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Microbial Cr(VI) reduction: role of electron donors, acceptors, and mechanisms, with special emphasis on Clostridium spp.

University of Florida Institutional Repository

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MICROBIAL Cr(VI) REDUCTION: ROLE OF ELECTRON DONORS, ACCEPTORS, AND MECHANISMS, WITH SPECIAL EMPHASIS ON CLOSTRIDIUM spp. By KANIKA SHARMA A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2002

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Copyright 2002 by Kanika Sharma

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For my parents, whose support and understanding has helped culminate my dream into a reality.

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ACKNOWLEDGMENTS I am grateful to my mentor, Dr. Andrew V. Ogram, for excellent supervision during the course of this dissertation. He has truly been a great source of inspiration, insight, and input. The knowledge he has imparted, and the patience he has displayed was vital to completing this study. I am thankful for the immense encouragement and financial support that he graciously provided during this study. I would like to express my sincere gratitude to the committee members, Drs. K. Hatfield, L. O. Ingram, K. R. Reddy, and R. D. Rhue, for each contributing in special and meaningful ways to my personal development and academic success. I also thank Drs. W. Harris, L.T. Ou, and H. Aldrich for all of their advice and help. A special word of thanks is due to Dr. Derek Lovley, and members of his lab at the University of Massachusetts, Amherst, for extending their lab facilities so that I might learn various anaerobic microbial techniques. At this time, I would also like to thank Dr. John Thomas, Bill Reve, and Irene Poyer for all their help with the analytical equipment. I am especially grateful to T. Van Pham and Lisa Stanley who provided me the extra pair of hands when work demanded. I thank Ilker Uz and Hector Castro for being wonderful friends and for being by my side through all happy and tough times. I sincerely thank members of my lab, past and present, Viji Ramakrishnan, Weiwei Chen, Drs. Yong Ping Duan, Milind A. Chavan, and Ashvini Chauhan. Without the advice and friendship of these colleagues, surviving the last 5 years would have been impossible. iv

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I acknowledge the support of the faculty, staff, and all the graduate students in the Soil and Water Science Department. I owe much of my academic and personal success to my parents and my brother, who, by example, provided me with the motivation and courage to pursue a Ph.D. degree. Special thanks go to my friends, near and far, for their love and support that made my stay at University of Florida so memorable. v

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TABLE OF CONTENTS page ACKNOWLEDGMENTS.................................................................................................iv LIST OF TABLES...........................................................................................................viii LIST OF FIGURES...........................................................................................................ix LIST OF ABBREVIATIONS AND ACRONYMS..........................................................xi ABSTRACT......................................................................................................................xii CHAPTER 1 GENERAL INTRODUCTION........................................................................................1 Chromium in Environment.............................................................................................1 Nutrition and Toxicity: Risks to Human Health.............................................................2 Animals....................................................................................................................2 Plants and Algae.......................................................................................................3 Microorganisms.......................................................................................................3 Environmental Chemistry...............................................................................................5 Reduction of Cr(VI).................................................................................................6 Oxidation of Cr(III) in Soils.....................................................................................7 Cr(VI) Remediation Strategies.......................................................................................7 Chromium Resistance in Bacteria...................................................................................9 Pathways for Chromium(VI) Reduction.......................................................................11 Direct Enzymatic Reduction of Cr(VI)..................................................................11 Bacterially Mediated Indirect Reduction of Cr(VI)...............................................13 Factors Affecting Microbial Chromium Reduction......................................................15 Outline of Dissertation..................................................................................................18 2 ENRICHMENT, ISOLATION, AND CHARACTERIZATION OF Cr(VI)REDUCING BACTERIA.............................................................................................27 Introduction...................................................................................................................27 Materials and Methods..................................................................................................29 Results and Discussion.................................................................................................33 Effect of Electron Donors and Acceptors on Cr(VI) Reduction............................33 Phylogenetic Analysis of Cr(VI) Reducing Bacteria.............................................36 vi

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3 Cr(VI) REDUCTION BY A CONSORTIUM OF GRAM POSITIVE FERMENTATIVE BACTERIA...................................................................................42 Introduction...................................................................................................................42 Materials and Methods..................................................................................................43 Results and Discussion.................................................................................................45 Composition of Fermentative Consortium GCAF.................................................45 Biotic versus Abiotic Reduction of Cr(VI)............................................................45 Kinetics of Cr(VI) Reduction.................................................................................46 Effect of Electron Donor on Cr(VI) Reduction.....................................................47 Effect of Cr(VI) Reduction on Cell Growth in Consortium GCAF.......................47 Effect of Cr(VI) on Metabolites.............................................................................47 4 IDENTIFICATION AND CHARACTERIZATION OF THE CHROMIUM REDUCING ISOLATE CLOSTRIDIUM sp. GCAF1..................................................58 Introduction...................................................................................................................58 Material and Methods...................................................................................................59 Results and Discussion.................................................................................................62 Chemotaxonomic Data...........................................................................................63 Phylogeny of Strain GCAF-1.................................................................................63 5 ELECTRON SHUTTLE-MEDIATED CHROMIUM REDUCTION BY CLOSTRIDIUM sp GCAF-1.........................................................................................79 Introduction...................................................................................................................79 Materials and Methods..................................................................................................80 Results and Discussion.................................................................................................82 Effect of Cr(VI) on Production of Metabolic Products of Strain GCAF-1............85 Proposed Mechanism of Cr(VI) Reduction by Strain GCAF-1.............................86 Environmental Relevance of Cr(VI) Reduction by Gram Positive Spore-forming Fermentative Species..............................................................87 6 SUMMARY AND CONCLUSIONS............................................................................97 APPENDIX MEDIA USED FOR ENRICHMENT STUDIES......................................103 LIST OF REFERENCES.................................................................................................104 BIOGRAPHICAL SKETCH...........................................................................................119 vii

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LIST OF TABLES Table page 1-1. Cr(VI) reducing bacteria described in literature.........................................................24 2-1. Combination of electron acceptors and donors supplemented in the media for anaerobic enrichment studies.................................................................................38 2-2. Accession numbers for 16S rDNA sequences used in this study...............................39 3-1. Cell growth in the presence of different electron acceptors.......................................56 3-2. Cr(VI) reduction by consortium GCAF in presence of varying concentrations of electron donor........................................................................................................56 3-3. Effect of Cr(VI) on the on the pattern of products of glucose fermentation by consortium GCAF..................................................................................................57 4-1. Cellular fatty acid composition of GCAF-1 grown with 10mM glucose...................76 4-2. Characteristics of GCAF-1.........................................................................................77 4-3. Sequence similarity between 16S rRNA gene of isolate GCAF-1 and type strains of the genus Clostridium showing closest similarity.................................................78 5-1. Effect of Cr(VI) on metabolites formed by strain GCAF-1.......................................96 viii

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LIST OF FIGURES Figure page 1-1. Chromium cycle in environment................................................................................19 1-2. Reduction potential diagram for chromium................................................................20 1-3. Eh-pH diagram for chromium-water system at standard state conditions..................21 1-4. Schematic diagram showing the possible pathways for anaerobic Cr(VI) reduction by bacteria..................................................................................................................22 1-5. Model showing reduction of Fe(III) mediated by humics..........................................23 1-6. Quinone model compound..........................................................................................23 2-1. Rate of reduction of Cr(VI) in enrichment cultures amended with different electron donors and electron acceptors......................................................................40 2-2. Phylogenetic tree constructed using maximum parsimony........................................41 3-1. Cr(VI) reduction and removal from the solution as an insoluble precipitate.............50 3-2. Glucose consumption by consortium GCAF-1 during reduction of Cr(VI)...............50 3-3. Acetate produced by oxidation of glucose by consortium GCAF.during the reduction of Cr(VI).....................................................................................................51 3-4. Butyrate produced by oxidation of glucose by consortium GCAF during the reduction of Cr(VI).....................................................................................................52 3-5. Lactate produced by oxidation of glucose by consortium GCAF during the reduction of Cr(VI).....................................................................................................53 3-6. SEMs showing the insoluble precipitates formed by consortium GCAF-1 during the reduction of Cr(VI) via Fe(II)and AQDS-mediated mechanisms......................54 3-7. EDX of precipitate formed by consortium GCAF-1 showing the position of Cr in the precipitate formed during Cr(VI) reduction...............................................................55 4-1. Scanning electron micrograph of isolate GCAF-1.....................................................66 ix

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4-2. Micrograph of spores of isolate GCAF-1...................................................................67 4-3. Negatively stained preparations of Cr(VI) reducing Clostridium sp. GCAF-1 showing peritrichous flagella.....................................................................................68 4-4. Electron micrograph of an ultra thin section of Clostridium sp. GCAF-1 showing the S-layer..................................................................................................................69 4-5. Electron micrographs of Cr(VI) reducing Clostridium sp. GCAF-1 showing the dividing cells containing terminal spores and glycogen inclusions in the cells.........70 4-6. Phylogenetic tree based on 16S rDNA comparisons showing the relative position of strain GCAF-1 among other species of genus Clostridium..................................71 4-7. Anaerobic growth curve of GCAF-1 in under various Cr(VI) concentrations...........72 4-8. Comparison of two 16S rRNA gene sequences from Clostridium sp. GCAF-1........75 5-1. Cr(VI) reduction by strain GCAF-1...........................................................................89 5-2. Glucose consumption during Cr(VI) reduction by strain GCAF-1............................90 5-3. Cell growth during Cr(VI) reduction by strain GCAF-1............................................91 5-3. Acetate production during oxidation of glucose by GCAF-1.....................................92 5-4. Butyrate production during oxidation of glucose by GCAF-1...................................93 5-5. Lactate production during oxidation of glucose by GCAF-1.....................................94 5-6. Schematic diagram of proposed metabolic pathway of formation of fermentative products by Clostridium sp. GCAF-1.........................................................................95 x

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LIST OF ABBREVIATIONS AND ACRONYMS 16S.............................................................................................................16 Svedburg unit AQDS...................................................................................Anthraquinone di-sulfonic acid ATP...................................................................................................adenosine triphosphate BM.....................................................................................................................Basal media Bp............................................................................................................................base pair CRB..........................................................................................chromium reducing bacteria FRB................................................................................................Fe(III) reducing bacteria G...............................................................................................................................Glucose GAF..............................................................................................Glucose +AQDS +Fe(III) GC...............................................................................................................Glucose+Cr(VI) GCA.............................................................................................Glucose +Cr(VI) +AQDS GCAF..............................................................................Glucose+ Cr(VI)+AQDS +Fe(III) GCF..............................................................................................Glucose +Cr(VI) +Fe(III) PCR.............................................................................................Polymerase chain reaction rDNA................................................................................ribosomal Deoxyribonucleic acid RNA ...........................................................................................................Ribonucleic acid X-gal..................................................5-bromo-4-chloro-3-indolyl-B-D-galactopyranoside xi

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Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy MICROBIAL Cr(VI) REDUCTION; ROLE OF ELECTRON DONORS, ACCEPTORS, AND MECHANISMS, WITH SPECIAL EMPHASIS ON CLOSTRIDIUM spp. By Kanika Sharma December, 2002 Chair: Dr. A.V. Ogram Major Department: Soil and Water Science Cr(VI) has been designated as a priority pollutant by the US Environmental Protection Agency (USEPA) due to its ability to cause mutations and cancer in humans. The risk associated with soil and groundwater contamination of chromium waste generated by many industries is high, and therefore Cr(VI) remediation is of critical importance. Using chemical and biological methods conjointly can decrease the cost of remediating contaminated sites. Microbial reduction of Cr(VI), an important aspect of biological remediation, requires the knowledge of microorganisms capable of reducing Cr(VI) and the mechanisms involved in the reduction processes. The overall objective of this study was to investigate the effect of various electron donors and acceptors on chromate reduction by indigenous Cr(VI)-reducing bacteria isolated from Cr(VI) contaminated sites and to understand the mechanism of Cr(VI) reduction by enriched bacterial consortium and the pure isolate. A series of bacterial enrichment cultures were established with a range of electron donors such as acetate, xii

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benzoate, lactate, citrate, and glucose, and electron acceptors such as Fe(III) and an humic acid analog, anthraquinone di-sulfonate (AQDS), to study their effects on the rates of Cr(VI) reduction. Results from this study demonstrated that the rates of Cr(VI) reduction in glucose and citrate enrichments were higher when compared with those of other electron donors. Enrichments amended with AQDS and Fe(III) showed enhanced rates of Cr(VI) reduction. GlucoseAQDS-Fe(III)-Cr(VI) enrichments (now on referred as GCAF) yielded the highest diversity of strains, which were distributed within the low G+C and high G+C groups of gram-positive bacteria. Phylogenetic analysis based on 16S rDNA studies revealed that isolates clustered with Bacillus, Cellulomonas, and Clostridium groups. Several strains were isolated from the consortium. Detailed kinetic studies with bacterial consortium and the pure strain GCAF-1 obtained from GCAF enrichment demonstrated an iron-promoted reduction of chromate. The presence of AQDS accelerated reduction of Cr(VI) only when Fe(III) was present in the medium. Analysis of fermentation metabolites produced by strain Clostridium sp. GCAF-1 revealed that the presence of Cr(VI) alters the acetate: butyrate and acetate: lactate ratios. Based on the overall results, direct and indirect (Fe (III) mediated) methods of reduction of Cr(VI) by Clostridium sp. GCAF-1 are proposed. xiii

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CHAPTER 1 GENERAL INTRODUCTION Chromium, the 24 th element on the periodic table, was first discovered in Siberian red lead ore (crocoite) in 1798 by the French chemist Nicholas-Louis Vauquelin. He named this new mineral chrom from the Greek word owing to the brilliant hues of the compound. Since then, chromium has found a variety of uses in the industries that exploit these colors and other characteristics such as its strength, hardness, corrosion resistance, and the oxidizing capabilities of certain chromium species (34). Chromium in Environment Chromium is found in many environments, including air, water, soil and all biota. It ranks 21 st among the elements in crustal abundance (74). The average concentration of chromium in the continental crust has been reported as 125 mg/kg (108). Concentrations in freshwater generally range from 0.1 to 6.0 g/L with an average of 1.0 g/L, while values for seawater average 0.3 g/L and range from 0.2 to 50 g/L (23). Freshwater chromium concentrations are dependent on soil chromium levels in the surrounding watershed areas. In addition, drainage water from irrigated agricultural areas with elevated amounts of soil chromium levels can have high chromium concentrations (as high as 800 g/L), as observed at various locations within San Joaquin Valley, CA (38, 50). Chromium is extracted from chromite ore [(Fe,Mg)O(Cr, Al, Fe) 2 O 3 ] that has largest deposits in South Africa, the Philippines, Southern Zimbabwe, and Turkey (100). The major users of chromium are the metallurgical, chemical, and refractory brick 1

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2 industries (78). Other industries that employ chromium include pigment manufacture, metal finishing, corrosion inhibition, organic synthesis, leather tanning, and wood preservation (57, 34, 174). Extensive industrial usage of chromium leads to generation of large volumes of chromium-containing wastes that are discharged into the environment. In addition to this waste, leakage due to improper handling and faulty storage containers also adds to the accumulation of chromium in the environment. Nutrition and Toxicity: Risks to Human Health Chromium is designated by the U.S. Environmental Protection Agency (USEPA) as a priority pollutant due to its ability to cause genetic mutations and cancer. Chromium is unique among regulated toxic elements in the environment because different species of chromium, specifically Cr(III) and Cr(VI), are regulated in different ways. Relying on the chemical, toxicological, and epidemiological evidence, regulation of Cr(VI) concentration is different from that of Cr(III). Trivalent chromium is the nutritionally useful form, while the hexavalent form is toxic and mutagenic. Cr(VI) is both a powerful epithelial irritant and confirmed human carcinogen (77, 120). On the contrary, Cr(III) is an essential element in animal physiology and plays a role in glucose and lipid metabolism (5, 103). Animals Cr (VI) is highly mobile in some soils, and contact with Cr(VI) maybe inevitable for aquatic and terrestrial organisms, including humans. In trace amounts, chromium is an essential component of human and animal nutrition (65, 102). It is associated with glucose metabolism (102) and has been shown to be an integral component of glucose tolerance factor (GTF), a factor required for maintaining normal glucose tolerance. Chromium functions by regulating and potentiating insulin action by increasing insulin

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3 binding to cell (4). Chromium is also known to be of importance in fat metabolism in animals (5). The biotoxicity of chromate is largely a function of its ability to cross biological membranes and its powerful oxidizing capabilities (NAS 1974). Humans can absorb Cr(VI) compounds through inhalation, dermal contact and ingestion. Human health effects of Cr(VI) include lung cancer, respiratory irritation, dermatitis, kidney and liver damage, and damage to various proteins and nucleic acids, leading to mutation and carcinogenesis (18). Plants and Algae Pratt (123) reported that low concentrations of chromium stimulated the growth of plants. However, a few years later it was demonstrated conclusively that chromium is not an essential component in plant nutrition (61). The effect of Cr(VI) was apparent on seed germination when more than 80% of the reduction in seed germination was observed in the presence of Cr(VI) when compared with those germinated in the absence of Cr(VI) (133). Cr(VI) concentrations of 5 to 60 mg/kg soil have been shown to retard plant growth due to root damage (7). Cr(VI) has been shown to affect the growth, photosynthesis, morphology, and enzyme activities in algae. Cr(VI) concentrations shown to be toxic to algae vary from 20 ppb to 10,000 ppb (7, 131, 135,143, 154). Microorganisms Hexavalent chromium is toxic and mutagenic to most bacteria. Among the visible effects reported in bacteria are cell elongation, cell enlargement, and inhibited cell division, which eventually leads to cell growth inhibition (31, 153). Changes in morphologies of gram-positive and gram-negative bacteria were also observed by

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4 Bondarenko et al (21). Few colonies of bacterial species such as Staphylococcus aureus, S. epidermidis, Bacillus cereus, and Bacillus subtilis were formed with degenerate cells that were reduced in size (20). Cr(VI) concentrations of 10-12 ppm were inhibitory to most soil bacteria in liquid media and, in general, gram-negative bacteria were more sensitive to Cr(VI) than were gram-positive bacteria (132). Increased content of Cr(VI) in soil was toxic to saprophytic and nitrifying bacteria. Lowered microbial biomass in soil was observed in the presence of high Cr(VI) in soil when it was determined using adenosine triphosphate (ATP) method (2, 178). Other bacteria such as E coli, Serratia marcescens and Enterobacter aerogenes were unable to grow in Cr(VI) concentrations of 1 mM.(8). .Metabolic effects of Cr(VI) on bacteria were evident by the observed changes in electron transport systems (124). Cr(VI) has been shown to cause mutagenic effects in Escherichia coli, Bacillus subtilis, and Salmonella typhimurium (113, 120, 162). The mutagenic effects of chromium are effective only when chromium crosses the cell membrane. Cr(VI) can easily diffuse across the cell membranes, unlike Cr(III) which can do so only under extreme conditions such as long incubations and high concentrations. Cell culture studies have shown that cellular uptake of chromate is at least 10 times greater than that of Cr(III) from equimolar solutions, (33, 55, 80). However, once inside the cell, most of Cr(VI) is reduced to Cr(III) by several reducing agents such as ascorbic acid, sodium sulfite, glutathione, NADPH and NADH (121). Based on several studies, it was concluded that trivalent chromium causes DNA-strand breaks (18, 19, 37, 80, 155, 156, 157). Cr(VI) causes genotoxic effects on bacterial cells, including frameshift mutations and base pair substitutions (120). DeFlora et al. (37) reported a more general effect of

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5 unbalanced nucleotide pools. These studies suggest that although Cr(III) form is the major agent responsible for molecular events leading to mutagenicity, it is Cr(VI) that poses the greater risk to human life due to its ability to easily enter the cell. Environmental Chemistry Chromium can exist in oxidation states ranging from 0 to 6 + The various chemical and biological changes that chromium undergoes in the environment depend on the conditions that govern its speciation and other activities. The solubility and adsorption by soil and sediments depend on the form of chromium species. Within the ranges of redox potentials and pH commonly found in soils, chromium exists predominantly as oxyanions of Cr(III) and Cr(VI). Cr(VI) is a strong oxidizer and exists only in oxygenated species that are very soluble and pH dependant according to the following equilibria (111). H 2 CrO 4 H + + HCrO 4 K a1 =10 0.6 HCrO 4 H + + CrO 4 2K a2 =10 .9 H 2 CrO 4 is a strong oxidizing agent and dominant species below pH -0.6 (32). Monohydrogen chromate, HCrO 4 exists between the pH values of 1 and 6. CrO 4 2predominates at or above pH 6. Cr 2 O 7 -2 dichromate ion is formed by dimerization of HCrO 4 ion at Cr(VI) concentrations above 10 -2 M (17, 86) Cr 2 O 7 2+ H 2 O 2HCrO 4 K=10 -2.2 Existence of dichromate ion is unlikely in the biologic systems as typical chromium concentrations in nature are considerably lower than 10-2 M, especially at physiological pH 7. Trivalent chromium is the more stable form. Due to its lower affinity for oxide and hydroxide ions, Cr(III) is known to form numerous complexes with both organic and inorganic ligands (99, 144). Due to chemical inertness, complex species of Cr(III) tend to

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6 be more stable in solution and can be isolated. The main aqueous Cr(III) species include Cr3+, Cr(OH)2+, Cr(OH) 3 and Cr(OH)4-(16), (14, 40). The Cr3+ species predominate at pH <3.6 (46), whereas Cr(OH)4 predominates at the pH >11.5 (126). At a slightly acidic to alkaline pH, ionic Cr(III) species precipitates as amorphous Cr(OH) 3 (126) or as a solid solution (Fe, Cr)(OH) 3 if Fe3+ is present (46) Cr(III) can also be chelated by organic molecules that are adsorbed to mineral surfaces (63, 64). In contrast, Cr(VI) compounds CrO 4 2, HCrO 4 Cr 2 O 7 2are very mobile in surface sediments because they are not strongly adsorbed to soils. Both oxidation and reduction of Cr(VI) can occur in geologic and aquatic environments ( Figure 1-1).The oxidation and reduction of chromium in soils depends on soil structure and on the redox conditions of the soil (73). Studies conducted to investigate the effect of adsorption of chromate and Cr(VI) on the clay sand mixture showed that clay was a suitable absorbent for chromate due to its high cation exchange capacity (CEC) and strong binding capability.(2). Chromium speciation in groundwater is affected by the pE (redox) and pH conditions (Figure 1-2). Reduction of Cr(VI) Reduction of Cr(VI) in soils depends largely on the presence of other electron acceptors such as the oxygen, nitrate, iron, and manganese that can act as electron sinks and accept electrons from the reactive organic and inorganic electron sources. Conditions will favor Cr(VI) reduction when electron donors are in excess and electron acceptors such as those mentioned above are low. Hexavalent chromium is a oxidizing agent and is readily reduced in the presence of appropriate electron donors, as shown in this equation: HCrO 4 + 7H + +3eCr 3+ +4H 2 O

