium nanocatalyst in the presence of Clostridium
pasteurianum BC1 shows the fastest rate of degradation of this class of azo dyes. Some of
the studies above used higher concentrations of dye compared to the present study, but
there is always the option to increase the palladium concentration and thereby allow an
increase in the total amount of dye that is degraded. 48
Some studies have shown high reduction reaction rates through chemical processes.
High degradation rates of 18.36hr‐1 have been achieved using Zero Valent Iron (ZVI); a
degradation rate that is 22 times more rapid than the one observed in this study [104]. This
reaction rate is accomplished with a starting contaminate concentration of 100ppm and a
starting ZVI concentration of 200ppm. It should be noted that ZVI is not a catalyst and is a
chemical reactant, as it will be consumed in the reaction and the concentration of ZVI will
decrease with increasing amount of azo dye reduction. Also, ZVI has to be prepared away
from the contaminated site and delivered to the site of contamination; however, ZVI is very
reactive and easily oxidizes in the atmosphere, which make storage, transportation and
delivery to contaminated site a challenge [72]. The use of Clostridium pasteurianum BC1 with bio‐nanopalladium, demonstrated in this study, allows for the application of our process in‐situ. The catalyst will be generated in‐situ at the site of contamination.
It should be emphasized that the cost of palladium is not a factor as concentrations used are in ppm levels. Further, use of Pd to treat groundwater contamination is acceptable as concentrations are very low and Pd is often found at these concentrations as along roadways as resultant of use of Pd in catalytic convertors in automobiles. Finally, any concern of residual Pd in groundwater appearing in our drinking water is unnecessary as most nanopalladium was found on cellular surface and municipal treatment of drinking water always filters microorganisms, and would thereby lead to removal of cell‐bound nanopalladium.
49
Chapter 4‐Conclusion
The catalytic properties of various metal nanoparticles have led to their use in environmental remediation applications. However, these remediation strategies are limited by their ability to deliver catalytic nanoparticles and hydrogen as a suitable electron donor to large treatment zones. Our aim was to develop and apply an efficient bioremediation method based on in‐situ biosynthesis of bio‐nanopalladium and hydrogen. C. pasteurianum
BC1 cells, loaded with bio‐nanopalladium, were used to effectively catalyze the reductive degradation and removal of Acid Orange 7 (AO7). While C. pasteurianum BC1 cells loaded with bio‐nanopalladium have been shown to catalyze the removal of inorganic contaminants, such as hexavalent chromium [82], this study represents the first instance of its application in the degradation of organic pollutants. Hydrogen produced fermentatively by the C. pasteurianum BC1 acted as the electron donor for the process, thus eliminating the need to add an external source as required by the process developed by De Windt et al. [77] and Lloyd et al. [83].
Bio‐nanopalladium was formed using cultures of C. pasteurianum BC1. The morphology of the bio‐nanopalladium was observed using a SEM to confirm the formation of nanoparticles. Samples containing Pd(0) and controls without Pd(0) were inoculated with AO7 and the residual concentration of AO7 was monitored with time. Living bacterial cultures containing bio‐nanopalladium showed significantly faster reductive degradation of the azo compound when compared to Pd(0)‐free or heat‐killed controls. Pd‐free bacterial cultures or control experiments conducted with heat‐killed cells showed negligible reduction of AO7. Our experiments also showed that the in‐situ biological production of H2 by C. pasteurianum BC1 was essential for the degradation of AO7, which suggests a novel 50
process where the in‐situ microbial production of hydrogen is directly coupled to the catalytic bio‐nanopalladium mediated reduction of AO7. These results are confirmed via analysis conducted using UV‐Vis spectroscopy. A full spectrum analysis from 190nm‐
900nm was done on all samples using UV‐Vis to determine absorbance. Concentration of
AO7 can be monitored via intensity of peak at 484nm. Concentration profiles were taken as a function of time in the presence of Pd(0) and without Pd(0). As time progressed, the intensity of absorbance at 484nm declined. Experiments containing 5ppm Pd showed a more rapid degradation of AO7 than those containing 1ppm and 3ppm Pd. For 50ppm AO7, the initial rates of reaction increased as Pd concentration increased; 0.63, 1.86 and 2.84hr‐1 were the initial rates of reaction for 1, 3 and 5ppm Pd, respectively. A first order kinetic model was used to illustrate the reaction kinetics of samples containing both living microbes and bio‐nanopalladium. Zero order curves were not modeled against experimental data, but were used in describing the rate of color change for samples without bio‐nanopalladium. AO7 concentrations of 50 and 100ppm were not fully degraded by cultures containing 1ppm Pd. The quickest degradation of 50ppm AO7 was achieved in experiments containing 5ppm Pd and was approximately 2hr. The spectrum showed the gradual decline in the 484nm peak as well as the increase in the 330nm, 286nm and 275nm peaks. The 330nm and 286nm peaks represented absorption peaks of 2‐naphthol, which were confirmed from the NIST database [96]. Other studies also found the same degradation products as found in this study [97, 98]. The minor peak at 275nm in the degradation products spectrum could indicate the presence of dibutyl phthalate, which is the autooxidation product of one of the two main hypothesized degradation products [100].