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7 Oxidation of Cr(III) in Soils Cr(III) is chemically more stable than Cr(VI). Initial studies convincingly showed that, in most cases, oxidation of Cr(III) does not occur in soils, regardless of the conditions (16). This was explained with chemical inertness of the Cr(III) and its complexes in the range of pH that normally exists in soils. However, it has since been determined that some Cr(III) can be oxidized to the hexavalent form in the presence of Mn(IV). The amount of Cr(III) oxidized to Cr(VI) was shown to be proportional to the amount of Mn(IV) reduced to Mn(II) (3, 15). Oxidation of chromium occurs in soils that are high in Mn(IV) and oxides and low in organic matter content (73). The conditions required for chromium oxidation are fairly specific and only a few cases of oxidation of Cr(III) oxidation are reported in literature. Oxidation of aqueous Cr (III) to Cr(VI) in soils does not occur over such a wide range as the reduction of aqueous Cr(VI). Also, Cr 3+ precipitates almost completely as Cr(OH) 3 often in conjunction with iron at pH values from 5.5 and 12.0 (40, 126). Cr 3+ + 3H 2 O Cr(OH) 3 (s) + 3H + Keq =10 -12 These factors are of great importance in assessing potential environmental hazards and remediation strategies for ecosystems with high levels of natural or anthropogenic chromium. Cr(VI) Remediation Strategies Remediation strategies are employed in order to minimize the risk of public exposure to chromium contaminated sites. Several common remediation strategies include the no action option, excavation and removal of contaminated soil, pump and treat strategies, and soil solidification and stabilization. In order to implement the optimal remediation strategy, an understanding of physical and chemical processes affecting the

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8 migration and chemical state of chromium is required. The no action option is adopted if the risk of exposure and potential impact to the environment is marginal. Knowledge of the type of soil reductants present is important for the implementation of this option. Excavation is no longer a very desirable method as it simply moves the contaminated soil from one place to another. Pump and treat is one of the most commonly used methods for aquifer remediation. The two main purposes are to remove contaminants from the subsurface for treatment and to maintain gradient control to prevent contaminants from migrating beyond compliance boundaries. Among the major concerns of employing this method is the residual concentration. The residual concentration is usually much higher than the maximum contaminant level (MCL) level set by EPA. Soil solidification process includes solidification of the contaminated soils by transforming Cr(VI) into an insoluble chemical form that is impermeable to the ground water. Traditional techniques for remediating chromate contaminated water also involve reduction of Cr(VI) to Cr(III) by chemical means (usually with Fe 2+ ) or electrochemical means at pH 5, followed by precipitation and filtration or sedimentation (41). The electrochemical Cr(VI) reduction process uses consumable iron electrodes and electrical current to generate ferrous ions that react with Cr(VI) to Cr(III) is given below. Increased quantity of resultant sludge by this method is one of the drawbacks. This method is often employed in combination with the pump and treat methods. 3Fe 2+ + CrO 4 2+ 4H 2 O 3Fe 3+ + Cr 3+ + 8OH These processes can be extremely reagent or energy intensive. Most of these methods take long periods of time to reach the regulatory level for remediating contaminated sites. The cost involved in these chemical enhanced remediation strategies

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9 is very high and this lowers the overall cost-benefit ratio. The discovery of microorganisms that can reduce metals has led to applications in the bioremediation which are potentially more cost effective than traditional methods. One of the major factors that decide the application of the bioremediation strategies is the bioavailability of the preferred electron donor by the indigenous microorganisms that are involved in metal reduction. For bioremediation of Cr(VI), stimulation of the existing microbial populations with bioavailable electron donors may result in increased metal reduction, thereby remediating the contaminated site. Although reduction of Cr(VI) to Cr(III) does not remove chromium from soils, it does limit the mobility and toxicity of chromium in the contaminated soils. Many potential remediation pathways are known for the chromate reduction, but the dominance of one pathway over another has not been established. Furthermore, coupled geochemical and microbiological processes have a potential to dominate the reduction of metals such as Cr(VI). Finally it must be recognized that there are many factors that effect the microbial reduction of Cr(VI) in soils. Clearly there is a need to understand the various groups of bacteria that reduce hexavalent chromium and the different mechanisms by which Cr(VI) is microbially reduced in soils. Chromium Resistance in Bacteria The persistent nature of some metals in environment has led to considerable modifications of the microbial community and their activities. Heavy metals have been shown to inhibit microbial growth and other enzymatic activities by blocking essential functional groups, displacing essential metal ions and modifying the conformations of the biological molecules, (49, 81, 171). In metal-contaminated environments, the responses of the microbial communities depend on the concentrations of the toxic agents they are

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10 exposed to among other factors such as nature of nutrients, chemical form of the toxic agent and so on. The resistance mechanisms proposed for heavy metal resistance in bacteria include exclusion by permeability barrier, exclusion by active transport, intracellular physical sequestration by the binding proteins of the cell, extracellular sequestration and detoxification by chemical modification of the toxic to non-toxic form of the metal. Microorganisms may adopt several strategies to reduce metal sensitivity to cellular targets: (i) mutations to decrease the sensitivity to the metal (ii) increased production of damaged cell component, (iii) increased efficiency of repair of damaged cell component, (iv) utilization of plasmid-encoded resistance mechanism. These mechanisms may either occur singly or in various combinations. Persistence of metal in environments selects for the resistant strains possessing either the resistant or the reduction capability. Organisms isolated from sediments of Cr(VI)-contaminated metal-processing waste evaporation ponds were found to be more Cr-tolerant compared with those found outside the ponds (85). Plasmid-associated bacterial resistance has been reported in Streptococcus lactis (42), Pseudomonas sp (148), and Alcaligenes eutrophus (27, 112, 119). Studies with Pseudomonas fluorsencens LB300 showed the loss of Cr(VI) resistance resulted with the loss of plasmid and transformation of the plasmidless strain done with the purified plasmid DNA resulted in regaining of the Cr(VI) resistant ability of the strain (21). Increased polysaccharide production has been reported in Pseudomonas sp. (1). Further studies with Pseudomonas ambigua and its Cr(VI) sensitive mutant S-1 led to the conclusion that the presence of thick membranes around the parent cell decreased the

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11 permeability of Cr(VI) of the cells and increased the resistance of the bacteria (59, 60). Enterobacter cloacae strain HO1 and yeast exhibited Cr(VI) resistance by decreased uptake of Cr(VI) (12, 116, 163). Pathways for Chromium(VI) Reduction Microorganisms obtain their energy for metabolism by participating in several oxidation-reduction reactions. In environments where the photosynthesis does not occur the transfer of electrons is the driving force that governs all the microbial processes. Depending on the environment the microorganisms have adapted and evolved the ability to be able to mediate various oxidation-reduction couples to conserve energy. Some Cr(VI) resistant bacteria are able to grow by reducing Cr(VI) to Cr(III). Cr(VI) reduction is considered to be a fortuitous reduction process that is employed by some bacteria as a mechanism of defense by detoxification of the environment they have to survive in. Most Cr(VI) reducing bacteria (CRB) reported so far are gram negative bacteria (12,45). Recently the ability to use Cr(VI) as terminal electron acceptor was demonstrated in a sulfate reducing bacterial consortium and Pantoea agglumerans (45, 152). Currently microbial reduction of Cr(VI) can be explained by two prevalent models: (i) direct enzymatic reduction, and (ii) indirect reduction. Distinguishing between these enzymatic and nonenzymatic Cr(VI) reductions is difficult. The direct enzymatic reduction refers to the reduction by the metal reductase system. Indirect mechanism refers to Cr(VI) reduction mainly by conditions provided by bacterial source such as the redox potential, or the bacterial metabolites. Direct Enzymatic Reduction of Cr(VI) Although CRB have been studied for many years now, little is known about the biochemistry and mechanism of Cr(VI) reduction. It still remains unclear if Cr(VI) is

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12 taken up by the cell and reduced in the cytoplasm or the periplasm or the electron are transferred to the outside of the cells or both. Direct contact between cells and the metal oxide has shown to be required for the energy conservation process (8). Enzymatic reduction of Cr(VI) has been observed in some CRB (10, 29, 52, 68, 114, 149, 173).The CRB are able to reduce Cr(VI) by either soluble enzyme systems or the membrane-bound system. Membrane-associated chromate reductase activity was first observed in Enterobacter cloacae HO1 where the insoluble form of reduced chromate precipitates was seen on the cell surface (164). In the presence of ascorbate reduced phenazine methosulfate (PMS) as electron donor, high chromate reduction was shown by right-side-out membrane vesicles of E. cloacae HO1 (164). Membrane-associated constitutive enzyme that mediated the transfer of electrons from NADH to chromate was later elucidated by Bopp et al. (21). In case of Shewanella putrefaciens MR-1 chromate reductase activity was associated with the cytoplasmic membrane of anaerobically grown cells (106). Formate and NADH served as electron donors for the reductase. No activity was observed when NADPH or L-lactate were provided as the electron donors. However, in Pseudomonas putida, unlike in Shewanella putrefaciens, NADPH served as an electron donor for this (117). Studies conducted by Shen and Wang (141) on E. coli suggested the presence of soluble chromate reductase. Cr(VI) reduction in another gram negative bacteria, Pseudomonas sp CRB5, was found to be mediated by a soluble enzyme contained in cytoplasm (101). In addition to gram-negative bacteria, soluble chromate reductases have also been observed in gram-positive strains. NADH was the preferred electron donor for the reduction of chromate by the soluble enzyme in Bacillus coagulans (122).

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13 Bacterially Mediated Indirect Reduction of Cr(VI) Redox potential-pH. Changes in pH and redox conditions are known to occur in medium during growth of bacterial cultures due to various biochemical reactions and the metabolites formed. These changes may indirectly affect the reduction of Cr(VI) in the medium. Lower redox and pH has been shown to favor reduction of Cr(VI)(36). Cr(VI) reduction occurs in a wide range of redox potentials. The optimum redox potential range has not been well established as yet. Reduction of Cr(VI) has been reported in redox conditions as high as +250mV (53). In the same culture, after 48 hours, Cr(VI) reduction was observed even when the redox potential dropped to mV. A higher rate of Cr(VI) reduction by Agrobacterium radiobacter was observed at mV compared with mV (82). In contrary, no reduction of Cr(VI) was observed with redox potential of mV for the first hour of incubation in cultures of Escherichia coli. Fe(III)-mediated reduction of Cr(VI). Fe(III) is the most abundant electron acceptor for anaerobic respiration in many sedimentary environments due to its ability to act as terminal electron acceptor for many organisms. Microbial reduction of Fe(III) significantly affects Cr(VI) biogeochemistry as reduced iron in sediments is one of the most significant electron donors for the reduction of Cr(VI). Three equivalents of Fe(II) are required for the reduction of one equivalent of Cr(VI). 3Fe(II) +Cr(VI)3Fe(III) + Cr(III). Therefore, Fe(III) reducing bacteria that are unable to support their growth on reduction of Fe(III) can indirectly reduce Cr(VI) via Fe(III) reduction (Figure 1-4). Reduction of chromate by dissimilatory iron-reducing bacteria was reported by Wielinga et al (169). They elucidated the reduction of Cr(VI) to Cr(III) via a closely coupled biotic-abiotic pathway under iron-reducing conditions.

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14 Quinone mediated reduction of Cr(VI). Humic substances are ubiquitous in the environment. They are heterogeneous organic high-molecular-weight macromolecules that are composed of many potentially reactive moieties. Humic substances were considered resistant to microbial degradation until recently, when the ability of humics to serve as electron acceptors and support bacterial growth under anaerobic conditions was reported (90). Humics function as primary electron acceptors for iron-reducing bacteria, and mediate transfer of electrons from humics to Fe(III) oxides, thereby stimulating the reduction of insoluble Fe(III) oxides (Figure 1-5) (90). Quinones serve as the primary electron-accepting moiety in the humic acids when they are reduced to hydroquinones by accepting two electrons, as shown in Figure 1-6. Scott et al. demonstrated the higher free-radical content of humic substances with higher electron accepting capacity with electron spin resonance measurements by showing a proportional increase in semiquinones and electron-accepting capacity of humic substances (137). To date several humic reducing bacteria have been isolated from a variety of environments (30). All iron-reducing bacteria that have been evaluated to date have shown the ability to transfer electrons to humic substances and other extracellular quinones.(91). Microbially reduced humics are also capable of reducing other metals, including manganese (IV) and technetium (VII) (83). Reduction of Tc(VII) mediated by Fe(III) was enhanced in the presence of anthraquinone di-sulfonate (AQDS), a humic acid analog that behaved as an electron shuttle (90). Although humic-mediated Cr(VI) reduction has not been reported so far thermodynamically, transfer of electron from humics ( E = 0.2mV) to Cr(VI) (E = 1.23mV) is plausible.

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15 Factors Affecting Microbial Chromium Reduction Cell density. Rate of Cr(VI) reduction has been shown to be a affected by cell density under both aerobic and anaerobic conditions. Wang et al. (164) reported increase in the rate of Cr(VI) reduction with increase in cell density under anaerobic conditions. Similar observations were made in both aerobic and anaerobic cultures of Escherichia coli. However, the rate of Cr(VI) reduction was not proportional to the increase in the cell density, and the specific rate of Cr(VI) reduction was higher at relatively lower cell densities (142). These observations were also documented in cultures of Enterobacter cloacae, Agrobacterium radiobacter, Pseudomonas fluorescens LB300, Bacillus coagulans, and Microbacterium sp. Initial Cr concentration. Depending upon the initial concentration of Cr(VI), its complete or incomplete reduction has been observed in Enterobacter cloacae HO1, (48, 71). Even though a decrease in cell viability was observed in the culture on addition of Cr(VI) to the growing culture (72, 163), the initial rate of Cr(VI) reduction increased with the increase in the initial rate of Cr(VI) in some cultures of Enterobacter cloacae (163), E. coli (139) P. flourescens (167) and Bacillus sp.(167). Similarly, initial specific rate of Cr(VI) reduction by cultures of E coli increased with increasing Cr(VI) concentrations. However, an increase in time required for complete reduction was also observed (142). Effect of other electron acceptors. Presence of oxygen does not completely inhibit Cr(VI) reduction in some bacteria but it represses it as in the case of Agrobacterium radiobacter EPS-916, E. coli ATCC 33456 and Pseudomonas stutzeri CMG463 (9, 71, 82, 139, 141, 165). Microbial reduction of Cr(VI) is completely inhibited in aerobic condition as in the case of E. cloacae HO1, even though cell growth was observed (48). Studies with enrichment microcosms showed only 41% reduction of

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16 Cr(VI) under aerobic conditions when compared with the 84% reduction observed in anaerobic conditions (97). Marsh et al. concluded that lower reducing conditions were required for Cr(VI) reduction because reduction of Cr(VI) was inhibited by oxygen and nitrate (97). Among other naturally occuring dominant electron acceptors, sulfate and nitrate have little effect on the Cr(VI) reduction upto concentrations of 10 mM and 16mM, respectively. The concentration of sulfate and nitrate to which microbial Cr(VI) reduction is not affected varies with the bacterial species. In the case of Cr(VI) reduction by Pseudomonas. putida Cr(VI), reduction was not affected by 1mM of sulfate and 0.2mM of nitrate. Concentrations of sulfate and nitrate, that did not affect Cr(VI) reduction, in case of Bacillus sp., were 10 mM and 16mM respectively, in case of E. coli were 83 mM sulfate and 129mM nitrate. Sulfate concentration as high as 50 mM did not affect the Cr(VI) reduction by Desulfovibrio vulgaris (93). In contrast, the chromate reduction by Enterobacter cloacae is inhibited by 32% in the presence of just 25M of sulfate and 84% in the presence of 5mM NaNO 3 Enrichment studies with alternative electron acceptors done by Marsh et al. showed that nitrate reduction preceeded Cr(VI) reduction. However, Fe(III) reduction and sulfate reduction always followed the Cr(VI) reduction. They supported their results by the Gibbs energy obtained by thermodynamic reactions (97). Temperature and pH effects. Optimum temperature and pH conditions reported for microbial Cr(VI) reduction strongly suggest that the reduction process is related to growth. Cr(VI) reduction was observed in cultures of Enterobacter cloacae at pH range of 6.0-8.5, and at pH range of 3.0 -8.0 in cultures of Escherichia coli and Bacillus

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17 coagulans. However the maximum initial specific rate of Cr(VI) reduction by all three bacteria was at pH 7.0, an optimal pH for most bacterial growth. Even though Cr(VI) reduction by E. coli and Enerobacter. cloacae occurred at a wide range of temperature of 10C to 50C, optimum temperature was found to be 36C and 30C respectively. These conditions were found to be optimal for the anaerobic growth of the bacteria. Studies with sediments have shown temperature optima of 22C and 50C and a pH optimum of 6.8 (97). Carbon sources. Studies have been conducted to try and establish the relationship between the electron donors and the rate of Cr(VI) reduction. Enrichment studies with the soils done by Marsh et al showed hydrogen to be an efficient electron donor for the reduction of Cr(VI). Addition of electron donors that increase the bioavailable hydrogen such as glucose, formate, and hydrogen stimulated the Cr(VI) reduction in the soils as compared with acetate and benzoate and lactate. The study also documented the dissolved hydrogen concentration in the Cr(VI)-reducing conditions. Based on the observation that very low hydrogen concentration was present under Cr(VI) reducing conditions similar to that reported under nitrateand manganese-reducing conditions, and the observation that Cr(VI) reduction occurs before iron or sulfate reduction it was concluded that very highly reducing conditions were not required for Cr(VI) reduction (98). Rege et al. reported the utilization of sucrose as a carbon source for Enterobacter cloacae HO1for reduction Cr(VI) (128).

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18 Outline of Dissertation The work presented in this dissertation was performed to get more insight into the diversity of electron donors and acceptors utilized by the indigenous chromium reducing bacteria (CRB). Special attention was paid to the kinetics of chromium reduction in the presence of alternative electron acceptors Fe(III) and AQDS. An attempt was made to explain the mechanism of Cr(VI) reduction by fermentative organisms. Chapter 2 describes the enrichment studies with indigenous CRB capable of utilizing various electron donors and acceptors. Difference in reduction of Cr(VI) is determined by the organisms enriched by the various electron donors chosen to represent the range of electron donors that naturally exist in nature. Effect of electron acceptors viz. Fe (III) oxides and anthraquinone di-sulfonate (AQDS), an humic acid analog on reduction of Cr(VI) is also investigated. Identification of the organisms isolated from the enrichments is also described based on the phylogenetic studies. Chapter 3 describes the isolation of the Cr(VI)-reducing consortium from the glucose enrichments and detailed kinetic studies of Cr(VI)-reduction by this consortium. In chapter 4 the isolation, identification, and detailed characterization of a novel species of chromium-reducing fermentative organism GCAF-1 is described. Chapter 5 includes detailed kinetic studies with fermentative isolate GCAF-1. It describes the possible mechanism adopted by Clostridium sp. GCAF1 reduce Cr(VI). Finally, the results presented in this dissertation are summarized and the implications of this research are discussed in Chapter 6.

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19 Figure 1-1. Chromium cycle in environment (174)

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20 Cr(VI)Cr(V)Cr (IV)Cr (III)Cr (II)Cr00.91V0.91V0.91V0.91V0.91V+1e+1e+1e+1e+1e +3e>1.2V1.41 V; E (pH 7.4) = 0.3-0.5 V+2e+2e>1.2V Figure 1-2. Reduction potential diagram for chromium. Positive E values favor the reduced form. E values for Cr(VI) and Cr(V) are dependant on the pH because the protons are involved in the reaction (111).

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21 Figure 1-3. Eh-pH diagram for chromium-water system at standard state conditions. Source: Dragun Figure (111)

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22 AnaerobicAerobicSurface Cr(VI)Cr(III) Fe(III)Fe(II) Humics (ox)Humics (red) Fe(III)Fe(II)FRBCRBHRB Figure 1-4. Schematic diagram showing the possible pathways for anaerobic Cr(VI) reduction by the three groups of bacteria, Fe(III) reducing bacteria (FRB); Cr(VI) reducing bacteria (CRB); humics reducing bacteria (HRB). Solid lines represent the biotic reduction of Cr(VI) and the abiotic reduction is represented by the dashed lines.

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23 Fe(II)Fe(III)Acetate HumicsreducedHumicsoxidizedCO2bacteria Figure 1-5. Model showing reduction of Fe(III) mediated by humics (90) Figure 1-6. Quinone model compound. The semiquinone species contains an unpaired electron (137)

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Table 1-1. Cr(VI) reducing bacteria described in literature Bacteria Redox potential Cr(VI) reduced Gram stain Reduction conditions Enzymatic reduction Carbon source Reference P. putida MK1 0.2mM Gram negative Anaerobic ND (117) Pseudomonas sp. CRB5 0.1mM Gram negative Aerobic and anaerobic Soluble reductase Does not require NADH (101) Pseudomonas dechromaticans ND 0.2mM Gram-negative Anaerobic ND Peptone / glucose (130) P. chromatophila ND Gram-negative Anaerobic ND ribose/ lactate/ acetate/ succinate/ butyrate/ glycerol/ fumarate (79) P. fluorescens LB300 ND 0.48mM Gram-negative Aerobic and to a lesser extent anaerobic Membrane associated, NADH dependant Glucose (22, 167) P. ambigua G-1 ND 0.4mM Gram-negative Aerobic NAD(P)H-dependant Nutrient broth (59) P. aeruginosa Gram-negative Anaerobic ND Acetate/ glucose (56) P. putida PRS2000 0.038mM Gram-negative Aerobi and anaerobic Soluble protein; NADH or NADPH dependent Glucose/ lactate (62) 24

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Table 1-1. continued Bacteria Redox potential Cr(VI) reduced Gram stain Reduction conditions Enzymatic reduction Carbon source Reference E. coli ATCC 33456 ND 0.3mM Gram-negative Anaerobic and aerobic; oxygen repressed Cr(VI) reduction. Majorly Soluble reductase little activity by membrane associated. Nutrient broth (141) Agrobacterium radiobacter EPS-916 (resting cells) -200mV 0.5mM under Eh -138mV Gram-negative Glucose Fructose lactose glutamate succinate (82) Desulfovibrio vulgaris Gram-negative (93) Micrococcus roseus Gram-positive (56) Streptomyces (Actinomycete) Gram-positive (35) Pantoea agglomerans SP1 NA 0.1mM Gram-negative (facultative anaerobe) Anaerobic. Cr(VI) used as terminal electron acceptor. NA Lactate, acetate, hydrogen (45) Achromobacter eurydice ND Gram-negative (56) 25

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Table 1-1. continued. Bacteria Redox potential Cr(VI) reduced Gram stain Reduction conditions Enzymatic reduction Electron donor Reference Dienococcus radiodurans R1 (Thermus group) NA 0.5mM;. Gram-positive Anaerobic and to a lesser extent in aerobic conditions NA lactate (47) Bacillus subtilis ND 0.1mM to 1mM Gram-positive Aerobic Soluble protein; NADH can act as electron donor (53) Enterobacter cloacae HO1 0.5 mM Gram-negative Anaerobic Membrane associated Acetate/ glycerol/ glucose (115) Rhodobacter sphaeroides ND 0.146Mol h -1 (aerobic) 1.6Mol h -1 Gram-negative Aerobic and Anaerobic Soluble fraction; NADH required. Succinic acid (110) Desulfotomaculum reducens ND Less than 0.20 mM Gram positive Anaerobic ND Butyrate/ lactate/ propionate/ pyruvate/ glucose (152) Thiobacillus ferroxidans ND 0.289mM Gram-negative Anaerobic abiotic sulfur (125) 26 ND:not determined -; no values were found in leterature.

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CHAPTER 2 ENRICHMENT, ISOLATION, AND CHARACTERIZATION OF CR(VI)REDUCING BACTERIA Introduction Understanding the microbe-metal interactions in the environment has gained considerable importance in the past decade and a half (67, 94, 160, 161, 170, 175, 177). Among the various aspects of the interactions studied, the role of microorganisms in remediating contaminated water, soils, and sediments is gaining much appreciation (47, 125, 158, 166, 176). Microorganisms can affect the solubility and the toxicity of metals and provide insitu remediation of contaminated fields. Field studies, conducted to exploit the ability of microbes to attenuate or remove contaminants from the environment by direct or indirect means, have shown stimulation of indigenous microorganisms to be an effective method of remediation. Microbial reduction of soluble Cr(VI) to its insoluble Cr(III) form is a cost-effective way to prevent the mobility of Cr(VI) beyond the compliance boundaries and to eliminate the risk of health hazards to humans. Microbial reduction of Cr(VI) is controlled by many factors, including cell density, initial concentration of Cr(VI), pH, and redox potential (97, 98, 176). Among the factors that contribute significantly to microbial reduction of Cr(VI) by influencing the activities of particular groups of soil bacteria are electron donors and acceptors present in the soil. Number and the activity of Cr(VI)-reducing bacteria in soil largely depend on the growth conditions and the organic compounds that serve as electron donors present in soil. 27

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28 In aerobic environments, oxygen is the most abundant electron acceptor. In the absence of oxygen, other electron acceptors such as NO 3 and SO 4 2can be utilized by the microorganisms to conserve energy. Several microorganisms also have the ability to couple the reduction of the metal oxides such as Fe(III) and Mn(IV) to the oxidation of the carbon source. Wide diversity of Fe(III)and Mn(IV)-reducing has been established to date (84, 152, 177). Although few bacteria with an ability to reduce Cr(VI) have been reported in literature, not many Cr(VI)-respiring bacteria are reported possibly due to two reasons (a) Cr(VI) is a mutagen and is toxic for most organisms, (b) presence of other electron acceptors that can support the growth of the organisms is much higher. Recently, a sulfate reducing-bacteria Desulfotomaculum sp and a consortium of sulfate-reducing bacteria with an ability to utilize Cr(VI) were reported (152). Presence of other electron acceptors can influence the reduction of Cr(VI). For instance, it has been shown in the past that Fe(II) abiotically reduces Cr(VI) under reducing conditions, and that soil organic matter with a high content of humics acts as an electron donor in Cr(VI) reduction. However, paucity in data exists regarding the importance of microorganisms in these transformations. The comprehensive study presented here addresses various aspects of microbial Cr(VI) reduction in soil such as the organisms involved in Cr(VI) reduction, and the role of electron donors and acceptors in Cr(VI) reduction. Comparative analysis of genetic sequences provides insights into the genealogical relationships of prokaryotes. Sequencing studies have used 5S rRNA (58), cytochrome c and ferredoxins (136) among others as possible genetic probes for phylogenetic analysis. However, phylogenetic patterns obtained are not congruent among each other and none of them match the branching patterns of those of 16S rRNA. The sequence of 16S rRNA

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29 genes (rDNA) is considered to be a valuable genetic marker for establishing phylogenetic relationships between organisms. The conserved character of these molecules together with regions of higher variability, their ubiquitous distribution, genetic stability, and functional constancy make them a suitable candidate for this application. The phylogenetic trees cluster organisms based on genetic makeup. Phenotypic characters do not have to be considered. Therefore, 16S rDNA was used in this study for identifying the various CRBs. Specific objectives of this study were to (i) maximize the diversity of the CRB to be isolated (ii) evaluate the contribution of various electron donors for Cr(VI) reduction, (iii) study the effect of alternative external electron acceptors on Cr(VI) reduction, and (iv) perform phylogenetic analysis of the CRB isolated. Materials and Methods Soil was obtained from a highly Cr(VI)-contaminated Superfund site in the Upper Peninsula of Michigan. This site is a wetland receiving Cr(VI) from effluents discharged from an adjacent leather-tanning facility. Soil was collected in sterile containers and immediately shipped, while being maintained below 4C, to our laboratory. Samples were stored under 4C until the work began. The concentration of chromium in the soil was determined to be approximately 17 g/ kg of soil. The iron content of these soil samples was determined to be 13 mg/kg. Enrichment media. Anaerobic enrichments were established with a variety of electron donors and electron acceptors in different combinations. Enrichments were prepared in bicarbonate buffered basal media composed of (per liter) KH 2 PO 4 (0.42 g); K 2 HPO 4 (0.22 g); NH 4 Cl (0.2 g); mineral mix (10ml); vitamin mix (15ml); KCl (0.38 g); NaCl (0.36 g), CaCl 2 .2H 2 O (0.04 g); MgSO 4. 7H 2 O (0.10 g); NaHCO 3 (1.8 g); Na 2 CO 3

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30 (0.5 g); 1mM Na 2 SeO 4 (1ml). vitamin mix was composed of (per liter) biotin (2.0 mg); folic acid (2.0 mg); pyridoxine HCl (10.0 mg); riboflavin (5.0 mg); thiamine (5.0 mg); nicotinic acid (5.0 mg); pantothenic acid (5.0 mg); vitamin B-12 (0.1 mg); p-aminobenzoic acid (5.0 mg); thioctic acid (5.0 mg); mineral mix was composed of (per liter) NTA (1.5g); MgSO 4 (3.0g); MnSO 4 .H 2 O (0.5g); NaCl (1.0g); FeSO 4 7H 2 O (0.1g); CaCl 2 .2H 2 O (0.1g); CoCl 2 .6H 2 O (0.1g); ZnCl 2 (0.13g); CuSO 4. 5H 2 0 (0.01g); AlK(SO 4 ) 2 .12H 2 O (0.01g); H 3 BO 3 (0.01g); Na 2 MoO 4 (0.025g); NiCl 2 .6H 2 O (0.024g); Na 2 WO 4 .2H 2 O (0.025g). 90 ml of this medium were dispensed in 117 ml serum bottles under gas (C0 2 :N 2 ::20:80) pressure and gassed for 30 min. When required, iron was added to the medium in the form of Fe(OH) 3 a from stock solution of 0.5M before autoclaving. After autoclaving, media were anaerobically amended with electron donors and acceptors. Sterile stock solutions of electron donors and acceptors were prepared separately under anaerobic conditions. Final concentrations of supplements in the medium were Cr(VI) (0.4mM); AQDS (0.1mM); and Fe(III) (5mM). Electron donors included 10mM each of acetate, benzoate, citrate, and glucose as required for individual enrichments (Table 2-1). Enrichments. Four sets of anaerobic enrichments were established with of different electron donors as described above. Each set contained 4 microcosms, each amended with a different combination of electron acceptors (Table2-1). Microcosms contained the following combinations of electron acceptors. Cr(VI) only; Cr(VI) and AQDS; Cr(VI) and Fe(III); Cr(VI), AQDS, and Fe(III). Each set also included one control to monitor abiotic reduction of Cr(VI). Controls were supplemented with all electron acceptors and appropriate donor inoculated with dead (autoclaved) cells.

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31 The inoculum was prepared by adding 10 g of soil to basal medium (10 ml). Soil in media was stirred under continuous flushing with nitrogen gas. After considerable stirring to break all soil aggregates, 10 ml of slurry were used as inoculum for enrichments. The enrichments were incubated at 30C in the dark without shaking. The concentration of Cr(VI) remaining in the microcosm was monitored at various times. On depletion of Cr(VI) from the enrichments, transfers were made with 10% inoculum to fresh medium containing appropriate electron acceptors and donor. Rates of Cr(VI) reduction were determined in all enrichments after the third transfer as described below. By the third transfer, soil particles were diluted out, thereby decreasing the likelihood of Cr(VI) reduction by chemicals other those supplemented in the medium. Initial Cr(VI) concentrations were measured in enrichments immediately following inoculation to assess chemical reduction of Cr(VI) by reduced components that carried over during transfers. Analytical methods. Concentrations of Cr(VI) were determined colorimetrically by UV/Visible spectrophotometer (perker) equipped with 1 cm cuvettes, using the diphenylcarbazide (DPC) assay as previously described (43, 159). 0.25ml solution, prepared with 0.025g of 1,5-diphenylcarbazide in 10 ml of acetone, were added to 10 ml of sample (diluted when necessary). After 15 minutes of incubation at room temperature (25C), absorbance at 540 nm was determined. This assay has a detection limit of 2 M All readings were conducted in triplicate. Strain isolation. Bacteria were isolated by a standard roll tube method, technique used for the isolation of the anaerobic bacteria. Roll tubes are anaerobic, sealed serum tubes that were prepared by rolling tubes containing sterile molten agar in medium with

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32 carbon source. The inoculum was added while the agar was still in molten form. The tubes were rolled till the agar layer was set along the walls of the tube. Isolated colonies appeared either embedded or on the surface of the agar layer. Each culture was passed through roll tubes several times until colonies with uniform morphology were obtained. Colonies selected and picked by sterile long stemmed pasteur pipettes were immediately transferred liquid basal media while maintaining the electron donors and acceptors. This set up was continuously maintained under nitrogen gas flow to keep it anaerobic. Isolates were obtained from enrichment cultures in which the fastest reduction of Cr(VI) was observed. Isolated colonies were tested for their ability to reduce Cr(VI). Isolates were tested for facultative and obligate anaerobic growth. Strains prefixed as GCAF were isolated from Glucose-Cr(VI) enrichment supplemented with Fe(III) and AQDS; Strains designated as GCF were from the glucose-Cr(VI) enrichment with Fe(III); GCA isolates were obtained from the glucose-Cr(VI) and AQDS enrichment. GC isolates were from the Glucose-Cr(VI) enrichment with no external electron acceptors. DNA isolation and amplification of 16S rDNA. Genomic DNA was extracted from each of the 12 isolates (1ml culture of pure isolates). Cells were harvested and lysed by boiling in 500 l of sterile water. 16S rRNA gene was amplified using universal bacterial primers 27f and 1492r (76)with Perkin Elmer thermocycler Model 240 (Norwalk, CT). Conditions for amplifications were as follows: 95 o C for 15 min, followed by 35 cycles of 94 o C for 30 seconds, 58 o C for 1 min and 72 o C for 30 sec. The final extension step was 7 minutes at 72 o C. The amplified product was purified using a commercially available kit (Qiagen, Inc.) and sequenced by the DNA Interdisciplinary Center for Biotechnology Research sequencing facility at the University of Florida.

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33 Phylogenetic analyses of the isolates 16S rDNA sequences were screened using the BLAST (ref.) program to identify organisms of highest similarity with 16S rDNA sequences of the various isolates obtained. Sequence alignment were either performed manually with sequences obtained from Ribosomal Database Project (96)or by using PILEUP function of GCG (Genetics Computer Group version ). CLUSTALX was used to view the alignment and finer adjustments were made manually using McCLADE version 3.0 (95) Phylogenetic trees were constructed using maximum parsimony analyses of the aligned sequences by PAUP 4.0b8 (150). Bootstrap values were assigned on 100 replicates after reweighing the characters by heuristic search strategy to assess the confidence level of various clades. The GenBank accession numbers for the sequences shown in Figure 2-2. Results and Discussion Effect of Electron Donors and Acceptors on Cr(VI) Reduction The results strongly suggested that the rate of Cr(VI) reduction by indigenous soil microorganisms was affected by the available electron donor and acceptors. Even though reduction of Cr(VI) was observed in all sets of enrichments, there was a difference in rate of Cr(VI) reduction. Concentrations of Cr(VI) remained constant in treatments that were not inoculated with cells, and insignificant amount of reduction was observed in treatments inoculated with heat killed cells (Figure 2-1). Cr(VI) was rapidly reduced in enrichments with glucose and citrate as electron donors. Cr(VI) concentrations fell below detection levels in less than a week in glucose enrichments. Higher turbidity was also observed in these enrichments. Enrichments with citrate as electron donor reduced Cr(VI) within 10 days. Bacterial enrichments amended with acetate and benzoate also showed loss of Cr(VI) although at a much lower rate. Cr(VI) in these enrichment cultures was

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34 completely reduced after three weeks. The pH of cultures was monitored and remained constant at 7.4. Enrichments with different electron donors were established in order to maximize the diversity of the CRB enriched. Acetate was chosen as an electron donor as it is abundant in nature and most metal reducing organisms have the ability to couple acetate oxidation to metal reduction (51, 109, 147). No loss of Cr(VI) and no bacterial growth was observed in enrichments with Cr(VI) as sole electron acceptor. There are three possible explanations; (i) toxicity of high concentrations of Cr(VI) (ii) absence of acetate utilizing bacteria that couple their growth to Cr(VI) reduction (iii) bacterial communities being among the 99.9% uncultivable bacteria. Benzoate and lactate were expected to support the growth of organisms that conserve energy by oxidizing aromatic compounds and fermentation products respectively. Bacteria with the ability to oxidize benzoate with Cr(VI) reduction has been reported (140). Citrate was one of the chosen electron donors for this study as it is a part of citric acid cycle that has major biosynthetic as well as energetic functions and many organisms have the ability to utilize it an electron donor and carbon source. Reduction of Cr(VI) in enrichment sets with the electron donors was observed only in the presence of other electron acceptors. Variation in rate of Cr(VI) reduction observed in different donor sets suggested diversity in organisms being enriched and possibly different mechanisms of Cr(VI) reduction. Slight turbidity was observed in glucose enrichment with Cr(VI) as sole electron acceptor but there was no reduction of Cr(VI) indicating growth of Cr(VI) resistant fermentative organisms.

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35 Reduction of Cr(VI) was observed in all enrichment set when the media were supplemented with additional electron acceptor AQDS or Fe(III). In presence of both Fe(III) and AQDS, an accelerated rate of Cr(VI) reduction was observed. These results can be explained by efficient channeling of electrons towards the reduction of Cr(VI) due to Fe(III) and AQDS acting as electron shuttles and the amount of electrons shuttled in these systems are much higher and therefore Cr(VI) is rapidly lost from the system. Microbially reduced form of AQDS can act as a shuttle by transferring electrons to insoluble Fe(III) and increasing the rate of Fe(III) reduction. Fe(II), in turn, can reduce Cr(VI) to Cr(III). In the absence of either of the electron acceptors, effective electron shuttle trains are broken, thereby lowering the rate of Cr(VI) reduction. Increased reduction of Cr(VI) is also indicative of the combined contribution of the AQDS respiring bacteria, Fe(III) reducing bacteria, and Cr(VI) reducing bacteria (Figure 1-4). The results also suggested that the presence of Fe(III) and AQDS in combination alleviate the toxicity of Cr(VI) to the bacteria more than when they are present alone with Cr(VI). Fastest rate of Cr(VI) was observed in enrichment culture with glucose as electron donor. In addition higher turbidity was also observed. Preference of glucose by the enriched indigenous CRB, over other electron donors used in the study was clearly evident. Anaerobic Cr(VI)-enrichment with glucose as electron donor was expected to enrich fermentative bacteria in addition to other CRB. In the environment fermentative organisms form the primary level where they oxidize more complex electron donors and form simpler metabolites that are used up by the secondary level microorganisms. Therefore the fermentative organisms that are resistant to Cr(VI) were expected to enrich

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36 first along with the other Cr(VI) resistant, non fermentative CRB. The fermentation metabolites formed could then be used by other non fermentative CRB that are unable to utilize glucose as carbon source. However, results from the phylogenetic study revealed that the enrichment culture that showed the fastest reduction of Cr(VI) was dominated by the fermentative gram positive bacteria. One of the possible explanation of the dominance of these bacteria in the enrichment culture is that the major indigenous CRB in the Cr(VI)-contaminated soil were fermentative bacteria, mainly Clostridium sp. and Celullomonas sp. At this point, it was not clear if Clostridium sp. reduces Cr(VI) directly or indirectly via Fe(III) and AQDS. The confirmation of this hypothesis and the elucidation of the mechanism of Cr(VI) reduction by these organism was the objective of the next study. Phylogenetic Analysis of Cr(VI) Reducing Bacteria Phylogenetic analysis of 16S rDNA sequences of the isolates obtained from enrichments with glucose as electron donor yielded organisms that belonged mostly to the high G+C gram positive Cellulomonas group and low G+C gram positive Clostridium group of bacteria. Several isolates belonging to Micrococcus, Bacillus, and Staphylococcus genera were also obtained. With exception of Bacillus and Micrococcus, attempts to grow these isolates aerobically were unsuccessful. Previously described CRB are capable of reducing Cr(VI) to Cr(III) and are phylogenetically diverse. Most CRB are gram-negative and facultative anaerobes. Few gram positive bacteria capable of reducing Cr(VI) have been reported to date. Reduction of Fe(III) by Clostridium sp has been documented previously, but no reports of Cr(VI) reduction by Cellulomonas sp or Clostridium sp have been published so far. Results from this study strongly suggest

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37 significant role played by the fermentative organisms in the reduction of Cr(VI) and perhaps other heavy metals. As more chromium reducing bacteria will be isolated from various environments it is likely that the diversity will continue to increase. It still remains to be seen if the capability to reduce Cr(VI) evolved independently and specifically in some organisms or if there are some organisms that can support their growth on the reduction of Cr(VI). For example, numerous studies suggest that NADH dependant-reductase enzyme was invariably involved in reduction of Cr(VI) to Cr(III) in E. coli and Pseudomonas sp. the finding that many other bacteria can reduce Cr(VI) without any enzyme indicates that there is more than one pathway for the reduction of Cr(VI). These studies emphasize that much study is required before the microorganisms in various environments will be known and before the mechanisms for Cr(VI) reduction will be understood.

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38 Table 2-1. Combination of electron acceptors and donors supplemented in the media for anaerobic enrichment studies Electron donors Electron acceptors Code Acetate Cr(VI) Cr(VI) and AQDS Cr(VI) and Fe(III) Cr(VI), AQDS and Fe(III) AC ACA ACF ACAF Benzoate Cr(VI) Cr(VI) and AQDS Cr(VI) and Fe(III) Cr(VI), AQDS and Fe(III) BC BCA BCF BCAF Citrate Cr(VI) Cr(VI) and AQDS Cr(VI) and Fe(III) Cr(VI), AQDS and Fe(III) CC CCA CCF CCAF Lactate Cr(VI) Cr(VI) and AQDS Cr(VI) and Fe(III) Cr(VI), AQDS and Fe(III) LC LCA LCF LCAF Glucose Cr(VI) Cr(VI) and AQDS Cr(VI) and Fe(III) Cr(VI), AQDS and Fe(III) GC GCA GCF GCAF

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39 Table 2-2. Accession numbers for 16S rDNA sequences used in this study Bacterial species Acession numbers* Clostridium cellasea X83804 Cellulomonas flavigena AF140036 Cellulomonas persica AF064701 Cellulomonas sp.strain 1533 Y09658 Cellulomonas hominis X82598 Oerskovia turbata X79454 Clostridium acetobutylicum X81021 Clostridium beijerinckii X68179 Clostridium roseum strain DSM 51 Y18171 Clostridium sp. (C.corinoforum) X76742 Clostridium sp. (C.favososporum) X76749 Clostridium .puniceum X71857 Clostridium butyricum X68176 Clostridium paraputrificum strain M-21 AB032556 Staphylococcus sp. strain LMG-19417 AJ276810 Bacillus megaterium D16273 Bacillus macroides X70312 Bacillus macroides AF157696 Bacillus subtilis N5 AF270793 Desulfotomaculum acetoxidans Y11566 Pseudomonas putida AF307869 *Gen Bank accession numbers except for those species sequenced used in this study

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40 Acetate enrichment02040608010001424Time(days)Cr(VI) remining (%) Glucose enrichment020406080100036Time (days)Cr(VI) remaining (%) Citrate enrichment0204060801000410Time(days)Cr(VI) remaining (%) Benzoate enrichment02040608010001020Time (days)Cr(VI) remaining (%) A D C B ca cf caf control ca cf caf control ca cf caf control ca cf caf control Figure 2-1. Rate of reduction of Cr(VI) in enrichment cultures amended with different electron donors and electron acceptors. A: acetate amended enrichments. B: benzoate amended enrichments; C: citrate amended enrichments, D: glucose amended enrichments. Each of the enrichment sets was supplemented with adiitional electron acceptors. ca :Cr(VI) and AQDS, cf: Cr(VI) and Fe(III); caf: Cr(VI),AQDS and Fe(III); control was set up with dead (autoclaved)cells amended with Cr(VI),AQDS and Fe(III) as electron acceptors. No cell control showed very insignificant reduction of Cr(VI).