Further, MALDI analysis confirmed the presence of 1‐amino 2‐naphthol, a constituent of
AO7 as a breakdown product, in line with our hypotheses. Also, among 34 studies on 51
microbial remediation of acid azo dyes, the process developed and presented in this thesis appears to have the fastest rate of degradation.
Implication of the Research
This is the first study to combine C. pasteurianum BC1 with Pd to catalytically degrade an organic compound. Azo dyes account for ~10,500 tons of contaminants in the world and a simple and quick degradation method as demonstrated in this study would be highly beneficial to the environment [6, 7]. This method could be used to degrade huge volumes of these compounds in bioreactors prior to discharge.
Other studies have shown degradation of azo compound through either reductive or oxidative electro‐chemical pathways and studies have been conducted that use bacteria to decolorized textile effluent streams, but none have attempted combining a hydrogen generating microbe with Pd and azo compounds. This study further proves the advantages of using C. pasteurianum BC1 as a self mediating catalyst support. Also, this study characterized some of the degradation products from the reductive degradation of AO7 and thereby conclusively proved the removal of azo dyes to occur via degradation of the azo dye, whereas most studies focused on the decolorization of the compounds. Decolorization could result from biosorption and does not necessarily indicate the degradation of the colorant.
Future Directions
The further scope of this work would be to investigate the ability of this microbe to produce nanoparticles of other cheaper catalysts for the reductive degradation of azo dyes, or for the reductive capabilities to reduce toxins in water streams. Catalysts that should be explored include nickel, iron and ruthenium. The formation of these catalysts may be confirmed using XRD and XANES. Another area of interest would be to investigate the 52
ability of this microbe and catalyst to reduce/remove other toxins/pollutants. Some other contaminants that will be of interest are different azo dyes, arsenic, fertilizer and other problematic water stream toxins.
Resources could also be spent determining the role of the metallothioneins peptides that attaches to toxins and helps the viability of the microbes. If high production of these peptides helps increase the rate of degradation, that would be favorable, but if they damage the cell by absorbing a high quantity of toxins then it would be ideal to suppress their production.
Once a favorable catalyst is adopted into use, laboratory‐scale column‐experiments should be conducted. These experiments will include the introduction of a carbon source and a catalyst into soil column to simulate real world conditions. A simulated ground water stream containing the contaminants will then be pumped through a cylinder of soil containing C. pasteurianum BC1 and the catalyst. Laboratory‐scale column‐experiments will demonstrate the ability of the microbe and catalyst to reduce pollutants at the site of contamination.
Batch reactors may also be researched as a possible contamination prevention step.
The process described throughout this paper can also be adapted for continual use in an industrial scale batch reactor. The microbes can be held in an anaerobic reactor vessel and the catalyst and contaminant could be added at various times. The effluent stream, containing catalytic material can be removed for the catalyst recycling and reintroduced to another batch of growing microbes. This process could be repeated with minimal losses to the catalyst.