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41 B Cellulomonas cellasea (X83804)gca10 Cellulomonas cellasea (AF140036)Cellulomonas persica (AF064701)gc9gc8gc7Cellulomonas sp. strain 1533 (Y09658)Cellulomonas hominis (X82598)Oerskovia turbata (X79454) gca14 gca13 Clostridium acetobutylicum (X81021)Clostridium beijerinckii (X68179)Clostridium roseum (Y18171) Clostridium coriniforum (X76742)Clostridium favososporum (X76749)Clostridium puniceum (X71857)Clostridium butyricum (X68176)gcaf7a gcaf7 gcaf9 gcaf5 gcaf1a gcf2Clostridium paraputrificum (AB032556)gca3gca4gca2gca7gca5Staphylococcus sp. (AJ276810)gc4Bacillus megaterium (D16273 gca8Bacillus macroides (NCD01661) (X70312)gc3Bacillus macroides (AF157696)(Bacillus subtilis) AF270793 cca1Desulfotomaculum acetoxidans (Y11566)Pseudomonas putida (AF307869)100 81649310089100739577100 100 81 7984911009173100901001009975100 10010010089100100100ootstrap Figure 2-2. Phylogenetic tree constructed using maximum parsimony (PAUP version 4.0b8). Numbers above the branches represent the bootstrap values (100 replicates). Codes for isolates are provided in Table 2-1.

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CHAPTER 3 CR(VI) REDUCTION BY A CONSORTIUM OF GRAM POSITIVE FERMENTATIVE BACTERIA Introduction Cr(VI) is mainly generated as an effluent by many industries. Due to its soluble nature it has been found in groundwater not only at the point source but also away from its source. Mutagenic and carcinogenic nature of Cr(VI) makes its contamination a matter of intense concern. Chemical processes that reduce Cr(VI) before it is discharged into the environment include chemical reduction, ion exchange and electrodepositing. The use of microbial reduction offers an inexpensive and long term alternative strategy to treat the Cr(VI)-contaminated soils and ground water. In order to implement the bioremediation strategy, it is important to understand microorganisms involved in Cr(VI) reduction and the mechanisms by which Cr(VI) is reduced. Data from enrichment studies (chapter 2) indicated that among the various carbon sources tested glucose was the preferred electron donor by the organisms to carry out the reduction of Cr(VI). Enrichments showing the fastest reduction of Cr(VI) had enriched gram positive bacteria belonging predominantly to Clostridium sp and Cellulomonas sp. Presence of fermentative bacteria like Clostridium sp. in metal contaminated sites has been described in the past, but no detailed study has been done to elucidate their role in metal reduction. Their major role in the metal contaminated environments has been described as being the provider for the carbon sources for other metal reducing bacteria. The metabolic products of fermentative 42

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43 bacteria act as electron donors for other microorganisms that are involved in metal reduction. Studies with consortium are significant as they provides a closer picture of what happens in the field where the microorganisms do not live in pure cultures. Pure culture studies are important to study the particular microbial activity and the consortium studies explain how the activity may be influenced by the presence of other organisms. Considering the above mentioned reasons the following study was done to understand the kinetics of Cr(VI) reduction by the cosortium GCAF isolated from glucose enrichment. The objectives of this study were to (i) monitor the kinetics of Cr(VI) reduction by the consortium GCAF, in the presence of Fe(III) and AQDS (ii) to elucidate the mechanism of Cr(VI) reduction by the consortium, (iii) to study the effect of Cr(VI) on the fermentation Materials and Methods Culture conditions for bacterial consortium. Fermentative consortium GCAF-1 was obtained during the previously conducted enrichment study (chapter 2). Glucose (10mM) was supplied as the electron donor. Cr(VI) (0.4 mM), Fe(III) (5 mM) and AQDS (0.1mM) were provided as electron acceptors. For inoculum purposes, the consortium was grown in the absence of any electron acceptor. All manipulations were made under an atmosphere of N 2 -CO 2 (80:20). Metal reduction experiments. Late log phase cultures were inoculated in fresh anaerobic medium with glucose as electron donor and incubated under 30C till early log phase was reached. The optical density of the inoculum was 0.6 (OD at 550nm). Media supplemented with appropriate electron donor and acceptors was inoculated with 1 % inoculum. Experimental set up consisted of eight different treatments.The treatments

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44 consisted of basal medium supplemented with (i) glucose and all three electron acceptors, (ii) glucose, Cr(VI) and Fe(III), (iii) glucose and Cr(VI), (iv) glucose, Fe(III) and AQDS, (vi) glucose and no electron acceptors, (vii) all three electron donors and no glucose and, (viii) glucose and all three electron acceptors and no bacterial inoculation. The concentration of glucose, Cr(VI), Fe(III) and AQDS were maintained as in the original consortium. All the addition of electron acceptors and donors was done separately from sterile stock solutions. After inoculation the experimental cultures were incubated at 30C throughout the study. Samples were taken with sterile syringe and needles that were flushed with N 2 to avoid any contamination of the cultures with oxygen at appropriate time intervals. Each test was performed in triplicates. Determination of cell numbers. Number of cells in the cultures were determined by direct count using acridine orange stain and the fluorescence microscope. Dilutions of the cultures were made wherever necessary to keep the cell count within the range of 50-100 per field area. 25% gluteraldehyde was used to fix the cells for counting. Cells were suspended in oxalate solution prior to counting to dissolve any insoluble Fe-oxides in the solution. Chemical analyses. Chromium analysis was performed by colorimetric method using UV/Vis spectrophotometer (Shimadzu) as mentioned previously (chapter 2). Glucose analyses was done by the colorimetric method using UV/Vis spectrophotometer at 490nm. Sample was filtered through 0.2m filter. 200l of 5% phenol was added to the equal amount 200l of sample. Immediately 1ml of conc. H 2 SO 4 was added to the mixture and gentally shaken. The reaction was kept stationary for 30 minutes to allow the solution to cool down. The solution was gently shaken before taking the reading at

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45 490nm. Precipitate analysis was done by EDX. Organis acids formed as he fermentation products were measured by High pressure liquid chromatography (Waters Co.) equipped with a UV detector (Waters, Co). Aminex HP 87H column was used as the separating column (300 X 7.5 mm). Sulfuric acid (5mM) was used as an eluent at the flow rate of 0.6 ml/ minute. Electron microscopy. Precipitates formed in the consortium was obtained by using the microcentrifuge (12000X g), rinsed twice with distilled water, and air dried on carbon coated mounts prior to viewing it via scanning electron microscopy. Results and Discussion Composition of Fermentative Consortium GCAF Active Cr(VI) reducing consortium isolated from the glucose enrichment was dominated by high G+C and low G+C gram positive bacteria. Fermentative consortium GCAF was unable to grow or reduce Cr(VI) when Cr(VI) was added as the sole electron acceptor to the medium. Growth of cells was inhibited by the toxicity of Cr(VI). However, in the presence of Fe(III) and AQDS, reduction of Cr(VI) was observed suggesting the possibiliy of Fe(III) alleviating the toxicity of the Cr(VI) to the cells of Consortium GCAF (Figure 3-1). Biotic versus Abiotic Reduction of Cr(VI) Cr(VI) reduction was a biotic process that did not occur in medium that was not inoculated with cells. No reduction of Cr(VI) was observed in the medium in the absence of the electron donor suggesting that the biotic reduction of Cr(VI) required the metabolically active cells.

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46 Kinetics of Cr(VI) Reduction Fermentative consortium GCAF was able to reduce Cr(VI) under anaerobic conditions. Presence of other electron acceptors had an effect on the amount of Cr(VI) reduced, the rate at which Cr(VI) was reduced, and the rate at which glucose was oxidized by the consortium GCAF. Media that were supplemented with other electron acceptors showed high turbidity indicative of bacterial growth. This was also confirmed by observing the sample under the microscope. Cr(VI) reduction was also observed in the active cultures. Complete reduction of Cr(VI) was observed in medium with AQDS and Fe(III) as additional electron acceptors. However, in the absence of AQDS only 66% of Cr(VI) was reduced. The rate of reduction of Cr(VI) was slower in the absence of AQDS. Similar trends were observed in the oxidation of glucose. In the absence of AQDS complete glucose (10mM) was not utilized and the rate of oxidation was much slower. These results suggested that the electrons being generated by oxidation of glucose, by the consortium were being transferred to Cr(VI) via Fe(III) and AQDS. In the absence of AQDS, the electrons were shuttled from the cells to the insoluble Fe(III). Soluble Fe(II) was then behaving as an electron shuttle and transferring the electrons to Cr(VI). Due to the requirement of contact between bacterium and insoluble Fe(III) to transfer electrons, the process was slow. This affected the overall reduction of Cr(VI). In presence of AQDS there is a higher turnover of the electrons and faster reduction of Fe(III). This in turn increases the rate of Cr(VI) reduction. AQDS is soluble unlike Fe(III) and it alleviates the need for contact between the cells and the metal. It accepts two electrons unlike Fe(III) that can accept only one electron at a time. Therefore higher reduction of Fe(III) occurs in the presence of AQDS which in turn augments the rate of Cr(VI) reduction. The higher rate of Cr(VI) reduction corresponds well to the high rate of glucose consumption in the

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47 presence of AQDS. In the absence of Cr(VI) the rate of glucose consumption was much higher suggesting that Cr(VI) effects the metabolic machinery of the cells. The initial color of the medium was yellow but with the reduction of Cr(VI) it became colorless. Insoluble amorphous Fe(III) was red in color that reduced to white precipitate that had disc shaped crystals when seen under a SEM microscope(Figure3-6). The X-ray diffraction analysis indicated this precipitate to be vivianite Effect of Electron Donor on Cr(VI) Reduction Concentration of electron donor in the medium affected the amount of Cr(VI) reduced (Table 3-2). However the rate of reduction of Cr(VI) was not depedant on the concentration of the electron donor. These results have implications in the field of bioremediation. Limitation of electron donors in the environment can impede the microbial reduction of Cr(VI) by the fermentative organisms. Effect of Cr(VI) Reduction on Cell Growth in Consortium GCAF The final cell concentration was lower by 47% in the medium in the presence of Cr(VI). Although presence of AQDS in the medium with Fe(III) increased the rate of Cr(VI) reduction, there was no significant difference in the cell numbers observed when compared with those in the absence of AQDS (Table 3-2). Effect of Cr(VI) on Metabolites Since consortium GCAF comprised mainly of fermentative bacteria effect of Cr(VI) on the fermentative products was determined. The variability in the rate of glucose oxidation during Cr(VI) reduction in presence of different electron acceptors suggested varied rates of product formation. Furthermore, reduction of external electron acceptors by the fermentative bacteria suggested some change in the fermentative metabolic products. Therefore the production of fermentation products of the consortium

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48 were monitored with time. The results observed showed that formation of products corresponded well with the time when oxidation of glucose started and the production carried on till glucose was consumed (Figure 3-3, 3-4, 3-5). The major fermentation products that were generated by the consortium GCAF when grown in presence of glucose were acetate, butyrate and lactate. No change in the proportions of products was observed when Fe(III) and AQDS was added to the medium. However, there was a shift in fermentation product pattern observed when Cr(VI) was added to the medium containing Fe(III) and AQDS as other external electron acceptors. Oxidation of glucose by the cells in consortium resulted in significantly less amount of butyrate, and more amount of acetate in the presence than in the absence of Cr(VI) (Table 3-3). These results suggested that the reduction of Cr(VI) effects the cells to shift the fermentation products to more oxidative forms. During the glucose fermentation by the fermentative cells, glucose is oxidized to pyruvate and generates two molecules of NADH (four reducing equivalents). The oxidative decarboxylation of pyruvate generates one molecule of formate and one molecule of acetyl CoA. Acetyl CoA has two alternative fates: it either forms acetate with a generation of ATP or can sacrifice the generation of energy by forming a more reduced form ethanol. The other possibilities for disposing reducing equivalents is by the formation of lactate. Lactate results in the reoxidation of one NADH. Butyrate formation is at the expense of 4 reducing equivalents (or 2 NADH). In the presence of Cr(VI) the drop in butyrate and lactate formation indicated that less of NADH were being reoxidized by those pathways. Increase in acetate could perhaps be the result of excess pyruvate being converted to acetate and formate. The energy generation step that is liked to acetate would prove beneficial to the

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49 cell as it has to survive in the toxic environment of Cr(VI) and reduce it. The excess NADH that were not reoxidized by lactate and butyrate were then channeled towards the reduction of Cr(VI).Whether reduction of Cr(VI) occurs inside the cell membrane or on the cell surface was not clear at this point. No difference in the pattern of the products formed in the medium in the presence of Fe(III) and AQDS and in their absence indicated that mechanism for reduction of Fe(III) and AQDS was different from the way Cr(VI) was reduced by the cells in consortium GCAF. So far there is no evidence that energy conservation by fermenting bacteria during reduction of Cr(VI). Although, reduction of Cr(VI) results in increased formation of acetate which is energetically favorable for fermenting bacteria. Mechanism of Cr(VI) reduction by a pure strain isolated from this consortium GCAF has been explained in chapter 5. Identification and characterization studies of the isolate have been conducted in order to explain the phylogenetic importance of the strain. GCAF may be a good candidate for the bioremediation of heavy metal laden waters and sediments.

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50 00.050.10.150.20.250.30.350.40.45050100150200250Time (hrs)Residual Cr(VI) (mM) GC NA NC NG S Figure 3-1. Cr(VI) reduction and removal from the solution as an insoluble precipitate. Glucose (10mM) was supplied as an electron donor. Fe(III) and AQDS were supplied as extra electron acceptors. Reduced insoluble Cr(VI) was removed from the solution by centrifugation prior to analysis. GC: glucose, Cr(VI); NA. Glucose, Cr(VI), Fe(III); NC. Glucose, Cr(VI), Fe(III), AQDS with no inoculation of cells; NG: Cr(VI), Fe(III), AQDS; S: glucose, Cr(VI), Fe(III) and AQDS. 02468101214050100150200250Time (hrs)Residual Glucose (mM) GC NA NC NCr NEA S Figure 3-2. Glucose consumption by consortium GCAF-1 during reduction of Cr(VI) (0.4mM) in the presence of Fe(III) (5mM) and AQDS.(0.1 mM). GC: glucose, Cr(VI); NA. Glucose, Cr(VI), Fe(III); NC. Glucose, Cr(VI), Fe(III), AQDS with no inoculation of cells; NCr; glucose, Fe(III) AQDS; NEA: glucose; S: glucose, Cr(VI), Fe(III) and AQDS.

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51 A 00.511.522.533.544.55050100150200250time (hrs)Acetate (mM) NA NCr NEA S 00.511.522.533.544.55024681012Glucose (mM)Acetate (mM) NA S NEA NCr B Figure 3-3. Acetate produced by oxidation of glucose by consortium GCAF.during the reduction of Cr(VI) in presence of added electron acceptors. (A) Acetate produced with respect to time. All treatments were set up in triplicates with eeror bars representing the standard error. (B) Acetate produced per mole of glucose consumed.NA. Glucose, Cr(VI), Fe(III); AQDS with no inoculation of cells; NCr; glucose, Fe(III) AQDS; NEA: glucose; S: glucose, Cr(VI), Fe(III) and AQDS.

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52 012345678910050100150200250time (hrs)Butyrate (mM) NA NCr NEA S 012345678910024681012Glucose (mM)Butyrate (mM) NA S NEA NCr Figure 3-4. Butyrate produced by oxidation of glucose by consortium GCAF during the reduction of Cr(VI) in presence of added electron acceptors. (A) Butyrate produced with respect to time. All treatments were set up in triplicates with eeror bars representing the standard error. (B) Butyrate produced per mole of glucose consumed.NA. Glucose, Cr(VI), Fe(III); AQDS with no inoculation of cells; NCr; glucose, Fe(III) AQDS; NEA: glucose; S: glucose, Cr(VI), Fe(III) and AQDS

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53 00.511.522.5050100150200250time (hrs)Lactate (mM) NA NCr NEA S 00.250.50.7511.251.51.7522.25024681012Glucose (mM)Lactate (mM) NA S NEA NCr Figure 3-5. Lactate produced by oxidation of glucose by consortium GCAF during reduction of Cr(VI) in presence of added electron acceptors. (A) Lactate produced with respect to time. All treatments were set up in triplicates with eeror bars representing the standard error. (B) Lactate produced per mole of glucose consumed. NA. Glucose, Cr(VI), Fe(III); AQDS with no inoculation of cells; NCr; glucose, Fe(III) AQDS; NEA: glucose; S: glucose, Cr(VI), Fe(III) and AQDS.

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54 Figure 3-6. SEMs showing the insoluble precipitates formed by consortium GCAF-1 during the reduction of Cr(VI) via Fe(II)and AQDS-mediated mechanisms

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55 Figure 3-7. EDX of precipitate formed by consortium GCAF-1 showing the distribution of Cr in the precipitate formed during Cr(VI) reduction.

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56 Table 3-1. Cell growth in the presence of different electron acceptors. Cells were grown in Basal Media supplemented with glucose as electron donor and Fe(III) as external electron acceptor. Cr(VI) and AQDS were added as described in the table. Cell counts were estimated by Acridine Orange Direct Count method using a fluorescence microscope. All treatments were set up in triplicates. Treatment Fe(III) AQDS Cr(VI) Cell numbers/ ml ( x 10 7 ) Reduction in Cell number (%) I + + 80.6 4.6 a II + + + 50.3 5.3 b 37.61 III + + 43.4 1.6 b 47.18 Table 3-2. Cr(VI) reduction by consortium GCAF in presence of varying concentrations of electron donor. Glucose Initial (mM) Glucose utilized (mM) Rate of Cr(VI) reduction (mM/day) Amount of Cr(VI) reduced (mM) 5.73 5.62 0.066 0.19 10.92 9.05 0.066 0.34 17.96 8.42 0.066 0.37

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57 Table 3-3. Effect of Cr(VI) on the on the pattern of products of glucose fermentation by consortium GCAF. Products formed (mM) AQDS Fe(III) Cr(VI) Concentration of substrate consumed (mM) Acetate Butyrate Lactate A _ 10 1.7 7.9 1.9 B + + 10 1.9 8.4 1.1 C + + + 10 4.1 5.5 0.9

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CHAPTER 4 IDENTIFICATION AND CHARACTERIZATION OF THE CHROMIUM REDUCING ISOLATE CLOSTRIDIUM SP. GCAF1 Introduction In the last few decades, microbial metal reduction has been identified as an important process for mineralization of organic compounds (92) and for detoxification and remediation of soils contaminated with toxic metals (6, 88, 89). Microbial Cr(VI) reduction was first demonstrated by Romanenko (129), following which a wide diversity of CRB have been isolated. Cr(VI) reduction by organisms belonging to the genera Bacillus, Escherichia, Pseudomonas, and Pantoea, among others.. This dissertation describes the involvement of Clostridium and Cellulomonas species (Chapter 2) in Cr(VI) reduction. The presence of fermentative bacteria in Cr(VI)-contaminated soils and sediments have been reported previously, no detailed studies on the direct involvement of these bacteria in Cr(VI) reduction have been documented to date. The genus Clostridium forms one of the largest gram-positive taxa and has significance in several fields. Many toxin producing pathogens (Clostridium perfringens, C. botulinum, C. tetani and C. difficile) and industrially important solvent producing fermentors (C. acetobutylicum, C. butyricum, C. aceticum) belong to this group (69, 145, 146, 172). This study reports the discovery of a new spore forming bacterium, Clostridium sp. GCAF-1. This strain has an ability to reduce Cr(VI) directly and indirectly via Fe(III-reduction). Detailed results of extensive analysis of 16S rRNA gene sequences, DNA58

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59 DNA homology and G+C content analyses are described here. Additional studies supporting the designation of GCAF-1 as a novel species of genus Clostridium are also reported. Material and Methods Source of sample and organism. Soil used as inocula for the enrichments originated from the Cr(VI) contaminated wetlands in Michigan. Tranfers were made several times into the fresh media to dissolve the soil particles out. transferred to Strain GCAF-1 was isolated from Cr(VI)-reducing anaerobic enrichment that was provided with glucose as electron donor and Fe(III) and AQDS as additional electron acceptors. The enrichments were amended with Fe(III) and AQDS as additional electron acceptors. Media and growth conditions. Standard anaerobic culture techniques were used during the preparation of the medium (11). A bicarbonate buffered mineral medium, as described in chapter 2, was used for the growth of the strain GCAF-1. The final pH was adjusted with HCl ca 7.0 and N 2 /CO 2 was bubbled through it to remove oxygen. The medium was dispensed into anaerobic pressure tubes or serum bottles under N 2 /CO 2, sealed with thick butyl rubber stoppers and then sterilized by autoclaving. Electron donor, glucose (10mM) and appropriate electron acceptor [Cr(VI) (400M); Fe(OH) 3 (5mM); AQDS (100M)] was added later from sterile anaerobic stocks. All incubations were done in the dark under 30C without any shaking. Isolation of strain GCAF-1. Strain GCAF-1 was isolated from the consortium enriched under glucose-Cr-Fe(III) and AQDS conditions. All procedures were carried out under the anoxic conditions in a glove box equipped with charcoal filter and with N 2 /CO 2 /H 2 (v/v) as gas phase. Traces of oxygen were removed by circulating the gases over the palladium catalyst and desiccant.