53
References
1. United States Environmental Protection Agency. Cleaning Up Our Land, Water and Air. http://www.epa.gov/cleanup/ (November 17, 2010), 2. United States Department of Energy. Office of Environmental Management. http://www.em.doe.gov/Pages/EMHome.aspx (November 14, 2010), 3. Zollinger, H., Color Chemistry: Synthesis, Properties and Applications of Organic Dyes and Pigments. 1st ed. ed.; VCH: New York, 1987. 4. Best Management Practices for Pollution Prevention in the Textile Industry. EPA Office of Research and Development, Office of Research and Development. 5. Snowden‐Swan, L. J., “Pollution Prevention in the Textile Industries,” in Industrial Pollution Prevention Handbook. Freeman, H.M. (Ed.) 1995, McGraw‐Hill, Inc, (New York). 6. Park, H.; Choi, W., Visible light and Fe(III)‐mediated degradation of Acid Orange 7 in the absence of H2O2. Journal of Photochemistry and Photobiology A: Chemistry 2003, 159, (3), 241‐247. 7. Vaidya, A. A.; Datye, K. V., Environmental pollution during chemical processing of synthetic fibres. Colourage 1982, 14, 3‐10. 8. Nilsson, R.; Nordlinder, R.; Wass, U.; Meding, B.; Belin, L., Asthma, rhinitis, and dermatitis in workers exposed to reactive dyes. British journal of industrial medicine 1993, 50, (1), 65‐70. 9. Brindley, L., July 8, 2009, "New solution for dye wastewater pollution"; http://www.rsc.org/chemistryworld/News/2009/July/08070901.asp; , Accessed November 14, 2010. 10. Arbroath, November 23, 2006, "Pollution turns Yellow River red;" http://arbroath.blogspot.com/2006/11/pollution‐turns‐yellow‐river‐red.html, Accessed November 14, 2010. 11. Jäger, I.; Hafner, C.; Schneider, K., Mutagenicity of different textile dye products in Salmonella typhimurium and mouse lymphoma cells. Mutation Research/Genetic Toxicology and Environmental Mutagenesis 2004, 561, (1‐2), 35‐44. 12. Sharma, M. K.; Sobti, R. C., Rec effect of certain textile dyes in Bacillus subtilis. Mutation Research/Genetic Toxicology and Environmental Mutagenesis 2000, 465, (1‐2), 27‐38. 13. Puvaneswari, N.; Muthukrishnan, J.; Gunasekaran, P., Toxicity assessment and microbial degradation of azo dyes. Indian Journal of Experimental Biology 2006, 44, 618‐626. 14. Joshi, M. M.; Labhsetwar, N. K.; Mangrulkar, P. A.; Tijare, S. N.; Kamble, S. P.; Rayalu, S. S., Visible light induced photoreduction of methyl orange by N‐doped mesoporous titania. Applied Catalysis A: General 2009, 357, (1), 26‐33. 15. Chatti, R.; Rayalu, S. S.; Dubey, N.; Labhsetwar, N.; Devotta, S., Solar‐based photoreduction of methyl orange using zeolite supported photocatalytic materials. Solar Energy Materials and Solar Cells 2007, 91, (2‐3), 180‐190. 16. Guettaï, N.; Ait Amar, H., Photocatalytic oxidation of methyl orange in presence of titanium dioxide in aqueous suspension. Part I: Parametric study. Desalination 2005, 185, (1‐3), 427‐437. 17. Sohn, Y. S.; Smith, Y. R.; Misra, M.; Subramanian, V., Electrochemically assisted photocatalytic degradation of methyl orange using anodized titanium dioxide nanotubes. Applied Catalysis B: Environmental 2008, 84, (3‐4), 372‐378. 54