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60 Biochemical analyses. The biochemical features of the strain GCAF-1 were determined to establish its species status. Production of riboflavin (yellow pigment) by GCAF-1, under anaerobic conditions, was tested by inoculating sterile milk under anaerobic conditions and incubating at 30C for 24 hours (70). Sensitivity to antibiotic rifampicin was tested and turbidity was monitored to determine the resistant and sensitive nature of the strain GCAF-1(70). Fatty acid analysis. Methyl esters of cellular fatty acids (FAME) were prepared according Metcalfe et al (105) and were analyzed using Microbial Identification System (MIDI, Inc., Newark, DE, USA). Fatty Acids were identified by comparison of retention times with those of commercial standards (Sigma Co., USA). Determination of G+C content of the DNA. The base composition of the DNA was determined by Deutsche Sammlung von Mikrooganismen und Zellkulturen (DSMZ, Germany). DNA was isolated by cell disruption with French pressure cell. DNA was purified on hydroxylapatite columns (26). The DNA was further hydrolyzed with P1 nuclease and nucleotides were dephosphorlized with bovine alkaline phosphatase (104). The resulting deoxiribonucleosides were analyzed by HPLC using column SelectaPore 90M, C18, 5 m (250 x 4.6mm) equipped with guard column 201gd54H (Vydac, Hesperia, CA 92345, USA). Chromatography conditions were as follows: temperature 45C, 10 l sample, solvent used was 0.3 M (NH 4 ) H 2 PO 4 / acetonitrile, 40:1 (v/v), pH 4.4, flow rate 1.3 ml / min (adapted from Tamaoka and Komagata) (151). Non-methylated lambda DNA (sigma) with GC content of 49.85 mol% (104) was taken as standard. GC ratio calculated from the ratio of deoxyguanosine (dG) and thymidine(dT) according to Mesbah (1989) (104).

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61 16S rRNA gene sequencing and phylogenetic analysis..Cells from actively growing culture of GCAF-1 were harvested by centrifuging at 3000xg. The pellet was washed three times and resuspended with a phosphate buffer saline. Suspended culture was lysed by boiling for fifteen minutes. The 16S rRNA gene was amplified by using the universal bacterial primers 27F (5`-AGAGTTTGATCCTGGCTCAG-3`) and 1492 R (5`-TACGGTTACCTTGTTACGACTT3`) (76). Purified PCR products were cloned using TOPO TA Cloning kit (Invitrogen, Life Technologies). 14 clones forming white colonies were chosen for further analysis. Insert DNA was amplified using M13 forward and reverse primers to under conditions described in appendix B. The insert was digested with Hae III and Alu I separately. Digestion reaction was set at 37C for 14 hours. The digestion pattern of the clones was compared on a 2% agar electrophoresis gel. Based on the results obtained, 16S rDNA amplification products of two clones were sent for sequencing. Sequencing was done at ICBR core sequencing facility at University of Florida with an automated sequencer. Sequences obtained were compared with those available from public databases using BLAST search and were aligned with type strains showing 98% or greater similarity using GCG. Phylogenetic tree was constructed based on 1400 bp 16S rRNA sequences using PAUP version 4.0. DNA-DNA hybridization. These studies were performed at the DSMZ with strains showing 98% (or higher 16S rDNA similarity) by thermal denaturation method described by DeLey et al (ref) with Gilford System 2600 spectrophotometer equipped with a Gilford 2527-R thermoprogrammer. DNA was isolated by chromatography on hydoxylapatite as described by Cashion et al. (26).

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62 Results and Discussion Cell morphology. GCAF-1 is a spore forming obligate anaerobe. Cells of strain GCAF-1 were rod shaped (4-7 long and 1-1.5 in diameter), occurring singly or in chains of 4-7cells (Figure 4-1). Terminal and subterminal endospores were observed under scanning electron microscope (SEM). Spores were oval in shape (1-1.5 by 0.5 ) (Figure 4-2). Spores constitute the dormant form of the bacterial cell that are produced during the advent of starvation. The spores are resistant to adverse conditions including high temperatures and organic solvents. Gram staining of GCAF-1 resulted in faint blue color that was regarded as positive. Cells were motile and motility could be demonstrated under electron microscopy when a freshly withdrawn sample was studied immediately Negative staining of GCAF-1 revealed the presence of peritrichous flagella and pili. Flagella represent the locomotory organelles of the cell. They are embedded in the cell membrane and extend through the cell envelope and project as a long strand. Flagella consist of many proteins including flagellin. Pili (synonym fimbriae) are hair like projections of the cell that may be involved in the sexual conjugation or may allow adhesion to the surfaces. Electron micrograph of the ultra thin section of the Cr(VI) reducing Clostridium sp GCAF-1 showed the presence of the S-layer. S-layers are the outer most component of the cell wall of several bacteria and archaea. They confer stability to the cell structure and protects cell from lytic enzymes. Biochemical characteristics. The Cr(VI) reducing strain GCAF-1 reduced higher concentrations of Cr(VI) (400M) completely in the presence of AQDS and Fe(III). In cultures with Cr(VI) as sole electron acceptor, complete reduction of Cr(VI) was observed only when Cr(VI) was present in low concentrations such as 20 M. Reduction of Cr(VI) occurred simultaneously with the growth of the organism. The generation time

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63 of this culture in the absence any electron acceptor was 2.04 Hrs. In the presence of 15M of Cr(VI) the time increased to 2.5 hrs. However, when the concentration of Cr(VI) was further increased to 35 and 50 M the generation time also increased to 4.0 and 4.5 hrs respectively (Figure 4-3). Other substrates that were tested for the chromium reduction but were not utilized were lactate, acetate, butyrate, formate and citrate. GCAF-1 was found to be resistant to rifampicin. It did not curdle the milk in 24 hrs and yellow pigment, riboflavin formation was not observed. This strain seemed closer to the C. beijerinckii than to C. acetobultylicum based on the few physiological properties tested. Chemotaxonomic Data. Fatty acid analysis. Cellular fatty acids profile of the isolate GCAF-1 is given in Table 4-1. The most prevalent fatty acids were 16 and 18 carbon atoms. Hexadecanoic acid was most abundant. Dimethylacetals (DMA) were found in the esterified preparation. DMA`s are the esterification products of plasmalogens, unique lipids found in anaerobes (118). DNA base composition. The G+C content of DNA from strain GCAF-1 was 30.7 mol %. The characteristics determined for strain GCAF-1 are summarized in Table 4-2 Phylogeny of Strain GCAF-1 Analyses of the 16S rRNA gene sequence. Comparative sequence searches of EMBL and Genbank databases revealed that 16S rDNA sequence of strain GCAF-1, was related to those of low G+C genus Clostridium of the gram-positive bacteria. Strain GCAF-1 clusters with group I organisms of genus Clostridium. Within this subphylum the highest sequence identity (98%) was obtained with 16S rRNA gene sequences of type strains of, C. beijerinckii, C. saccharybutylicum, C. saccharoperbutylacetonicum, C. butyricum, C. roseum (Table 4-3) (Figure 4-4).

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64 Clostridium sp. GCAF-1 contains at least two 16S rRNA genes. Restriction patterns obtained with HaeIII were identical for all DNA fragments obtained from 14 clones selected. However, two kinds of restriction patterns obtained with Alu I indicated the presence of more than one kind of 16S rRNA gene operon. Sequencing of the selected clones revealed the difference of 5 base pairs, one being at the site of Alu I. The results obtained from the cloned 16S rDNA of isolate GCAF-1 suggests the possibility of at least two types of 16S r RNA gene operons, but may be greater. A difference of 5 bases (0.36%) can result from PCRintroduced errors (Figure 4-6). Alternatively there is a possibility of sequence heterogeneity between 16S rRNA gene operons as previously described in E. coli and Clostridium paradoxum (25, 107). Presence of multiple 16S rRNA genes with heterogeneous intervening sequences has been described in Clostridium paradoxum (127). Such results may have an implication on the microbial ecology studies, wherein the group of highly related environmental 16S rDNA clone sequences obtained from many environments may represent not a group of separate, phylogenetically highly related strain but rather the sequence heterogeneity of the 16S rDNA contained within one strain. DNA -DNA hybridization. As described previously, the 16S rRNA gene of 5 Clostridial species showed similarity of greater than 97% with that of GCAF-1. DNADNA hybridization was carried out as with these 5 type strains of genus Clostridium (Table 4-3). Determination of genomic similarities revealed that strain GCAF-1 had only 23% reassociation values with Clostridium beijerinckii.

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65 Based on the general conclusion that strains with more than 97% 16S rRNA gene-sequence similarities that do not exhibit DNA-DNA homologies of 70% of more are accepted as being representatives of a single species (66, 168). GCAF-1 is a novel species in the cluster I of Clostridium. The 16S rDNA sequence is being submitted tot he Genbank. In summary, isolate GCAF-1, obtained from a glucose-oxidizing Cr(VI) reducing enrichment was identified as a novel species belonging to genus Clostridium. The name Clostridium chromoreductans sp. nov is proposed. Other biochemical and chemotaxonomic data further describes the isolate GCAF-1. The Cr(VI) reducing ability of the organism is investigated in chapter 5 in detail. This strain can be used as a model organism to provide an insight to the role of fermentative bacteria in metal reduction

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66 A B Figure 4-1. Scanning electron micrograph of isolate GCAF-1.A. Rod shaped cells. Scale bar, 10M. B. Cells occur in chains. Scale bar, 5m.

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67 Spores A Spore B Figure 4-2. Micrograph of spores of isolate GCAF-1. A. Differential Interference image of sporulating cells with subterminal spores. B. Scanning electron microscopy of GCAF-1 spores. Scale bar 5m.

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68 Flagella A Pili Flagella B Figure 4-3. Negatively stained preparations of Cr(VI) reducing Clostridium sp. GCAF-1 showing peritrichous flagella. A. Bacterium showing peritrichous flagella. B. Dividing cells still maintain the flagella.

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69 Spore CM CW S-layer Figure 4-4. Electron micrograph of an ultra thin section of Clostridium sp. GCAF-1 showing the S-layer. Due to slight plasmolysis, the protoplast has drawn away from the cell wall. CM, cell membrane; CW, cell wall.

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70 Spores Figure 4-5. Electron micrographs of Cr(VI) reducing Clostridium sp. GCAF-1 showing the dividing cells containing terminal spores and glycogen inclusions in the cells.

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71 B Clostridium cellulosi (L09177) Clostridium leptum (AJ305238) Clostridium cellobioparum (X71856)Clostridium cellulolyticum (X71847)Clostridium aldrichii (X71846) Clostridium kluyveri (M59092) Clostridium ljungdahlii (M59097) Clostridium histolyticum (M59094) Clostridium limosum (M59096) Clostridium proteolyticum (X73448)Clostridium roseum (Y18171) Clostridium beijerinckii (X68179) GCAF1Clostridium saccharobutylicum (U16147)Clostridium saccaroperbutyacetonicum (U16122)Clostridium favososporum (X76749)Clostridium corinoforum (X76742) Clostridium puniceum (X71857) Clostridium butyricum (X68176) Clostridium paraputrificum (X73445)Clostridium acetobutylicum (X78071)Clostridium tetani (X74770) Lactobacillus casei (D16551) Enterobacter aerogenes (AF395913)99 100 100100100100 10056 1008594 98 100 85986998709095ootstrap Figure 4-6. Phylogenetic tree based on 16S rDNA comparisons showing the relative position of strain GCAF-1 among other representative species of genus Clostridium. The branching pattern was generated by the neighbour joining method and the bootstrap values, shown at the nodes were calculated from 100 replicates. Bootstrap values less than 50% are omitted from the figure. Bar, 0.01 substitutions per nucleotide position. The gen bank acession numbers for the 16S r RNA sequences are given after the strain names.

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72 0.010.111005101520Time (Hrs)Absorbance 550nm Figure 4-7. Anaerobic growth curve of GCAF-1 in under various Cr(VI) concentrations. --in absence of Cr(VI); ---in presence of 0.015 mM Cr(VI); ---0.032 mM Cr(VI);-----0.050 mM Cr(VI) ; ----0.070 mM Cr(VI). a Values are represented by the average of the duplicates. b Growth rate was determined in cultures kept at 30C in dark without shaking.

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Figure 4-8. Comparison of two 16S rRNA gene sequences from Clostridium sp. GCAF-1. Two sequences are represented by CLS-1 and CLS-2. highlight the different bases.

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74 Identities = 1378/1383 (99%) Strand = Plus / Plus CLS-1: 13 gttccttcgggaacggattagcggcggacgggtgagtaacacgtgggtaacctgcctcat 72 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| CLS-2: 1 gttccttcgggaacggattagcggcggacgggtgagtaacacgtgggtaacctgcctcat 60 CLS-1: 73 agaggggaatagcctttcgaaaggaagattaataccgcataagattgtagtttcgcatga 132 ||||||||||||||||||||||||||||||||||||||||||||||||||||| |||||| CLS-2: 61 agaggggaatagcctttcgaaaggaagattaataccgcataagattgtagttttgcatga 120 CLS-1: 133 aacagcaattaaaggagtaatccgctatgagatggacccgcgtcgcattagctagttggt 192 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| CLS-2: 121 aacagcaattaaaggagtaatccgctatgagatggacccgcgtcgcattagctagttggt 180 CLS-1: 193 gaggtaacggctcaccaaggcgacgatgcgtagccgacctgagagggtgatcggccacat 252 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| CLS-2: 181 gaggtaacggctcaccaaggcgacgatgcgtagccgacctgagagggtgatcggccacat 240 CLS-1: 253 tgggactgagacacggcccagactcctacgggaggcagcagtggggaatattgcacaatg 312 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| CLS-2: 241 tgggactgagacacggcccagactcctacgggaggcagcagtggggaatattgcacaatg 300 CLS-1: 313 ggggaaaccctgatgcagcaacgccgcgtgagtgatgacggtcttcggattgtaaaactc 372 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| CLS-2: 301 ggggaaaccctgatgcagcaacgccgcgtgagtgatgacggtcttcggattgtaaaactc 360 CLS-1: 373 tgtctttggggacgataatgacggtacccaaggaggaagccacggctaactacgtgccag 432 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| CLS-2: 361 tgtctttggggacgataatgacggtacccaaggaggaagccacggctaactacgtgccag 420 CLS-1: 433 cagccgcggtaatacgtaggtagcaagcgttgtccggatttactgggcgtaaagggagcg 492 ||||||||||||||||||||| |||||||||||||||||||||||||||||||||||||| CLS-2: 421 cagccgcggtaatacgtaggtggcaagcgttgtccggatttactgggcgtaaagggagcg 480 CLS-1: 493 taggtggatatttaagtgggatgtgaaatactcgggcttaacctgagtgctgcattccaa 552 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| CLS-2: 481 taggtggatatttaagtgggatgtgaaatactcgggcttaacctgagtgctgcattccaa 540 CLS-1: 553 actggatatctagagtgcaggagaggaaagtagaattcctagtgtagcggtgaaatgcgt 612 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| CLS-2: 541 actggatatctagagtgcaggagaggaaagtagaattcctagtgtagcggtgaaatgcgt 600 CLS-1: 613 agagattaggaagaataccagtggcgaaggcgactttctggactgtaactgacactgagg 672 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| CLS-2: 601 agagattaggaagaataccagtggcgaaggcgactttctggactgtaactgacactgagg 660 CLS-1: 673 ctcgaaagcgtggggagcaaacaggattagataccctggtagtccacgccgtaaacgatg 732 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| CLS-2: 661 ctcgaaagcgtggggagcaaacaggattagataccctggtagtccacgccgtaaacgatg 720

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75 CLS-1: 733 aatactaggtgtaggggttgtcatgacctctgtgccgccgctaacgcattaagtattccg 792 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| CLS-2: 721 aatactaggtgtaggggttgtcatgacctctgtgccgccgctaacgcattaagtattccg 780 CLS-1: 793 cctggggagtacggtcgcaagattaaaactcaaaggaattgacgggggcccgcacaagca 852 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| CLS-2: 781 cctggggagtacggtcgcaagattaaaactcaaaggaattgacgggggcccgcacaagca 840 CLS-1: 853 gcggagcatgtggtttaattcgaagcaacgcgaagaaccttacctagacttgacatctcc 912 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| CLS-2: 841 gcggagcatgtggtttaattcgaagcaacgcgaagaaccttacctagacttgacatctcc 900 CLS-1: 913 tgaattacccttaatcggggaagcccttcggggcaggaagacaggtggtgcatggttgtc 972 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| CLS-2: 901 tgaattacccttaatcggggaagcccttcggggcaggaagacaggtggtgcatggttgtc 960 CLS-1: 973 gtcagctcgtgtcgtgagatgttgggttaagtcccgcaacgagcgcaacccttattgtta 1032 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| CLS-2: 961 gtcagctcgtgtcgtgagatgttgggttaagtcccgcaacgagcgcaacccttattgtta 1020 CLS-1: 1033 gttgctaccatttagttgagcactctagcgagactgcccgggttaaccgggaggaaggtg 1092 || ||||||||||||||||||||||||||||||||||||||||||||||||||||||||| CLS-2: 1021 gtcgctaccatttagttgagcactctagcgagactgcccgggttaaccgggaggaaggtg 1080 CLS-1: 1093 gggatgacgtcaaatcatcatgccccttatgtctagggctacacacgtgctacaatggct 1152 ||||||||||||||||||||||||||||||||||||||| |||||||||||||||||||| CLS-2: 1081 gggatgacgtcaaatcatcatgccccttatgtctagggccacacacgtgctacaatggct 1140 CLS-1: 1153 ggtacagagagatgctaaaccgcgaggtggagccaaacttcaaaaccagtctcagttcgg 1212 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| CLS-2: 1141 ggtacagagagatgctaaaccgcgaggtggagccaaacttcaaaaccagtctcagttcgg 1200 CLS-1: 1213 attgtaggctgaaactcgcctacatgaagctggagttgctagtaatcgcgaatcagaatg 1272 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| CLS-2: 1201 attgtaggctgaaactcgcctacatgaagctggagttgctagtaatcgcgaatcagaatg 1260 CLS-1: 1273 tcgcggtgaatacgttcccgggccttgtacacaccgcccgtcacaccatgagagttggca 1332 |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| CLS-2: 1261 tcgcggtgaatacgttcccgggccttgtacacaccgcccgtcacaccatgagagttggca 1320 CLS-1: 1333 atacccaaagttcgtgagctaaccgcaaggaggcagcgacctaaggtagggtcagcgatt 1392 ||||||||||||||||||||||||| |||||||||||||||||||||||||||||||||| CLS-2: 1321 atacccaaagttcgtgagctaaccgtaaggaggcagcgacctaaggtagggtcagcgatt 1380 CLS-1: 1393 ggg 1395 ||| CLS-2: 1381 ggg 1383

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76 Table 4-1. Cellular fatty acid composition of GCAF-1 grown with 10mM glucose. Component Fatty acids in profile (area%) 14:0 FAME 5.44 16:0 ALDE 1.14 16:1 CIS 7 FAME 4.25 16:1 CIS 9 FAME 6.73 16:0 FAME 29.46 16:1 CIS 9 DMA 2.37 16:0 DMA 5.44 18:1 CIS 9 FAME 22.58 18:1 CIS 13 FAME 0.57 18:0 FAME 2.97 18:1 CIS 9 DMA 3.42 18:1 CIS 11 DMA 1.73 20:1 CIS 11 FAME 0.52 ECL Unknown 14:762 (15:2 FAME, 15:1 CIS 7) 1.61 15:0 ANTEI 3 OH FAME, 16:1 CIS 7 DMA 2.08 ECL Unknown 16:760 (17:2 FAME 17:1 CIS 8 FAME) 0.96 ECL Unknown 16:801 (17:1 CIS 9 FAME, 17:2 FAME) 0.56 ECL Unknown 17:834 (18:1 c11/t9/t6/ FAME) 5.84 ECL Unknown 18:622 (19:0 ISO FAME) 0.63 The cellular fatty acid compositions were analyzed by Microbial ID, Inc. In fatty acid designation the first and second numbers indicate the number of carbon atoms, and the number of double bonds, respectively. Peaks lower than 0.5% in area are not represented. a FAME, fatty acid methyl ester; b DMA, dimethyl acetyl; c ISO, iso; d CIS, cis form double bond; e c, cis form; f ECL, Equivalent chain length; g ALDE, aldehyde; h ANTEI, anteiso; i xOH indicates the position of hydoxylation; j t, trans from double bond.

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77 Table 4-2. Characteristics of GCAF-1 Organism GCAF-1 Genus Clostridium cluster I G+C Mol content 30:7% Carbon sources not utilized Acetate, Lactate, butyrate, Lactate Resistance to Rifampicin + Riboflavin formation in milk Facultative growth Spore forming + Spore size 1-1.5 by 0.5 Length of rods 4.0-7.0 y 1.5 MIC of Cr(VI) > 0.1 mM Major fermentation products Acetate, butyrate, lactate a Motility motile a Fermentation products as determined by HPLC equipped by UV /vis detector:

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78 Table 4-3. Sequence similarity between 16S rRNA gene of isolate GCAF-1 and type strains of the genus Clostridium showing closest similarity. Species Strain Sequence similarity of 16S rDNA to isolate (%) DNA DNA hybridization with the isolate (%) Reference C. acetobutylicum a ATCC 824, b DSM 792, c NCIB, 8052 91 d ND Mc Coy Emend Keis C. butyricum ATCC 19398 DSM 10702, DSM 552 94 ND Prazmowski C. beijerinckii ATCC 25752, DSM, 791, NCIB 9362 98 23.5 Donker Emend Keis C. paraputrificum ATCC 25780, DSM 2630, NCIB 10671 96 ND Bienstock snyder C. puniceum DSM 2619, NCIB 11596 98 32.9 Lund C. roseum ATCC 17797, DSM 2619, NCIB 11596 98 33.6 Mc Coy Cato C. saccharobutylicum ATCC BAA-117, DSM 13864 98 e ND Keis C. sacchaoperbutylicum ATCC 27021, DSM 14923 98 36.3 keis Similarity higher than 97% is indicative of sequences belonging to same genus. a ATCC American Type Culture Collection, Manassas, VA, USA b DSM: Cultures from Deutsche Sammlung von Mikroorganismen und Zelkulturen, Braunschweig, Germany c NCIMB: National Collections of Industrial, Food and Marine Bacteria, Abeerdeen, UK. d ND: Not determined e ND: Not determined. Biochemical test conducted.