18. Huang, M.; Xu, C.; Wu, Z.; Huang, Y.; Lin, J.; Wu, J., Photocatalytic discolorization of methyl orange solution by Pt modified TiO2 loaded on natural zeolite. Dyes and Pigments 2008, 77, (2), 327‐334. 19. Li, Y.; Li, X.; Li, J.; Yin, J., Photocatalytic degradation of methyl orange by TiO2‐coated activated carbon and kinetic study. Water Research 2006, 40, (6), 1119‐1126. 20. Wang, S.; Gong, Q.; Liang, J., Sonophotocatalytic degradation of methyl orange by carbon nanotube/TiO2 in aqueous solutions. Ultrasonics Sonochemistry 2009, 16, (2), 205‐208. 21. Kim, Y.; Lee, J.; Jeong, H.; Lee, Y.; Um, M.‐H.; Jeong, K. M.; Yeo, M.‐K.; Kang, M., Methyl orange removal over Zn‐incorporated TiO2 photo‐catalyst. Journal of Industrial and Engineering Chemistry 2008, 14, (3), 396‐400. 22. Konstantinou, I. K.; Albanis, T. A., TiO2‐assisted photocatalytic degradation of azo dyes in aqueous solution: kinetic and mechanistic investigations: A review. Applied Catalysis B: Environmental 2004, 49, (1), 1‐14. 23. Gomes, J. A.; Cocke, D. L.; Mahmud, M. A.; Moreno, H.; Peterson, E.; Mollah, M. Y.; Parga, J. R., Treatment of Orange II Azo Dye Using Electrochemical and Photo‐Oxidative Methods. ECS Transactions 2008, 6, (18), 29‐41. 24. Chahbane, N.; Lenoir, D.; Souabi, S.; Collins, T. J.; Schramm, K. W., FeIII‐TAML‐Catalyzed Green Oxidative Decolorization of Textile Dyes in Wastewater. CLEAN – Soil, Air, Water 2007, 35, (5), 459‐464. 25. Punj, S.; John, G. H., Purification and identification of an FMN‐dependent NAD(P)H azoreductase from Enterococcus faecalis. Curr Issues Mol Biol 2009, 11, (2), 59‐65. 26. Tauber, M. M.; Gübitz, G. M.; Rehorek, A., Degradation of azo dyes by oxidative processes ‐ Laccase and ultrasound treatment. Bioresource Technology 2008, 99, (10), 4213‐4220. 27. Robinson, T.; McMullan, G.; Marchant, R.; Nigam, P., Remediation of dyes in textile effluent: a critical review on current treatment technologies with a proposed alternative. Bioresource Technology 2001, 77, (3), 247‐255. 28. Barragán, B. E.; Costa, C.; Carmen Márquez, M., Biodegradation of azo dyes by bacteria inoculated on solid media. Dyes and Pigments 2007, 75, (1), 73‐81. 29. D., O. O.; Osuntoki, A. A.; Gbenle, G. O., Textile effluent biodegradation potentials of textile effluent‐adapted and non‐adapted bacteria. African Journal of Biotechnology 2006, 5, (20), 1980‐1984. 30. Kirby, N. Bioremediation of textile industry wastewater by white rot fungi. University of Ulster, Coleraine, 1999. 31. Pagga, U.; Taeger, K., Development of a method for adsorption of dyestuffs on activated sludge. Water Research 1994, 28, (5), 1051‐1057. 32. Ghodake, G.; Jadhav, S.; Dawkar, V.; Govindwar, S., Biodegradation of diazo dye Direct brown MR by Acinetobacter calcoaceticus NCIM 2890. International Biodeterioration & Biodegradation 2009, 63, (4), 433‐439. 33. Khalid, A.; Arshad, M.; Crowley, D. E., Biodegradation potential of pure and mixed bacterial cultures for removal of 4‐nitroaniline from textile dye wastewater. Water Research 2009, 43, (4), 1110‐1116. 34. Joshi, T.; Iyengar, L.; Singh, K.; Garg, S., Isolation, identification and application of novel bacterial consortium TJ‐1 for the decolourization of structurally different azo dyes. Bioresource Technology 2008, 99, (15), 7115‐7121. 55
35. Khalid, A.; Arshad, M.; Crowley, D. E., Accelerated decolorization of structurally different azo dyes by newly isolated bacterial strains. Applied Microbiology and Biotechnology 2008, 78, (2), 361‐369. 36. Khalid, A.; Arshad, M.; Crowley, D. E., Decolorization of azo dyes by Shewanella sp under saline conditions. Applied Microbiology and Biotechnology 2008, 79, (6), 1053‐1059. 37. Deng, D. Y.; Guo, J.; Zeng, G. Q.; Sun, G. P., Decolorization of anthraquinone, triphenylmethane and azo dyes by a new isolated Bacillus cereus strain DC11. International Biodeterioration & Biodegradation 2008, 62, (3), 263‐269. 38. Kolekar, Y. M.; Pawar, S. P.; Gawai, K. R.; Lokhande, P. D.; Shouche, Y. S.; Kodam, K. M., Decolorization and degradation of Disperse Blue 79 and Acid Orange 10, by Bacillus fusiformis KMK5 isolated from the textile dye contaminated soil. Bioresource Technology 2008, 99, (18), 8999‐9003. 