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CHAPTER 5 ELECTRON SHUTTLE-MEDIATED CHROMIUM REDUCTION BY CLOSTRIDIUM sp GCAF-1 Introduction Strictly anaerobic fermentative organisms, although ubiquitous in the environment, have not been studied extensively in terms of heavy metal remediation. Direct metal reduction by fermentative organisms is not generally considered to be significant, however, there are a few reports documenting the ability of these obligate anaerobes to reduce metals such as iron and selenium (39, 118). The currently accepted hypothesis regarding the role of fermentative bacteria in metal reduction is that complete oxidation of fermentable compounds to carbon dioxide is coupled to Fe(III) reduction by the cooperative activity of fermentative and Fe(III) respiring bacteria (92) that utilize the fermentative products as electron donors. This does not consider that fermentative organisms may play a significant role in direct reduction of metals. Thereby it disregards any ecologically significant role of fermentative bacteria in direct reduction of metals. Many bacterial strains have been shown to mediate reduction of Cr(VI) to Cr(III) both aerobically (22, 59, 75) and anaerobically (28, 93, 152, 163). Most of these organisms belong to the gram-negative group of bacteria. Cr(VI) reducing gram-positive bacteria reported to date, belong to the genera Bacillus (122), Staphylococcus (134) and Micrococcus (9). Although studies based on the preliminary screening of organisms isolated from Cr(VI) contaminated soil have reported the presence of gram-positive 79

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80 fermentative bacteria in metal contaminated sites, there is no report documenting the active participation of fermentative organisms in Cr(VI) reduction. Results from our enrichment studies (Chapter 2) show that the most rapid rate of Cr(VI) reduction was observed in enrichments amended with glucose as electron donor. Even though acetate is considered to be one of the most preferred electron donors for most metal reducing bacteria, our data indicated efficient chromium reducers to be among glucose utilizers. Isolation and identification studies revealed that the predominant enriched microorganisms belonged to the genus Clostridium. Cr(VI) reducing isolate GCAF-1 was obtained from this enriched culture. In order to understand the mechanisms adopted by GCAF-1 to reduce Cr(VI), detailed kinetic studies were undertaken. In doing so, direct and Fe-dependant indirect pathways for Cr(VI) reduction were identified. These results suggest that the selective advantage of strain GCAF-1 in Cr(VI) contaminated environments, due to its ability to grow in the presence of typically toxic concentrations of Cr(VI) and to reduce high concentrations of Cr(VI) in presence of Fe(III) and humics, is of potential environmental relevance. This study not only adds to the growing list of organisms involved in the Cr(VI) reduction, but also suggests the significant role played by fermentative organisms in the reduction of Cr(VI). Materials and Methods Source of organism and isolation. Strain GCAF-1 was isolated from anaerobic enrichment cultures that were initiated with the soil sample from a Cr(VI) contaminated wetland in Michigan. Enrichment cultures were supplemented with glucose as electron donor and Cr(VI), AQDS and Fe(III) as electron acceptors. Strain GCAF-1 was isolated by using standard anaerobic technique of roll tube.

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81 Cultivation of strain GCAF-1. Strict anaerobic techniques were used throughout the course of study as described previously (87). All incubations were in dark at 30C unless specified. Medium was prepared in serum bottles and bubbled with (N 2 / CO 2 :: 80: 20) to remove the dissoled oxygen. These bottles were then capped with blue butyl rubber stoppers and aluminum crimp seals under (N 2 / CO 2 :: 80: 20). Media was sterilized by autoclaving it for 30 minutes. Appropriate electron donor and acceptors were added from sterile stock solutions. For routine maintenance of cultures, 10 ml of medium were dispensed into anaerobic pressure tubes (Bellco glass, Inc., Vineland, N.J) and sparged with appropriate gas mixture for 10 minutes before sterilizing the medium by autoclaving. Cell growth and kinetics study. Growth of cells was monitored during the kinetic study by cell counts with acridine orange direct count. For the kinetics study, 48 ml of basal medium was dispensed into 120 ml serum bottles and sparged for 30 minutes with appropriate gas mixture to remove traces of oxygen from the medium. Medium was sterilized by autoclaving it for 30 minutes. Glucose was added as electron donor for all treatments except in controls that were set up with no electron donor. To determine the effect of Fe(III) and AQDS on Cr(VI) reduction, several treatments were set up with different combinations of electron acceptors: (i) Cr(VI) with Fe(III)and AQDS (ii) Cr(VI) with Fe(III), (iii) Cr(VI) with AQDS, and (iv) Cr(VI). In order to account for the effect of Fe(III) and AQDS on the metabolism of strain GCAF-1 another treatment with Fe(III) and AQDS in the absence of Cr(VI) was set up as a control. Fe(OH) 3 was synthesized by titrating a solution of FeCl 3 .6H 2 O with 10% NaOH to pH of 9.0. Cr(VI) was provided in the form of K 2 Cr 2 O 7. Anthraquinone di-sulfonate (AQDS), humic acid analog (Sigma)

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82 was used to mimic the effect of humic acids on Cr(VI) reduction. Appropriate addition of electron donor and acceptors were made to the bottles to get a final concentration of glucose (10 mM), Cr(VI) (0.4 mM), Fe(III) (5 mM), and AQDS (0.1mM) in the media. Samples were taken from serum bottles with a syringe. Analytical techniques. Cell enumeration was done by staining the cells with acridine orange and counting the cells under epifluorescence microscope as previously described (87). 0.1 ml of cell culture was taken from the serum bottle and fixed with 0.1 ml of 25% gluteraldehyde for 10 minutes. 0.8ml of PBS was added to make up the volume to 1 ml. Samples were diluted in oxalate solution in order to dissolve the particulate forms of iron. Sample was passed through 0.2 m millipore filter and stained with the acridine orange (5 drops of 0.1 M). After washing off the excess stain with filtersterlized distilled water, the filter was placed on the glass slide and viewed under the microscope. Cr(VI) concentrations were measured by colorimetric methods using UV spectrophotometer as described in chapter 2 (159). Glucose concentrations were determined spectrophotometrically at 450nm. Fermentation products were analyzed with high pressure liquid chromatography (HPLC, Waters) equipped with an Aminex HPX-87H column (7.8 by 300 mm column) (Biorad) and UV detector (Waters) at 210 nm. Sulfuric acid (5mM) was used as the eluent buffer. Flow rate was maintained at 0.6 ml min -1 Results and Discussion Strain GCAF-1 was isolated from a Cr(VI) reducing enrichment culture that was supplemented with glucose as electron donor and Fe(III) and AQDS as additional electron acceptors. Earlier studies with Cr(VI) reduction have reported Fe(II) to be one of its ecologically significant reductant in soils (13, 24, 54, 138). Also the ability of

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83 Clostridium beijerinckii and C. butyricum (118) to reduce Fe(III) to Fe(II) made Fe(III) a potential candidate to study the effect of Fe(III) on Cr(VI) reduction. The Biphasic Mechanisms for Cr(VI) Reduction by Clostridium sp GCAF-1. Strain GCAF1 was capable of direct and Fe-dependant indirect reduction of Cr(VI). It reduced 400 M of Cr(VI) in the presence of Fe(III) and AQDS. In the absence of these additional electron acceptors strain GCAF-1 was able to reduce up to 100M of Cr(VI). Cell growth in GCAF-1 cultures supplemented with Cr(VI) as sole electron acceptors, with concentrations higher than 100M, was negligible. Toxicity of the heavy metal at higher concentrations may have prevented the cell proliferation. Growth rate of the cells of strain GCAF-1 decreased as the initial Cr(VI) concentration in the medium increased (Figure4-?). The fermentative growth rate of GCAF-1 was similar to that when 16M of Cr(VI) was present in the medium (Figure 5-1). However, slight increase in lag phase is observed indicating that cells need to get acclimatized to Cr(VI) before they enter the logarithmic growth phase. Cr(VI) was completely reduced during growth. No growth was observed in medium with Cr(VI) concentration of 200M. GCAF-1 was a fermentative spore-forming organism and formation of spores was observed in the fermentative culture in 24 hours. In presence of Cr(VI) delayed sporulation was observed and cells exhibited altered morphology. The cells appeared longer and much thinner than those grown in absence of Cr(VI) suggesting the effect of Cr(VI) on cell division of the cells as documented by Theodotou et al. (153). Negligible Cr(VI) reduction was noted in the absence of electron donor or in the absence of cells. These results indicated that reduction of Cr(VI) was biotic and was carried out by metabolically active cells.

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84 In the presence of Fe(III) and AQDS, strain GCAF-1 was able to reduce higher concentrations of Cr(VI). An interesting observation was that reduction of Cr(VI) in medium containing both AQDS and Fe(III) (GCAF medium) was faster than the Cr(VI) reduction in the medium containing only Fe(III) (GCF medium). These results suggested that Fe(III) acted as an electron shuttle for the reduction of Cr(VI). In the presence of Fe(III), AQDS also formed a part of the train of shuttles transferring electrons to Cr(VI). Besides acting as electron shuttle, Fe(III) also played an important role in alleviating the Cr(VI) toxicity to the cells of GCAF-1. This was evident by the absence of cell growth and negligible reduction of Cr(VI) in culture medium supplemented with AQDS only. It must be noted that since strain GCAF-1 is fermentative it is capable of growing in the absence of electron acceptor and the therefore, the absence of Fe(III) should not have been the limiting factor for its growth. Toxicity of Cr(VI) concentration used in this study however, has been established earlier. Reduction of Cr(VI) corresponded well to the oxidation of glucose by strain GCAF-1 (Figure 5-2). There was negligible change in the glucose concentrations in the absence of cells. No oxidation of glucose was observed in cultures that showed no reduction of Cr(VI). Glucose oxidation by strain GCAF-1 was observed during its fermentative growth in the absence of any external electron acceptor. When the rate of glucose oxidation in GCF and GCAF media was compared there was no significant difference observed. The cell numbers in GCAF cultures were slightly higher when compared with those in GCF cultures (Figure 5-3). Cell growth in both the cultures ceased with the complete oxidation of glucose. However, complete reduction of Cr(VI) was only observed in GCAF medium. Incomplete and slower rate of Cr(VI) reduction was noted in the GCF medium. This indicated that electrons generated during glucose

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85 oxidation by GCAF-1 are channeled more efficiently towards Cr(VI) reduction in the presence AQDS and Fe(II). This can be explained as follows. Reduction of Cr(VI) in the presence of Fe(III) depended on the reduction of Fe(III) by the cells. Fe(III) in this case acted as an electron shuttle. Under the prevailing pH conditions Fe(III) oxide is insoluble and the microbial reduction of Fe(III) requires the cells to be in contact with the metal oxide as described previously (ref). Presence of AQDS in the medium stimulates the reduction of Fe(III) by alleviating the need for the contact by the cells. AQDS is soluble and is capable of shuttling electrons from the cells to Fe(III) oxide. Reduced AQDS is oxidized once the electrons are transferred to metal oxide and it is ready to accept electrons again from the cells. Fe(II) generated directly or indirectly by microbial activity then reduces Cr(VI). Therefore the rate of Cr(VI) reduction was dependant on the reduction of Fe(III). Cr(VI) reduction by strain GCAF-1 was possible by four different pathways: (i) by Fe(II) that is directly generated by the cells, (ii) by Fe(II) that is reduced by reduced AQDS (iii) by reduced form of AQDS (iv) directly by the cells. Effect of Cr(VI) on Production of Metabolic Products of Strain GCAF-1 In order to determine the effect of Cr(VI) on the metabolism of strain GCAF-1, the metabolites in different media were compared. Formation of metabolic products (Figure 5-4, 5-5, 5-6) corresponded well with the oxidation of glucose (Figure 5-2) and the reduction of Cr(VI) (Figure 5-1). Metabolites were detected in the spent growth medium of cells. As expected, absence of fermentation products was observed in media that did not show any oxidation of glucose. Oxidation of glucose by strain GCAF-1 in the absence of any external electron acceptor yielded acetate, butyrate and lactate as the three major fermentative products (Table 5-1). In the absence of Cr(VI), no change in the fermentation products was observed in growth media supplemented with Fe(III) and

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86 AQDS. However, the effect of Cr(VI) on the metabolic products was apparent by the change in concentrations of acetate, butyrate, and lactate formed per 10 mM of glucose in the presence of Cr(VI) in the medium (Table 5-1). There was an increase in the acetate concentrations noted along with a decrease in the butyrate concentrations (Figure 5-4, 5-5) in GCAF in GCF cultures. This indicated the possibility that (i) Cr(VI) was directly reduced by strain GCAF-1, and (ii) Fe(III) and AQDS were reduced by strain GCAF-1 by a pathway that was different from that by which Cr(VI) was reduced. Proposed Mechanism of Cr(VI) Reduction by Strain GCAF-1. Based on the results presented above a hypothesis elucidating the pathway for Cr(VI) reduction by fermentative strain GCAF-1 is proposed. Generally, in glycolytic pathways the oxidation of one mole of glucose by Embden-Meyerhof-Parnas pathway yields two moles of pyruvate. This pathway also produces two NADH, plus two ATP molecules. The pyruvate is further decarboxylated to acetyl CoA, CO2, and h2 using pyruvate-ferredoxin oxidoreductase and hydrogenase. The acetyl CoA has two fates. Some of it is condensed to form acetoacetyl-CoA, which is reduced to -hydroxybutyryl-CoA using one of the two NADHs. This product is reduced to butyryl-CoA using the second NADH. CoASH is displaced by inorganic phosphate and butyryl phosphate donates phosphoryl group to ADP and forms ATP and butyrate. Some aetylCoA is also converted to acetate via acetyl -P in a reaction that yields an additional ATP. Twice as much ATP is generated per acetate produced as opposed to butyrate. Mass balance of fermentation products formed (within 10% error) by Clostridium sp. GCAF-1 during oxidation of 10mM of glucose in the absence of Cr(VI) showed that 20 NADH produced by the oxidation of 10 mM of glucose, were used as follows: 15 NADH for butyrate, 1 NADH for lactate and 2 NADH used for NADH:ferredoxin oxidoreductase. In presence

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87 of Cr(VI), 11 NADH were used for the formation of butyrate, 1 NADH was utilized for lactate formation and 1.2 for Cr(VI) reduction. 5.2 NADH was utilized for the reduction of Fe(III) and AQDS. In presence of Cr(VI), decrease in concentration of butyrate would result due to the channeling of electrons from NADH towards reduction of Cr(VI) and more ATP formation per acetyl CoA. Reduction of Cr(VI) was perhaps one of the defense mechanism adopted by the cell to detoxify the environment for its survival. Therefore, NADH was perhaps acting as electron source for the reduction of Cr(VI). This speculation is further supported by several reports of microbial NADH dependant enzymatic reduction of Cr(VI) (10, 44, 52, 117, 149). The electrons from NADH are transferred through the electron transport chain and transported via a cytochrome to Cr(VI). Presence of cytochromes has been reported in some members of Clostridium sp. Decrease in formation of butyrate content was coupled with the increase in acetate concentrations. Increase in acetate concentrations are advantageous for the cell as it requires to make more energy to compensate for the energy expended for activating its defense mechanisms against the toxic Cr(VI) molecules. Whether Cr(VI) is used as an electron acceptor by the strain GCAF-1 is currently unclear. Location of Cr(VI) reduction by the cell is also not clear Environmental Relevance of Cr(VI) Reduction by Gram Positive Spore-forming Fermentative Species Presence of fermentative organisms in soil is ubiquitous. These organisms generally form the primary group of bacteria that oxidize the complex carbon sources into simple carbon forms that are utilized as electron donors by the known metal reducing bacteria. In anaerobic Cr(VI)contaminated environments like the wetlands fermentative bacteria such as Clostridium sp GCAF-1 are active participants in reduction of Cr(VI) as

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88 they can reduce directly reduce Cr(VI) in addition to providing carbon sources for most metal reducers. The spore forming ability of the organism is advantageous in the advent of nonconducive conditions for its survival. In environments with low concentrations of Fe(III) and humics, Cr(VI) can still be reduced by strain GCAF-1. These results suggest that microbial Cr(VI) reduction by Clostridium sp. GCAF-1 via Fe(II) is of potential environmental relevance. Further investigation of Cr(VI) reduction by fermentative organisms is warranted so as to determine the environmental significance of this group of bacteria in field of metal reduction.

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89 00.050.10.150.20.250.30.350.40.4504590135180225Time (hrs)Residual Cr(VI) (mM) GC GCA GCAF GCF Figure 5-1. Cr(VI) reduction by strain GCAF-1. G, glucose (10mM) was provided as electron donor. Electron acceptors included A, AQDS (0.1mM); C, Cr(VI) (0.4mM); F, Fe(OH) 3 (5mM). All treatments were set up in triplicates with error bars representing the standard error

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90 0246810120153045607590Time (hrs)Resudual Glucose (mM) G GCA GC GCAF GCF Figure 5-2. Glucose consumption during Cr(VI) reduction by strain GCAF-1. G, glucose (10mM) was provided as electron donor. Electron acceptors included A, AQDS (0.1mM); C, Cr(VI) (0.4mM); F, Fe(OH) 3 (5mM). All treatments were set up in triplicates with error bars representing the standard error.

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91 02468101214160153045607590105Time (hrs)Cells ( x 106 ml-1) GCAF GCF Figure 5-3. Cell growth during Cr(VI) reduction by strain GCAF-1. G, glucose (10mM) was provided as electron donor. Electron acceptors included A, AQDS (0.1mM); C, Cr(VI) (0.4mM); F, Fe(OH) 3 (5mM). All treatments were set up in triplicates with error bars representing the standard error.

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92 01234567020406080Time (hrs)Acetate (mM) G GCAF GCF 0123456702468101214Glucose (mM)Acetate (mM) GCF G GCAF A B Figure 5-3. Acetate production during oxidation of glucose by GCAF-1. G, glucose (10mM) was provided as electron donor. Electron acceptors included A, AQDS (0.1mM); C, Cr(VI) (0.4mM); F, Fe(OH) 3 (5mM).

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93 024681012020406080Time (hrs)Butyrate (mM) G GCAF GCF A 02468101202468101214Glucose (mM)Butyrate (mM) GCF G GCAF B Figure 5-4. Butyrate production during oxidation of glucose by GCAF-1. G, glucose (10mM) was provided as electron donor. Electron acceptors included A, AQDS (0.1mM); C, Cr(VI) (0.4mM); F, Fe(OH) 3 (5mM).

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94 00.20.40.60.811.2020406080Time (hrs)Lactate (mM) G GCAF GCF A 0.00.20.40.60.81.01.202468101214Glucose (mM)Lactate (mM) GCF G GCAF B Figure 5-5. Lactate production during oxidation of glucose by GCAF-1. G, glucose (10mM) was provided as electron donor. Electron acceptors included A, AQDS (0.1mM); C, Cr(VI) (0.4mM); F, Fe(OH) 3 (5mM).

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95 Glucose ADP+P ATP Butyrate 2NADH 2NAD+ NADH NAD+ Lactate Acetate Pyruvate Figure 5-6. Schematic diagram of proposed metabolic pathway of formation of fermentative products by Clostridium sp. GCAF-1. Presence of Cr(VI) causes the cell to form lower concentrations of butyrate as NADH serves as an electron donor for the reduction of Cr(VI). Increase in acetate concentrations is coupled with the energy forming step.

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96 Table 5-1. Effect of Cr(VI) on metabolites formed by strain GCAF-1. Glucose oxidation by strain GCAF-1 in medium containing glucose as electron donor and Fe(III) and AQDS as additional electron acceptors formed the following fermentation products in the presence and absence of Cr(VI). Fermentation products Without Cr(VI) With Cr(VI) mM / 10mM of glucose Acetate 2.01 5.01 Lactate 0.74 0.83 Butyrate 7.57 5.41 Ratios Acetate: Lactate 2.72 6.06 Acetate: Butyrate 0.27 0.93

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CHAPTER 6 SUMMARY AND CONCLUSIONS Cr(VI) contamination in soils is not uncommon, especially near industries involved in glass work, chrome plating, and leather tanning. The mutagenic and carcinogenic properties of Cr(VI) necessitate effective remedial processes. Difficulties associated with chemical and physical techniques to remediate a Cr(VI)-contaminated site to EPA recommended levels (50ppb), in addition to the higher costs involved, assert the need for bioremedial measures. Implementation of these techniques requires knowledge of the following factors: the organisms involved; the factors that govern the optimum reductive ability of these organisms; and the mechanisms involved in Cr(VI) reduction This dissertation addresses the above mentioned factors and investigates the Cr(VI)-reducing ability of a previously undescribed fermentative strain, thereby adding a representative of the genus Clostridium to the growing list of Cr(VI)-reducing bacterial groups. This work provides a greater understanding of processes by which microorganisms reduce Cr(VI) in nature. Soils contaminated with Cr(VI) for over 5 decades were used in this study to enrich a diversity of indigenous Cr(VI)-reducing microorganisms. To maximize the diversity of the bacteria enriched and to ascertain the preferred electron donor by these bacteria, enrichments were amended with different electron donors and electron acceptors. The donors were chosen to represent those found in anaerobic soils. Among the various electron donors tested, enrichment cultures supplemented with glucose showed the fastest microbial Cr(VI) reduction, suggesting: (i) many rapidly growing Cr97

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98 reducing bacteria preferred glucose as electron donor; and (ii) organisms enriched by glucose were more efficient at reducing Cr(VI) than those enriched by other electron donors in our systems. Enrichments with Cr(VI) as sole electron acceptor showed insignificant microbial growth and Cr(VI) reduction. The effect of additional electron acceptors was apparent when a higher rate of Cr(VI) reduction was observed in the presence of Fe(III) or anthraquinone di sulfonate (AQDS), a humic acid analog. In the presence of both AQDS and Fe(III), the rate of Cr(VI) reduction was further augmented. These results indicated the following: (i) microbially-mediated reduction of Cr(VI) by the cooperative activity of Fe(III)-reducing, AQDS-reducing and Cr(VI) reducing bacteria (Figure 1-4), (ii) the toxicity of Cr(VI) is alleviated in the presence of Fe(III) or AQDS. Characterization of cultivable members of the glucose-oxidizing consortium revealed the dominance of the genera Cellulomonas and Clostridium. Few isolates belonging to Staphylococcus, Micrococcus and Bacillus were also obtained. The presence of fermentative bacteria was not surprising owing to their ability to ferment glucose; however, their dominance in the Cr(VI) amended culture suggested their significant role in Cr(VI) reduction. Detailed kinetic studies were conducted with the enriched Cr(VI)-reducing consortium that was dominated by organisms belonging to Clostridium sp. Reduction of Cr(VI) was observed only in the presence of an additional electron acceptor (Fe(III) or AQDS). The rate of Cr(VI) reduction observed was higher when both Fe(III) and AQDS were present in the medium, compared to when only Fe(III) was present. The presence of AQDS, a humic acid analog, has been shown to act as an electron shuttle and enhance the

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99 Fe(III) reduction (90). Therefore, the lower rate of Cr(VI) reduction in system with no AQDS may be attributed to the slow rate of Fe(III) reduction. We speculate that microbially generated Fe(II) acts as an electron shuttle for the reduction of Cr(VI). In the presence of AQDS, the rate of Cr(VI) reduction is faster due to the stimulatory effect of AQDS on Fe(III) reduction. These results supported the role of AQDS as electron shuttle to enhance the Fe(III) reduction. Another possibility could be absence of AQDS-reducing organisms in the culture in absence of AQDS. Similar studies with a pure isolate were undertaken to further elucidate the mechanism of Cr(VI) reduction by the Clostridium sp and to compare the rate of Cr(VI) reduction by the consortium and a pure isolate. An isolate, GCAF-1, obtained from this consortium was used for further studies. Studies with both consortium and the pure isolate showed comparable rate of Cr(VI) reduction in the presence of Fe(III) and AQDS. Results suggested that the Cr(VI) reducing ability of the organism is maintained at the same level when in isolation or when in a consortium with other organisms. Studies further demonstrated the requirement of Fe(III) by Clostridium sp. GCAF-1 to reduce Cr(VI) concentrations as high as 20 ppm. It was also observed that the amount of Cr(VI) reduced per unit of glucose in Fe(III) systems was lower than when AQDS was present along with Fe(III) in the medium. This study also demonstrated the ability of GCAF-1 to directly reduce Cr(VI) in addition to Fe(III)-mediated Cr(VI) reduction as described above. GCAF-1 was capable of reducing 5 ppm Cr(VI) directly. Whether Cr(VI) was being used an electron acceptor with growth by Clostridium sp. GCAF-1 is not clear. At higher Cr(VI) concentrations in the absence of other electron acceptors, no bacterial growth and no reduction of Cr(VI)

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100 were observed. Reduction of Cr(VI) by GCAF-1 was also found to be dependant on the presence of glucose. In the absence of glucose no reduction of Cr(VI) was observed. The concentration of glucose, however, was not a rate limiting factor for the reduction of Cr(VI) under the experimental conditions. These results may have great implications in the environments with much lower electron donor concentrations. Under glucose-limiting conditions, reduction of Cr(VI) may be effected negatively. Analysis of the metabolic products of the consortium and GCAF-1 revealed that acetate, lactate and butyrate were the major fermentation products formed during glucose oxidation. No difference was observed in the ratios of metabolic products of cultures grown in the absence of an external electron acceptor and those grown in the presence of Fe(III) and AQDS. However, cultures grown with Fe(III), AQDS and Cr(VI) showed an increase in the acetate: butyrate and acetate: lactate ratios. These changes in cell metabolism may serve two purposes: (i) Energy generation by adenosine tri-phosphate (ATP) formation; and/or (ii) reduction of Cr(VI) to detoxify the immediate environment. Formation of acetate in glycolytic pathway is coupled with ATP production, and an increase in acetate concentration is perhaps a strategy adopted by strain GCAF-1 to generate more energy to survive in toxic Cr(VI) environments. A decrease in butyrate and lactate concentrations was accompanied by reduction of Cr(VI), suggesting that NADH, electron donor for the reduction of pyruvate to butyrate and lactate, was channeling its electrons towards the reduction of Cr(VI) via the electron transport chain. Identification and phylogenetic characterization of isolate GCAF-1 revealed this strain to belong to the genus Clostridium. DNA-DNA hybridization with all type strains showing 98% similarity in 16S rDNA indicated the strain to be a novel Clostridium

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101 species. GCAF-1 also exhibited the presence of at least two 16S rRNA copies. Other detailed biochemical analyses of GCAF-1 are also documented in this study. In view of the ability of this strain GCAF-1 to reduce Cr(VI), the species name Clostridium chromoreductans sp. nov. is proposed. Studies presented in this dissertation provide significant implications for bioremediation of soils and water contaminated with toxic metals such as Cr(VI). In order to implement bioremediation in soils use of bacterial strain with minimal growth limiting requirements can decrease costs and be more beneficial for the working. GCAF-1 is one such easy maintenance organism. In anoxic soils, GCAF-1 can directly reduce Cr(VI) in soils thereby limiting its mobility. GCAF-1 is a fermentative organism that can grow in the absence of an external electron acceptor, unlike most other known Cr(VI) reducing bacteria, Enterobacter cloacae, Pseudomonas putida, Desulfovibrio, Desufotomaculum sp. Pantoea agglumorens. Environments that are rich in Fe(III) and humic acids may be dominated by FRB. Microbial reduction of Cr(VI) in these environments may predominantly be due to FRB. Under Fe(III)and humicslimiting conditions, Cr(VI) reduction by FRM and HRM is insignificant. In such conditions organisms such as GCAF-1 play a significant role in reducing Cr(VI). As demonstrated in this study, GCAF-1 can reduce Cr(VI) significantly by using Fe(III) and AQDS as electron shuttles. The spore-forming ability of GCAF-1 offers another advantage over non-spore forming organisms. This ability enables these organisms to persist in the environment under unfavorable conditions. Among the advantages offered by the use of this strain is the source of carbon for the growth of this organism. Wastes such as molasses from sugar

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102 industries may be provided as the carbon source, thereby making the implementation of bioremediation by these organisms economical. Future studies with Clostridium sp. GCAF-1 to test its ability to reduce other toxic metals may reveal the full potential of this organism. Field studies of the application of these concepts may provide a more accurate picture of the potential importance of Clostridium GCAF-1 in the environment.

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APPENDIX MEDIA USED FOR ENRICHMENT STUDIES NB BASAL MEDIA Chemicals L -1 Q H 2 O 900ml KH 2 PO 4 0.42 g K 2 HPO 4 0.22 g NH 4 Cl 0.2 g NB Mineral elixir 10ml Vitamin mix(DL) 15ml KCl 0.38 g NaCl 0.36 g MgSO 4 7H 2 O 0.10 g NaHCO 3 1.8 g Na 2 CO 3 0.5 g 1mM Na 2 SeO 4 1ml Bring to final volume to 1L and bubble out media with 80/20 N2/CO2, Final pH ca. 7.0-7.2 Donors and acceptors should be added anaerobically and aseptically to the media after autoclaving. Adjust all donors and acceptors to pH 7.0. VITAMIN MIX MINERAL MIX mg/L g/L biotin 2.0 NTA 1.5 folic acid 2.0 MgSO 4 3.0 pyridoxine HCl 10.0 MnSO 4 H 2 O 0.5 riboflavin 5.0 NaCl 1.0 thiamine 5.0 FeSO 4 7H 2 O 0.1 nicotinic acid 5.0 CaCl 2 2H 2 O 0.1 pantothenic acid 5.0 CoCl 2 6H 2 O 0.1 B-12 0.1 ZnCl 2 0.13 p-aminobenzoic acid 5.0 CuSO 4 5H 2 0 0.01 thioctic acid 5.0 AlK(SO 4 ) 2 12H 2 O 0.01 H 3 BO 3 0.01 Na 2 MoO 4 0.025 NiCl 2 6H 2 O 0.024 Na 2 WO 4 2H 2 O 0.025 103

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LIST OF REFERENCES 1. Aislabie, J., and M. W. Loutit. 1986. Accumulation of Cr(III) by bacteria isolated from polluted sediment. Mar. Environ. Res. 20:221-232. 2. Ajmal, M., A. A. Nomani, and A. Ahmad. 1984. Acute toxicity of chrome electroplating wastes to microorganisms adsorption of chromate and chromium(VI) on a mixture of clay and sand. Water Air Soil Poll. 23:119-127. 3. Amacher, M. C., and D. E. Baker. 1982. Redox reactions involving chromium. plutonium and manganese in soils., p. 166. Institute for research on land and Water Resources, Pennsylvania State University and U. S. Department of Energy, Las Vegas, Nevada. 4. Anderson, R. A., M. M. Polansky, N. A. Bryden, S. J. Bhathena, and J. Canary. 1987. Effects of supplemental chromium on patients with symptoms of reactive hypoglycemia. Metabolism 36:351-355. 5. Anderson, R. A. 1989. Essentiality of chromium in humans. Sci. Tot. Environ. 86:75-81. 6. Anderson, R. T., and D. R. Lovley. 1997. Ecology and biogeochemistry of in situ groundwater bioremediation. Adv. Microb Ecol, 15:289-350. 7. Anon. 1974. Medical and Biological effects of pollutants: chromium. National Academy Press, Washington. 8. Arnold, R., T. DiChristina, and M. R. Hoffman. 1988. Reductive dissolution of Fe (III) oxides by Pseudomonas sp 200. Biotechnol. Bioeng. 32:1081-1096. 9. Badar, U., N. Ahmed, A. J. Beswick, P. Pattanapitpaisal, and L. E. Macaskie. 2000. Reduction of chromate by microorganisms isolated from metal contaminated sites of Karachi, Pakistan. Biotechnol. Lett 22:829-836. 10. Bae, W., T. Kang, J. Jung, C. Park, S. Choi, and B. Jeong. 2000. Purification and characterization of NADH-dependent Cr(VI) reductase from Escherichia coli ATCC 33456. J Microbiol Biotechnol 10:580-586. 11. Balch, W. E., G. E. Fox, L. J. Margrum, and R. S. Woese. 1979. Methanogens: reevaluation of a unique biological group. Microbiol Rev. 43:1153-1157. 104

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BIOGRAPHICAL SKETCH Kanika Sharma was born in Sagar, Madhya Pradesh, India, on 23 rd April 1974, to Nishchint and Amarnath Sharma. She had an excellent opportunity to visit a lot of places and make a lot of friends owing to her father`s job as defense personnel. In 1994, she graduated from Miranda House, the University of Delhi, with a first class and received a Bachelor of Science degree in botany (honors). She obtained a master`s degree in biochemical technology in 1995. Kanika went back to graduate school to get another masters degree in agrochemical and pest management from the University of Delhi, India, in 1997. In January 1998, Kanika joined the University of Florida to pursue doctoral studies in environmental microbiology. Her research was supported by a part of the Department of Energy (DOE) research grant awarded to Dr. Andrew Ogram. Amidst the friendly and easygoing members of the Ogram lab and the department, it did not take long for her to make several everlasting friendships. In future, Kanika hopes to pursue an academic career in science full time and to hone the scientific skills acquired during the course of her graduate studies. 119


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MICROBIAL Cr(VI) REDUCTION: ROLE OF ELECTRON DONORS, ACCEPTORS,
AND MECHANISMS, WITH SPECIAL EMPHASIS ON CLOSTRIDIUM spp.













By

KANIKA SHARMA


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA


2002




























Copyright 2002

by

Kanika Sharma




























For my parents, whose support and understanding has helped culminate my dream

into a reality.















ACKNOWLEDGMENTS

I am grateful to my mentor, Dr. Andrew V. Ogram, for excellent supervision

during the course of this dissertation. He has truly been a great source of inspiration,

insight, and input. The knowledge he has imparted, and the patience he has displayed was

vital to completing this study. I am thankful for the immense encouragement and

financial support that he graciously provided during this study.

I would like to express my sincere gratitude to the committee members, Drs. K.

Hatfield, L. O. Ingram, K. R. Reddy, and R. D. Rhue, for each contributing in special and

meaningful ways to my personal development and academic success. I also thank Drs. W.

Harris, L.T. Ou, and H. Aldrich for all of their advice and help.

A special word of thanks is due to Dr. Derek Lovley, and members of his lab at

the University of Massachusetts, Amherst, for extending their lab facilities so that I might

learn various anaerobic microbial techniques. At this time, I would also like to thank Dr.

John Thomas, Bill Reve, and Irene Poyer for all their help with the analytical equipment.

I am especially grateful to T. Van Pham and Lisa Stanley who provided me the

extra pair of hands when work demanded. I thank Ilker Uz and Hector Castro for being

wonderful friends and for being by my side through all happy and tough times. I sincerely

thank members of my lab, past and present, Viji Ramakrishnan, Weiwei Chen, Drs. Yong

Ping Duan, Milind A. Chavan, and Ashvini Chauhan. Without the advice and friendship

of these colleagues, surviving the last 5 years would have been impossible.









I acknowledge the support of the faculty, staff, and all the graduate students in the

Soil and Water Science Department.

I owe much of my academic and personal success to my parents and my brother,

who, by example, provided me with the motivation and courage to pursue a Ph.D. degree.

Special thanks go to my friends, near and far, for their love and support that made my

stay at University of Florida so memorable.
















TABLE OF CONTENTS
page

A C K N O W L E D G M E N T S ................................................................................................. iv

LIST OF TABLES .................................................... .......... .............. viii

LIST O F FIG U RE S .... ........................... ............ ix

LIST OF ABBREVIATIONS AND ACRONYMS ................................. .............. xi

A B S T R A C T ........................................................... ............... x ii

CHAPTER

1 GEN ER AL IN TR O D U CTION ....................................... .......................................

Chrom ium in Environm ent ....................................................... ................. .............. 1
Nutrition and Toxicity: Risks to Human Health......................................................... 2
A n im als ................................................................................................... . 2
Plants and Algae............................................. ......... 3
M icroorganism s ........................................ ............ ....................... ........ 3
Environm mental Chem istry ............................... ................ ................................. 5
R education of Cr(V I) ................................................ ....... .. .......... .. 6
O xidation of C r(III) in Soils........................................................................ ....... 7
Cr(VI) Rem edition Strategies ........................... ....... .................................... 7
Chrom ium R resistance in B acteria.......................................... ............................. 9
Pathways for Chromium(VI) Reduction.................................. ......................... 11
Direct Enzym atic Reduction of Cr(VI)....... ............................... ................. 11
Bacterially Mediated Indirect Reduction of Cr(VI)............................................. 13
Factors Affecting Microbial Chromium Reduction................................................... 15
Outline of Dissertation ........................ ....... ........ 18

2 ENRICHMENT, ISOLATION, AND CHARACTERIZATION OF Cr(VI)-
R ED U C IN G B A C TE R IA ..................................................................... ..................27

Introduction............................... ........... .......... 27
M materials and M ethods................... ............................................. .................... ...... 29
R results and D discussion .............................................. .... ................ .... ............... 33
Effect of Electron Donors and Acceptors on Cr(VI) Reduction............................ 33
Phylogenetic Analysis of Cr(VI) Reducing Bacteria................................... 36









3 Cr(VI) REDUCTION BY A CONSORTIUM OF GRAM POSITIVE
FERM EN TA TIV E BA CTERIA ........................................................ .....................42

Introduction............................... ........... .......... 42
M materials and M ethods................... ............................................. .................... ...... 43
R results and D discussion ............ .... .. ... ........ ................ ... ........... ......... 45
Composition of Fermentative Consortium GCAF .............................................. 45
Biotic versus Abiotic Reduction of Cr(VI)........................................................ 45
Kinetics of Cr(VI) Reduction................................................ 46
Effect of Electron Donor on Cr(VI) Reduction ..................................................... 47
Effect of Cr(VI) Reduction on Cell Growth in Consortium GCAF....................... 47
Effect of Cr(V I) on M etabolites ............................................................... ....... 47

4 IDENTIFICATION AND CHARACTERIZATION OF THE CHROMIUM
REDUCING ISOLATE CLOSTRIDIUM sp. GCAF 1 ....................... ...............58

Introduction............................... ........... .......... 58
M material and M methods .............. ............................................................ .............. 59
Results and Discussion ........................................... ................ 62
Chem otaxonom ic D ata ...................... .... ................................... .............. 63
P hylogeny of Strain G C A F -1 ........................................................................ ... 63

5 ELECTRON SHUTTLE-MEDIATED CHROMIUM REDUCTION BY
CLOSTRIDIUM sp GCAF-1 ..........................................................................79

Introduction....................... ............... ..... ............. 79
M materials and M ethods................... ............................................. .................... ...... 80
R results and D discussion ............... .................. .. ........ .... .... ....... ................... 82
Effect of Cr(VI) on Production of Metabolic Products of Strain GCAF-1 ........... 85
Proposed Mechanism of Cr(VI) Reduction by Strain GCAF-1........................... 86
Environmental Relevance of Cr(VI) Reduction by Gram Positive
Spore-form ing Ferm entative Species.................................... .................... 87

6 SUM M ARY AND CON CLU SION S ................................................. .....................97

APPENDIX MEDIA USED FOR ENRICHMENT STUDIES ..................................... 103

L IST O F R E F E R E N C E S ...................................................................... ..................... 104

BIOGRAPHICAL SKETCH ........................................................... ........119
















LIST OF TABLES


Table page

1-1. Cr(VI) reducing bacteria described in literature.............. .... .................24

2-1. Combination of electron acceptors and donors supplemented in the media for
anaerobic enrichm ent studies.......................................... ........................... 38

2-2. Accession numbers for 16S rDNA sequences used in this study..............................39

3-1. Cell growth in the presence of different electron acceptors....................................56

3-2. Cr(VI) reduction by consortium GCAF in presence of varying concentrations of
electron donor .......................................................................56

3-3. Effect of Cr(VI) on the on the pattern of products of glucose fermentation by
consortium G CA F ......................................... ............... .. ........ .... 57

4-1. Cellular fatty acid composition of GCAF-1 grown with 10mM glucose...................76

4-2 C characteristics of G C A F -1 .............................................................. .....................77

4-3. Sequence similarity between 16S rRNA gene of isolate GCAF-1 and type strains of
the genus Clostridium showing closest similarity ...........................................78

5-1. Effect of Cr(VI) on metabolites formed by strain GCAF-1 .......................................96
















LIST OF FIGURES


Figure p

1-1. Chromium cycle in environment .............. ............. ............... 19

1-2. Reduction potential diagram for chromium..................................... ............... 20

1-3. Eh-pH diagram for chromium-water system at standard state conditions..................21

1-4. Schematic diagram showing the possible pathways for anaerobic Cr(VI) reduction
b y b bacteria .. ................................................................................ 2 2

1-5. Model showing reduction of Fe(III) mediated by humics .......................................23

1-6. Q uinone m odel com pound............................................................... .....................23

2-1. Rate of reduction of Cr(VI) in enrichment cultures amended with different
electron donors and electron acceptors. ........................................ ............... 40

2-2. Phylogenetic tree constructed using maximum parsimony. .....................................41

3-1. Cr(VI) reduction and removal from the solution as an insoluble precipitate. ............50

3-2. Glucose consumption by consortium GCAF-1 during reduction of Cr(VI) ..............50

3-3. Acetate produced by oxidation of glucose by consortium GCAF.during the
reduction of Cr(V I). ........... .. .... .............................. .......... .. .. ........ .... 51

3-4. Butyrate produced by oxidation of glucose by consortium GCAF during the
reduction of Cr(V I). ................ ............................... .............. 52

3-5. Lactate produced by oxidation of glucose by consortium GCAF during the
reduction of Cr(V I). ........... .. .... .............................. .......... .. .. ........ .... 53

3-6. SEMs showing the insoluble precipitates formed by consortium GCAF-1 during
the reduction of Cr(VI) via Fe(II)- and AQDS-mediated mechanisms....................54

3-7. EDX of precipitate formed by consortium GCAF-1 showing the position of Cr in the
precipitate formed during Cr(VI) reduction. ................................... ............... 55

4-1. Scanning electron micrograph of isolate GCAF-1. ............................................. 66









4-2. M icrograph of spores of isolate GCAF-1. ........................................ ...............67

4-3. Negatively stained preparations of Cr(VI) reducing Clostridium sp. GCAF-1
show ing peritrichous flagella. ........................................ ......................................68

4-4. Electron micrograph of an ultra thin section of Clostridium sp. GCAF-1 showing
th e S -lay e r ...................................... ................................. ................ 6 9

4-5. Electron micrographs of Cr(VI) reducing Clostridium sp. GCAF-1 showing the
dividing cells containing terminal spores and glycogen inclusions in the cells.........70

4-6. Phylogenetic tree based on 16S rDNA comparisons showing the relative position
of strain GCAF-1 among other species of genus Clostridium ............................ 71

4-7. Anaerobic growth curve of GCAF-1 in under various Cr(VI) concentrations..........72

4-8. Comparison of two 16S rRNA gene sequences from Clostridium sp. GCAF-1........75

5-1. Cr(V I) reduction by strain G CAF-1 ........................................ ....................... 89

5-2. Glucose consumption during Cr(VI) reduction by strain GCAF-1 ..........................90

5-3. Cell growth during Cr(VI) reduction by strain GCAF-1 ........................................91

5-3. Acetate production during oxidation of glucose by GCAF-1...................................92

5-4. Butyrate production during oxidation of glucose by GCAF-1.............................. 93

5-5. Lactate production during oxidation of glucose by GCAF-1 ...............................94

5-6. Schematic diagram of proposed metabolic pathway of formation of fermentative
products by Clostridium sp. GCAF-1.................. ....... .................................. 95
















LIST OF ABBREVIATIONS AND ACRONYMS

16S ........................ ....... ................................. 16 Svedburg unit

AQD S ................................................... ......................... Anthraquinone di-sulfonic acid

A T P ................................................. .......................... ..................adenosine triphosphate

BM ................................... .............................. ................ Basal m edia

B p ................... ................... ................... ............................................................. b a se p a ir

CRB .................................... .............. chromium reducing bacteria

FR B ................................ ............... ........................................... F e(III) reducing bacteria

G .................................................................. ........ .... G lu c o se

GAF...... .................................... Glucose +AQDS +Fe(III)

G C .............. .......... ............ ...... ............................. ............ .. G lucose+ C r(V I)

G CA .... ........................... ................................................ G lucose +Cr(V I) +A QD S

GCAF..... .... ..................................................... ....... Glucose+ Cr(VI)+AQDS +Fe(III)

GCF ............. ......... ............ ....................... .............. Glucose +Cr(VI) +Fe(III)

PCR .................................................................. ... ...... ...... ... Polym erase chain reaction

rDNA..................................................... ribosomal Deoxyribonucleic acid

R N A ...... ..................................................................................... . ..R ibonucleic acid

X-gal ............................................ 5-bromo-4-chloro-3-indolyl-B-D-galactopyranoside















Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

MICROBIAL Cr(VI) REDUCTION; ROLE OF ELECTRON DONORS, ACCEPTORS,
AND MECHANISMS, WITH SPECIAL EMPHASIS ON CLOSTRIDIUM spp.