39. Gopinath, K. P.; Sahib, H. A. M.; Muthukumar, K.; Velan, M., Improved biodegradation of Congored by using Bacillus sp. Bioresource Technology 2009, 100, (2), 670‐675. 40. Mabrouk, M.; Yousef, H., Decolorization of fast Red by Bacillus subtilis HM. J Appl Sci Res 2008, 4, 262‐268. 41. Dave, S. R.; Dave, R. H., Isolation and characterization of Bacillus thuringiensis for Acid red 119 dye decolourisation. Bioresource Technology 2009, 100, (1), 249‐253. 42. Bafana, A.; Chakrabarti, T.; Devi, S. S., Azoreductase and dye detoxification activities of Bacillus velezensis strain AB. Applied Microbiology and Biotechnology 2008, 77, (5), 1139‐1144. 43. Wang, H.; Su, J. Q.; Zheng, X. W.; Tian, Y.; Xiong, X. J.; Zheng, T. L., Bacterial decolorization and degradation of the reactive dye Reactive Red 180 by Citrobacter sp CK3. International Biodeterioration & Biodegradation 2009, 63, (4), 395‐399. 44. Bafana, A.; Krishnamurthi, K.; Devi, S. S.; Chakrabarti, T., Biological decolourization of CI direct black 38 by E‐gallinarum. Journal of Hazardous Materials 2008, 157, (1), 187‐193. 45. Chang, J. S.; Kuo, T. S.; Chao, Y. P.; Ho, J. Y.; Lin, P. J., Azo dye decolorization with a mutant Escherichia coli strain. Biotechnology Letters 2000, 22, (9), 807‐812. 46. Isik, M.; Sponza, D. T., Effect of oxygen on decolorization of azo dyes by Escherichia coli and Pseudomonas sp and fate of aromatic amines. Process Biochemistry 2003, 38, (8), 1183‐1192. 47. Liu, G. F.; Zhou, J. T.; Wang, J.; Zhou, M.; Lu, H.; Jin, R. F., Acceleration of azo dye decolorization by using quinone reductase activity of azoreductase and quinone redox mediator. Bioresource Technology 2009, 100, (11), 2791‐2795. 48. Guo, J.; Zhou, J.; Wang, D.; al, e., The new incorporation bio‐treatment technology of bromoamine acid and azo dyes wastewater under high salt conditions. Biodegr 2008, 19, 93‐98. 49. Guo, J.; Zhou, J.; Wang, D.; Tian, C.; Wang Ping, M.; Udden, S., A novel moderately halophilic bacterium for decolorizing azo dye under high salt conditions. Biodegr 2008, 19, (15‐19). 50. Guo, J.; Fang, M.; Jiang, K.; Cui, D., Bioaugmentation combined with biofilm process in the treatment of petrochemical wastewater at low temperatures. J Wat Resour Protect 2008, 1, 1‐65. 51. Asad, S.; Amoozegar, M. A.; Pourbabaee, A. A.; Sarbolouki, M. N.; Dastgheib, S. M. M., Decolorization of textile azo dyes by newly isolated halophilic and halotolerant bacteria. Bioresource Technology 2007, 98, (11), 2082‐2088. 56
52. Vijaykumar, M. H.; Vaishampayan, P. A.; Shouche, Y. S.; Karegoudar, T. B., Decolourization of naphthalene‐containing sulfonated azo dyes by Kerstersia sp strain VKY1. Enzyme and Microbial Technology 2007, 40, (2), 204‐211. 53. Franciscon, E.; Zille, A.; Fantinatti‐Garboggini, F.; Silva, I. S.; Cavaco‐Paulo, A.; Durrant, L. R., Microaerophilic‐aerobic sequential decolourization/biodegradation of textile azo dyes by a facultative Klebsiella sp strain VN‐31. Process Biochemistry 2009, 44, (4), 446‐ 452. 54. Seesuriyachan, P.; Takenaka, S.; Kuntiya, A.; Klayraung, S.; Murakmi, S.; Aoki, K., Metabolism of azo dyes by Lactobacillus casei TISTR 1500 and effects of various factors on decolorization. Water Research 2007, 41, (5), 985‐992. 55. Moosvi, S.; Kher, X.; Madamwar, D., Isolation, characterization and decolorization of textile dyes by a mixed bacterial consortium JW‐2. Dyes and Pigments 2007, 74, (3), 723‐ 729. 56. Saratale, R. G.; Saratale, G. D.; Kalyani, D. C.; Chang, J. S.; Govindwar, S. P., Enhanced decolorization and biodegradation of textile azo dye Scarlet R by using developed microbial consortium‐GR. Bioresource Technology 2009, 100, (9), 2493‐2500. 57. Hu, T. L., Kinetics of azoreductase and assessment of toxicity of metabolic products from azo dyes by Pseudomonas luteola. Water Science and Technology 2001, 43, (2), 261‐269. 58. Silveira, E.; Marques, P. P.; Silva, S. S.; Lima, J. L.; Porto, A. L. F.; Tambourgi, E. B., Selection of Pseudomonas for industrial textile dyes decolourization. International Biodeterioration & Biodegradation 2009, 63, (2), 230‐235. 59. Kalme, S.; Jadhav, S.; Jadhav, M.; Govindwar, S., Textile dye degrading laccase from Pseudomonas desmolyticum NCIM 2112. Enzyme and Microbial Technology 2009, 44, (2), 65‐71. 60. Chen, B. Y.; Chen, S. Y.; Lin, M. Y.; Chang, J. S., Exploring bioaugmentation strategies for azo‐dye decolorization using a mixed consortium of Pseudomonas luteola and Escherichia coli. Process Biochemistry 2006, 41, (7), 1574‐1581. 61. Ben Mansour, H.; Mosrati, R.; Corroler, D.; Ghedira, K.; Barillier, D.; Chekir, L., In vitro mutagenicity of Acid Violet 7 and its degradation products by Pseudomonas putida mt‐ 2: Correlation with chemical structures. Environmental Toxicology and Pharmacology 2009, 27, (2), 231‐236. 62. Kalyani, D. C.; Telke, A. A.; Dhanve, R. S.; Jadhav, J. P., Ecofriendly biodegradation and detoxification of Reactive Red 2 textile dye by newly isolated Pseudomonas sp SUK1. Journal of Hazardous Materials 2009, 163, (2‐3), 735‐742. 63. Wang, X. Z.; Cheng, X.; Sun, D. H.; Qi, H., Biodecolorization and partial mineralization of Reactive Black 5 by a strain of Rhodopseudomonas palustris. Journal of Environmental Sciences‐China 2008, 20, (10), 1218‐1225. 64. Xu, M. Y.; Guo, J.; Sun, G. P., Biodegradation of textile azo dye by Shewanella decolorationis S12 under microaerophilic conditions. Applied Microbiology and Biotechnology 2007, 76, (3), 719‐726. 65. Xu, M. Y.; Guo, J.; Cen, Y. H.; Zhong, X. Y.; Cao, W.; Sun, G. P., Shewanella decolorationis sp nov., a dye‐decolorizing bacterium isolated from activated sludge of a waste‐water treatment plant. International Journal of Systematic and Evolutionary Microbiology 2005, 55, 363‐368. 57
66. Pearce, C. I.; Christie, R.; Boothman, C.; von Canstein, H.; Guthrie, J. T.; Lloyd, J. R., Reactive azo dye reduction by Shewanella strain j18 143. Biotechnology and Bioengineering 2006, 95, (4), 692‐703. 67. Fan, L.; Zhu, S. N.; Liu, D. Q.; Ni, J. R., Decolorization of 1‐amino‐4‐bromoanthraquinone‐ 2‐sulfonic acid by a newly isolated strain of Sphingomonas herbicidovorans. International Biodeterioration & Biodegradation 2009, 63, (1), 88‐92. 68. Epolito, W. J.; Yang, H.; Bottomley, L. A.; Pavlostathis, S. G., Kinetics of zero‐valent iron reductive transformation of the anthraquinone dye Reactive Blue 4. Journal of Hazardous Materials 2008, 160, (2‐3), 594‐600. 69. Chang, S. H.; Chuang, S. H.; Li, H. C.; Liang, H. H.; Huang, L. C., Comparative study on the degradation of IC Remazol Brilliant Blue R and IC Acid Black 1 by Fenton oxidation and Fe‐0/air process and toxicity evaluation. Journal of Hazardous Materials 2009, 166, (2‐ 3), 1279‐1288. 70. Chang, S. H.; Wang, K. S.; Chao, S. J.; Peng, T. H.; Huang, L. C., Degradation of azo and anthraquinone dyes by a low‐cost Fe‐0/air Process. Journal of Hazardous Materials 2009, 166, (2‐3), 1127‐1133. 71. Fan, J.; Guo, Y. H.; Wang, J. J.; Fan, M. H., Rapid decolorization of azo dye methyl orange in aqueous solution by nanoscale zerovalent iron particles. Journal of Hazardous Materials 2009, 166, (2‐3), 904‐910. 72. Sohn, K.; Kang, S. W.; Ahn, S.; Woo, M.; Yang, S.‐K., Fe(0) Nanoparticles for Nitrate Reduction: Stability, Reactivity, and Transformation. Environmental Science & Technology 2006, 40, (17), 5514‐5519. 73. Wang, J.; Jiang, Z.; Zhang, Z. H.; Xie, Y. P.; Wang, X. F.; Xing, Z. Q.; Xu, R.; Zhang, X. D., Sonocatalytic degradation of acid red B and rhodamine B catalyzed by nano‐sized ZnO powder under ultrasonic irradiation. Ultrasonics Sonochemistry 2008, 15, (5), 768‐774. 74. Hayat, K.; Gondal, M. A.; Khaled, M. M.; Ahmed, S., Kinetic study of laser‐induced photocatalytic degradation of dye (alizarin yellow) from wastewater using nanostructured ZnO. Journal of Environmental Science and Health Part a‐ Toxic/Hazardous Substances & Environmental Engineering 2010, 45, (11), 1413‐1420. 75. Rupa, A. V.; Manikandan, D.; Divakar, D.; Sivakumar, T., Effect of deposition of Ag on TiO2 nanoparticles on the photodegradation of Reactive Yellow‐17. Journal of Hazardous Materials 2007, 147, (3), 906‐913. 76. Sobana, N.; Muruganadham, M.; Swaminathan, M., Nano‐Ag particles doped TiO2 for efficient photodegradation of Direct azo dyes. J Mol Catal a‐Chem 2006, 258, (1‐2), 124‐ 132. 77. Windt, W. D.; Aelterman, P.; Verstraete, W., Bioreductive deposition of palladium (0) nanoparticles on Shewanella oneidensis with catalytic activity towards reductive dechlorination of polychlorinated biphenyls. Environmental Microbiology 2005, 7, (3), 314‐325. 78. Lloyd, J. R., Microbial reduction of metals and radionuclides. Fems Microbiology Reviews 2003, 27, (2‐3), 411‐425. 79. Vyjayanthi, J. P.; Suresh, S., Decolorization of Drimarene Red Dye Using Palladized Bacterial Cellulose in a Reactor. Water Environment Research 2010, 82, (7), 601‐609. 80. Chidambaram, D.; Hennebel, T.; Taghavi, S.; Mast, J.; Boon, N.; Verstraete, W.; van der Lelie, D.; Fitts, J. P., Concomitant Microbial Generation of Palladium Nanoparticles and 58
Hydrogen To Immobilize Chromate. Environmental Science & Technology 2010, 44, (19), 7635‐7640. 81. Dodge, C. J.; Francis, A. J., Anaerobic microbial dossolution of transition and heavy metal toxins. Applied Environmental Microbiology 1988, 54, 1009‐1014. 82. Chidambaram, D.; Hennebel, T.; Taghavi, S.; Mast, J.; Boon, N.; Verstraete, W.; Van Der Lelie, D.; Fitts, J., A one step process for in situ biogenic palladium nanoparticle formation and chromate reduction by Clostridium pasteurianum BC1. Environmental Science & Technology 2010. 83. Lloyd, J. R.; Yong, P.; Macaskie, L. E., Enzymatic Recovery of Elemental Palladium by Using Sulfate‐Reducing Bacteria. Appl. Environ. Microbiol. 1998, 64, (11), 4607‐4609. 84. Mutafov, S.; Avramova, T.; Stefanova, L.; Angelova, B., Decolorization of Acid Orange 7 by bacteria of different tinctorial type: a comparative study. World Journal of Microbiology and Biotechnology 2007, 23, 417‐422. 85. Knapp, J.; Newbym, P., The microbiological decolorization of an industrial effluent containing a diazo‐linked chromophore. Water Research 1995, 29 1807–1809. 86. Joe, M.‐H.; Lim, S.‐Y.; Kim, D.‐H.; Lee, I.‐S., Decolorization of reactive dyes by Clostridium bifermentans SL186 isolated from contaminated soil. World Journal of Microbiology and Biotechnology 2008, 24, (10), 2221‐2226. 87. Méndez‐Paz, D.; Omil, F.; Lema, J. M., Anaerobic treatment of azo dye Acid Orange 7 under batch conditions. Enzyme and Microbial Technology 2005, 36, (2‐3), 264‐272. 88. Ye, S. Y.; Chan, S. L. I.; Ouyang, L. Z.; Zhu, M., Hydrogen storage and structure variation in Mg/Pd multi‐layer film. Journal of Alloys and Compounds 2010, 504, (2), 493‐497. 89. Xu, Z. H.; Bae, W.; Mulchandani, A.; Mehra, R. K.; Chen, W., Heavy metal removal by novel CBD‐EC20 sorbents immobilized on cellulose. Biomacromolecules 2002, 3, (3), 462‐465. 90. Guo, J.; Kang, L.; Wang, X.; Yang, J., Decolorization and Degradation of Azo Dyes by Redox Mediator System with Bacteria. In Biodegradation of Azo Dyes, Atacag Erkurt, H., Ed. Springer Berlin / Heidelberg: 2010; Vol. 9, pp 85‐100. 91. Bardi, L.; Marzona, M., Factors Affecting the Complete Mineralization of Azo Dyes. In Biodegradation of Azo Dyes, Atacag Erkurt, H., Ed. Springer Berlin / Heidelberg: 2010; Vol. 9, pp 195‐210. 92. Zielonka, J.; Podsiadly, R.; Czerwinska, M.; Sikora, A.; Sokolowska, J.; Marcinek, A., Color changes accompanying one‐electron reduction and oxidation of the azo dyes. Journal of Photochemistry and Photobiology A: Chemistry 2004, 163, (3), 373‐379. 93. Neta, P.; Levanon, H., Spectrophotometric study of the radicals produced by the reduction of syn‐ and anti‐azobenzene. The Journal of Physical Chemistry 1977, 81, (24), 2288‐2292. 94. Sharma, K. K.; Rao, B. S. M.; Mohan, H.; Mittal, J. P.; Oakes, J.; O'Neill, P., Free‐Radical‐ Induced Oxidation and Reduction of 1‐Arylazo‐2‐naphthol Dyes: A Radiation Chemical Study. The Journal of Physical Chemistry A 2002, 106, (12), 2915‐2923. 95. Abbott, L. C.; Batchelor, S. N.; Smith, J. R. L.; Moore, J. N., Reductive Reaction Mechanisms of the Azo Dye Orange II in Aqueous Solution and in Cellulose: From Radical Intermediates to Products. The Journal of Physical Chemistry A 2009, 113, (21), 6091‐ 6103. 96. Forster, T., "Die pH‐Abhangigkeit der fluoreszenz von naphthalinderivaten" in NIST Chemistry WebBook, NIST Standard Reference Database Number 69, Eds. P.J. Linstrom 59
and W.G. Mallard, National Institute of Standards and Technology, Gaithersburg MD, 20899, http://webbook.nist.gov, (retrieved November 5, 2010). 54, 531‐535. 97. Liu, H.; Li, G.; Qu, J.; Liu, H., Degradation of azo dye Acid Orange 7 in water by Fe0/granular activated carbon system in the presence of ultrasound. Journal of Hazardous Materials 2007, 144, (1‐2), 180‐186. 98. Kayan, B.; Gözmen, B.; Demirel, M.; Gizir, A. M., Degradation of acid red 97 dye in aqueous medium using wet oxidation and electro‐Fenton techniques. Journal of Hazardous Materials 2010, 177, (1‐3), 95‐102. 99. Fihtengolts, V. S., et al, "Atlas of UV Absorption Spectra of Substances Used in Synthetic Rubber Manufacture" in NIST Chemistry WebBook, NIST Standard Reference Database Number 69, Eds. P.J. Linstrom and W.G. Mallard, National Institute of Standards and Technology, Gaithersburg MD, 20899, http://webbook.nist.gov, (retrieved November 12, 2010). 163. 100. Kudlich, M.; Hetheridge, M. J.; Knackmuss, H.‐J.; Stolz, A., Autoxidation Reactions of Different Aromatic o‐Aminohydroxynaphthalenes That Are Formed during the Anaerobic Reduction of Sulfonated Azo Dyes. Environmental Science & Technology 1999, 33, (6), 896‐901. 101. Parshetti, G. K.; Telke, A. A.; Kalyani, D. C.; Govindwar, S. P., Decolorization and detoxification of sulfonated azo dye methyl orange by Kocuria rosea MTCC 1532. Journal of Hazardous Materials 2010, 176, (1‐3), 503‐509. 102. Tasaki, T.; Wada, T.; Fujimoto, K.; Kai, S.; Ohe, K.; Oshima, T.; Baba, Y.; Kukizaki, M., Degradation of methyl orange using short‐wavelength UV irradiation with oxygen microbubbles. Journal of Hazardous Materials 2009, 162, (2‐3), 1103‐1110. 103. Daneshvar, N.; Rasoulifard, M. H.; Khataee, A. R.; Hosseinzadeh, F., Removal of C.I. Acid Orange 7 from aqueous solution by UV irradiation in the presence of ZnO nanopowder. Journal of Hazardous Materials 2007, 143, (1‐2), 95‐101. 104. Fan, J.; Guo, Y.; Wang, J.; Fan, M., Rapid decolorization of azo dye methyl orange in aqueous solution by nanoscale zerovalent iron particles. Journal of Hazardous Materials 2009, 166, (2‐3), 904‐910.