By

Kanika Sharma

December, 2002


Chair: Dr. A.V. Ogram
Major Department: Soil and Water Science

Cr(VI) has been designated as a priority pollutant by the US Environmental

Protection Agency (USEPA) due to its ability to cause mutations and cancer in humans.

The risk associated with soil and groundwater contamination of chromium waste

generated by many industries is high, and therefore Cr(VI) remediation is of critical

importance. Using chemical and biological methods conjointly can decrease the cost of

remediating contaminated sites. Microbial reduction of Cr(VI), an important aspect of

biological remediation, requires the knowledge of microorganisms capable of reducing

Cr(VI) and the mechanisms involved in the reduction processes.

The overall objective of this study was to investigate the effect of various electron

donors and acceptors on chromate reduction by indigenous Cr(VI)-reducing bacteria

isolated from Cr(VI) contaminated sites and to understand the mechanism of Cr(VI)

reduction by enriched bacterial consortium and the pure isolate. A series of bacterial

enrichment cultures were established with a range of electron donors such as acetate,









benzoate, lactate, citrate, and glucose, and electron acceptors such as Fe(III) and an

humic acid analog, anthraquinone di-sulfonate (AQDS), to study their effects on the rates

of Cr(VI) reduction. Results from this study demonstrated that the rates of Cr(VI)

reduction in glucose and citrate enrichments were higher when compared with those of

other electron donors. Enrichments amended with AQDS and Fe(III) showed enhanced

rates of Cr(VI) reduction. Glucose- AQDS-Fe(III)-Cr(VI) enrichments (now on referred

as GCAF) yielded the highest diversity of strains, which were distributed within the low

G+C and high G+C groups of gram-positive bacteria. Phylogenetic analysis based on 16S

rDNA studies revealed that isolates clustered with Bacillus, Cellulomonas, and

Clostridium groups. Several strains were isolated from the consortium. Detailed kinetic

studies with bacterial consortium and the pure strain GCAF-1 obtained from GCAF

enrichment demonstrated an iron-promoted reduction of chromate. The presence of

AQDS accelerated reduction of Cr(VI) only when Fe(III) was present in the medium.

Analysis of fermentation metabolites produced by strain Clostridium sp. GCAF-1

revealed that the presence of Cr(VI) alters the acetate: butyrate and acetate: lactate ratios.

Based on the overall results, direct and indirect (Fe (III) mediated) methods of reduction

of Cr(VI) by Clostridium sp. GCAF-1 are proposed.














CHAPTER 1
GENERAL INTRODUCTION

Chromium, the 24th element on the periodic table, was first discovered in Siberian

red lead ore (crocoite) in 1798 by the French chemist Nicholas-Louis Vauquelin. He

named this new mineral chrom from the Greek word ypco/a, owing to the brilliant hues

of the compound. Since then, chromium has found a variety of uses in the industries that

exploit these colors and other characteristics such as its strength, hardness, corrosion

resistance, and the oxidizing capabilities of certain chromium species (34).

Chromium in Environment

Chromium is found in many environments, including air, water, soil and all biota.

It ranks 21st among the elements in crustal abundance (74). The average concentration of

chromium in the continental crust has been reported as 125 mg/kg (108). Concentrations

in freshwater generally range from 0.1 to 6.0 [g/L with an average of 1.0 gg/L, while

values for seawater average 0.3 [g/L and range from 0.2 to 50 [g/L (23). Freshwater

chromium concentrations are dependent on soil chromium levels in the surrounding

watershed areas. In addition, drainage water from irrigated agricultural areas with

elevated amounts of soil chromium levels can have high chromium concentrations (as

high as 800 gg/L), as observed at various locations within San Joaquin Valley, CA (38,

50).

Chromium is extracted from chromite ore [(Fe,Mg)O(Cr, Al, Fe)203] that has

largest deposits in South Africa, the Philippines, Southern Zimbabwe, and Turkey (100).

The major users of chromium are the metallurgical, chemical, and refractory brick









industries (78). Other industries that employ chromium include pigment manufacture,

metal finishing, corrosion inhibition, organic synthesis, leather tanning, and wood

preservation (57, 34, 174). Extensive industrial usage of chromium leads to generation of

large volumes of chromium-containing wastes that are discharged into the environment.

In addition to this waste, leakage due to improper handling and faulty storage containers

also adds to the accumulation of chromium in the environment.

Nutrition and Toxicity: Risks to Human Health

Chromium is designated by the U.S. Environmental Protection Agency (USEPA)

as a priority pollutant due to its ability to cause genetic mutations and cancer. Chromium

is unique among regulated toxic elements in the environment because different species of

chromium, specifically Cr(III) and Cr(VI), are regulated in different ways. Relying on the

chemical, toxicological, and epidemiological evidence, regulation of Cr(VI)

concentration is different from that of Cr(III). Trivalent chromium is the nutritionally

useful form, while the hexavalent form is toxic and mutagenic. Cr(VI) is both a powerful

epithelial irritant and confirmed human carcinogen (77, 120). On the contrary, Cr(III) is

an essential element in animal physiology and plays a role in glucose and lipid

metabolism (5, 103).

Animals

Cr (VI) is highly mobile in some soils, and contact with Cr(VI) maybe inevitable

for aquatic and terrestrial organisms, including humans. In trace amounts, chromium is an

essential component of human and animal nutrition (65, 102). It is associated with

glucose metabolism (102) and has been shown to be an integral component of glucose

tolerance factor (GTF), a factor required for maintaining normal glucose tolerance.

Chromium functions by regulating and potentiating insulin action by increasing insulin









binding to cell (4). Chromium is also known to be of importance in fat metabolism in

animals (5).

The biotoxicity of chromate is largely a function of its ability to cross biological

membranes and its powerful oxidizing capabilities (NAS 1974). Humans can absorb

Cr(VI) compounds through inhalation, dermal contact and ingestion. Human health

effects of Cr(VI) include lung cancer, respiratory irritation, dermatitis, kidney and liver

damage, and damage to various proteins and nucleic acids, leading to mutation and

carcinogenesis (18).

Plants and Algae

Pratt (123) reported that low concentrations of chromium stimulated the growth of

plants. However, a few years later it was demonstrated conclusively that chromium is not

an essential component in plant nutrition (61). The effect of Cr(VI) was apparent on seed

germination when more than 80% of the reduction in seed germination was observed in

the presence of Cr(VI) when compared with those germinated in the absence of Cr(VI)

(133). Cr(VI) concentrations of 5 to 60 mg/kg soil have been shown to retard plant

growth due to root damage (7).

Cr(VI) has been shown to affect the growth, photosynthesis, morphology, and

enzyme activities in algae. Cr(VI) concentrations shown to be toxic to algae vary from 20

ppb to 10,000 ppb (7, 131, 135,143, 154).

Microorganisms

Hexavalent chromium is toxic and mutagenic to most bacteria. Among the visible

effects reported in bacteria are cell elongation, cell enlargement, and inhibited cell

division, which eventually leads to cell growth inhibition (31, 153). Changes in

morphologies of gram-positive and gram-negative bacteria were also observed by









Bondarenko et al (21). Few colonies of bacterial species such as Staphylococcus aureus,

S. epidermidis, Bacillus cereus, and Bacillus subtilis were formed with degenerate cells

that were reduced in size (20). Cr(VI) concentrations of 10-12 ppm were inhibitory to

most soil bacteria in liquid media and, in general, gram-negative bacteria were more

sensitive to Cr(VI) than were gram-positive bacteria (132). Increased content of Cr(VI) in

soil was toxic to saprophytic and nitrifying bacteria. Lowered microbial biomass in soil

was observed in the presence of high Cr(VI) in soil when it was determined using

adenosine triphosphate (ATP) method (2, 178). Other bacteria such as E coli, Serratia

marcescens and Enterobacter aerogenes were unable to grow in Cr(VI) concentrations of

1 mM.(8). .Metabolic effects of Cr(VI) on bacteria were evident by the observed changes

in electron transport systems (124).

Cr(VI) has been shown to cause mutagenic effects in Escherichia coli, Bacillus

subtilis, and Salmonella typhimurium (113, 120, 162). The mutagenic effects of

chromium are effective only when chromium crosses the cell membrane. Cr(VI) can

easily diffuse across the cell membranes, unlike Cr(III) which can do so only under

extreme conditions such as long incubations and high concentrations. Cell culture studies

have shown that cellular uptake of chromate is at least 10 times greater than that of

Cr(III) from equimolar solutions, (33, 55, 80). However, once inside the cell, most of

Cr(VI) is reduced to Cr(III) by several reducing agents such as ascorbic acid, sodium

sulfite, glutathione, NADPH and NADH (121). Based on several studies, it was

concluded that trivalent chromium causes DNA-strand breaks (18, 19, 37, 80, 155, 156,

157). Cr(VI) causes genotoxic effects on bacterial cells, including frameshift mutations

and base pair substitutions (120). DeFlora et al. (37) reported a more general effect of









unbalanced nucleotide pools. These studies suggest that although Cr(III) form is the

major agent responsible for molecular events leading to mutagenicity, it is Cr(VI) that

poses the greater risk to human life due to its ability to easily enter the cell.

Environmental Chemistry

Chromium can exist in oxidation states ranging from 0 to 6+. The various

chemical and biological changes that chromium undergoes in the environment depend on

the conditions that govern its speciation and other activities. The solubility and

adsorption by soil and sediments depend on the form of chromium species. Within the

ranges of redox potentials and pH commonly found in soils, chromium exists

predominantly as oxyanions of Cr(III) and Cr(VI).

Cr(VI) is a strong oxidizer and exists only in oxygenated species that are very

soluble and pH dependant according to the following equilibria (111).

H2Cr04 H+ + HCrO4- Kai =10 0.6

HCrO4- H + Cr042- Ka2 =10 5.9

H2Cr04- is a strong oxidizing agent and dominant species below pH -0.6 (32).

Monohydrogen chromate, HCrO4-, exists between the pH values of 1 and 6. Cr04 2-

predominates at or above pH 6. Cr207-2 dichromate ion is formed by dimerization of

HCrO-4 ion at Cr(VI) concentrations above 10-2 M (17, 86)

Cr2072- + H20 2HCrO4 K=10-22

Existence of dichromate ion is unlikely in the biologic systems as typical chromium

concentrations in nature are considerably lower than 10-2 M, especially at physiological

pH 7. Trivalent chromium is the more stable form. Due to its lower affinity for oxide and

hydroxide ions, Cr(III) is known to form numerous complexes with both organic and

inorganic ligands (99, 144). Due to chemical inertness, complex species of Cr(III) tend to









be more stable in solution and can be isolated. The main aqueous Cr(III) species include

Cr3+, Cr(OH)2+, Cr(OH)3, and Cr(OH)4-(16), (14, 40). The Cr3+ species predominate at

pH <3.6 (46), whereas Cr(OH)4- predominates at the pH >11.5 (126). At a slightly acidic

to alkaline pH, ionic Cr(III) species precipitates as amorphous Cr(OH)3 (126) or as a

solid solution (Fe, Cr)(OH)3 ifFe3+ is present (46) Cr(III) can also be chelated by organic

molecules that are adsorbed to mineral surfaces (63, 64). In contrast, Cr(VI) compounds

CrO42-, HCrO4, Cr2072- are very mobile in surface sediments because they are not

strongly adsorbed to soils.

Both oxidation and reduction of Cr(VI) can occur in geologic and aquatic

environments ( Figure 1-1).The oxidation and reduction of chromium in soils depends on

soil structure and on the redox conditions of the soil (73). Studies conducted to

investigate the effect of adsorption of chromate and Cr(VI) on the clay sand mixture

showed that clay was a suitable absorbent for chromate due to its high cation exchange

capacity (CEC) and strong binding capability.(2). Chromium speciation in groundwater is

affected by the pE redoxx) and pH conditions (Figure 1-2).

Reduction of Cr(VI)

Reduction of Cr(VI) in soils depends largely on the presence of other electron

acceptors such as the oxygen, nitrate, iron, and manganese that can act as electron sinks

and accept electrons from the reactive organic and inorganic electron sources. Conditions

will favor Cr(VI) reduction when electron donors are in excess and electron acceptors

such as those mentioned above are low. Hexavalent chromium is a oxidizing agent and is

readily reduced in the presence of appropriate electron donors, as shown in this equation:

HCrO4 + 7H+ +3e-0 Cr3+ +4H20









Oxidation of Cr(III) in Soils

Cr(III) is chemically more stable than Cr(VI). Initial studies convincingly showed

that, in most cases, oxidation of Cr(III) does not occur in soils, regardless of the

conditions (16). This was explained with chemical inertness of the Cr(III) and its

complexes in the range of pH that normally exists in soils. However, it has since been

determined that some Cr(III) can be oxidized to the hexavalent form in the presence of

Mn(IV). The amount of Cr(III) oxidized to Cr(VI) was shown to be proportional to the

amount of Mn(IV) reduced to Mn(II) (3, 15). Oxidation of chromium occurs in soils that

are high in Mn(IV) and oxides and low in organic matter content (73). The conditions

required for chromium oxidation are fairly specific and only a few cases of oxidation of

Cr(III) oxidation are reported in literature.

Oxidation of aqueous Cr (III) to Cr(VI) in soils does not occur over such a wide

range as the reduction of aqueous Cr(VI). Also, Cr3+ precipitates almost completely as

Cr(OH)3 often in conjunction with iron at pH values from 5.5 and 12.0 (40, 126).

Cr3+ + 3H20 K= Cr(OH)3 (s) + 3H+ Keq =10-12

These factors are of great importance in assessing potential environmental hazards

and remediation strategies for ecosystems with high levels of natural or anthropogenic

chromium.

Cr(VI) Remediation Strategies

Remediation strategies are employed in order to minimize the risk of public

exposure to chromium contaminated sites. Several common remediation strategies

include the no action option, excavation and removal of contaminated soil, pump and

treat strategies, and soil solidification and stabilization. In order to implement the optimal

remediation strategy, an understanding of physical and chemical processes affecting the









migration and chemical state of chromium is required. The no action option is adopted if

the risk of exposure and potential impact to the environment is marginal. Knowledge of

the type of soil reductants present is important for the implementation of this option.

Excavation is no longer a very desirable method as it simply moves the contaminated soil

from one place to another. Pump and treat is one of the most commonly used methods for

aquifer remediation. The two main purposes are to remove contaminants from the

subsurface for treatment and to maintain gradient control to prevent contaminants from

migrating beyond compliance boundaries. Among the major concerns of employing this

method is the residual concentration. The residual concentration is usually much higher

than the maximum contaminant level (MCL) level set by EPA. Soil solidification process

includes solidification of the contaminated soils by transforming Cr(VI) into an insoluble

chemical form that is impermeable to the ground water. Traditional techniques for

remediating chromate contaminated water also involve reduction of Cr(VI) to Cr(III) by

chemical means (usually with Fe2+) or electrochemical means at pH 5, followed by

precipitation and filtration or sedimentation (41). The electrochemical Cr(VI) reduction

process uses consumable iron electrodes and electrical current to generate ferrous ions

that react with Cr(VI) to Cr(III) is given below. Increased quantity of resultant sludge by

this method is one of the drawbacks. This method is often employed in combination with

the pump and treat methods.

3Fe2+ + CrO42- + 4H20 3Fe 3++ +Cr3+ + 80H-

These processes can be extremely reagent or energy intensive. Most of these

methods take long periods of time to reach the regulatory level for remediating

contaminated sites. The cost involved in these chemical enhanced remediation strategies









is very high and this lowers the overall cost-benefit ratio. The discovery of

microorganisms that can reduce metals has led to applications in the bioremediation

which are potentially more cost effective than traditional methods. One of the major

factors that decide the application of the bioremediation strategies is the bioavailability of

the preferred electron donor by the indigenous microorganisms that are involved in metal

reduction. For bioremediation of Cr(VI), stimulation of the existing microbial populations

with bioavailable electron donors may result in increased metal reduction, thereby

remediating the contaminated site. Although reduction of Cr(VI) to Cr(III) does not

remove chromium from soils, it does limit the mobility and toxicity of chromium in the

contaminated soils. Many potential remediation pathways are known for the chromate

reduction, but the dominance of one pathway over another has not been established.

Furthermore, coupled geochemical and microbiological processes have a potential to

dominate the reduction of metals such as Cr(VI).

Finally it must be recognized that there are many factors that effect the microbial

reduction of Cr(VI) in soils. Clearly there is a need to understand the various groups of

bacteria that reduce hexavalent chromium and the different mechanisms by which Cr(VI)

is microbially reduced in soils.

Chromium Resistance in Bacteria

The persistent nature of some metals in environment has led to considerable

modifications of the microbial community and their activities. Heavy metals have been

shown to inhibit microbial growth and other enzymatic activities by blocking essential

functional groups, displacing essential metal ions and modifying the conformations of the

biological molecules, (49, 81, 171). In metal-contaminated environments, the responses

of the microbial communities depend on the concentrations of the toxic agents they are









exposed to among other factors such as nature of nutrients, chemical form of the toxic

agent and so on. The resistance mechanisms proposed for heavy metal resistance in

bacteria include exclusion by permeability barrier, exclusion by active transport,

intracellular physical sequestration by the binding proteins of the cell, extracellular

sequestration and detoxification by chemical modification of the toxic to non-toxic form

of the metal.

Microorganisms may adopt several strategies to reduce metal sensitivity to

cellular targets: (i) mutations to decrease the sensitivity to the metal (ii) increased

production of damaged cell component, (iii) increased efficiency of repair of damaged

cell component, (iv) utilization of plasmid-encoded resistance mechanism. These

mechanisms may either occur singly or in various combinations. Persistence of metal in

environments selects for the resistant strains possessing either the resistant or the

reduction capability. Organisms isolated from sediments of Cr(VI)-contaminated metal-

processing waste evaporation ponds were found to be more Cr-tolerant compared with

those found outside the ponds (85). Plasmid-associated bacterial resistance has been

reported in Streptococcus lactis (42), Pseudomonas sp (148), and Alcaligenes eutrophus

(27, 112, 119). Studies with Pseudomonasfluorsencens LB300 showed the loss of Cr(VI)

resistance resulted with the loss of plasmid and transformation of the plasmidless strain

done with the purified plasmid DNA resulted in regaining of the Cr(VI) resistant ability

of the strain (21).

Increased polysaccharide production has been reported in Pseudomonas sp. (1).

Further studies with Pseudomonas ambigua and its Cr(VI) sensitive mutant S-1 led to the

conclusion that the presence of thick membranes around the parent cell decreased the









permeability of Cr(VI) of the cells and increased the resistance of the bacteria (59, 60).

Enterobacter cloacae strain HO1 and yeast exhibited Cr(VI) resistance by decreased

uptake of Cr(VI) (12, 116, 163).

Pathways for Chromium(VI) Reduction

Microorganisms obtain their energy for metabolism by participating in several

oxidation-reduction reactions. In environments where the photosynthesis does not occur

the transfer of electrons is the driving force that governs all the microbial processes.

Depending on the environment the microorganisms have adapted and evolved the ability

to be able to mediate various oxidation-reduction couples to conserve energy. Some

Cr(VI) resistant bacteria are able to grow by reducing Cr(VI) to Cr(III). Cr(VI) reduction

is considered to be a fortuitous reduction process that is employed by some bacteria as a

mechanism of defense by detoxification of the environment they have to survive in. Most

Cr(VI) reducing bacteria (CRB) reported so far are gram negative bacteria (12,45).

Recently the ability to use Cr(VI) as terminal electron acceptor was demonstrated in a

sulfate reducing bacterial consortium and Pantoea\ uhtn (45, 152).

Currently microbial reduction of Cr(VI) can be explained by two prevalent

models: (i) direct enzymatic reduction, and (ii) indirect reduction. Distinguishing between

these enzymatic and nonenzymatic Cr(VI) reductions is difficult. The direct enzymatic

reduction refers to the reduction by the metal reductase system. Indirect mechanism

refers to Cr(VI) reduction mainly by conditions provided by bacterial source such as the

redox potential, or the bacterial metabolites.

Direct Enzymatic Reduction of Cr(VI)

Although CRB have been studied for many years now, little is known about the

biochemistry and mechanism of Cr(VI) reduction. It still remains unclear if Cr(VI) is









taken up by the cell and reduced in the cytoplasm or the periplasm or the electron are

transferred to the outside of the cells or both. Direct contact between cells and the metal

oxide has shown to be required for the energy conservation process (8). Enzymatic

reduction of Cr(VI) has been observed in some CRB (10, 29, 52, 68, 114, 149, 173).The

CRB are able to reduce Cr(VI) by either soluble enzyme systems or the membrane-bound

system. Membrane-associated chromate reductase activity was first observed in

Enterobacter cloacae HO1 where the insoluble form of reduced chromate precipitates

was seen on the cell surface (164). In the presence of ascorbate reduced phenazine

methosulfate (PMS) as electron donor, high chromate reduction was shown by right-side-

out membrane vesicles of E. cloacae HO1 (164). Membrane-associated constitutive

enzyme that mediated the transfer of electrons from NADH to chromate was later

elucidated by Bopp et al. (21). In case of lii t ll //t putrefaciens MR-1 chromate

reductase activity was associated with the cytoplasmic membrane of anaerobically grown

cells (106). Formate and NADH served as electron donors for the reductase. No activity

was observed when NADPH or L-lactate were provided as the electron donors. However,

in Pseudomonas putida, unlike in ,\/henii //l putrefaciens, NADPH served as an

electron donor for this (117).

Studies conducted by Shen and Wang (141) on E. coli suggested the presence of

soluble chromate reductase. Cr(VI) reduction in another gram negative bacteria,

Pseudomonas sp CRB5, was found to be mediated by a soluble enzyme contained in

cytoplasm (101). In addition to gram-negative bacteria, soluble chromate reductases have

also been observed in gram-positive strains. NADH was the preferred electron donor for

the reduction of chromate by the soluble enzyme in Bacillus coagulans (122).









Bacterially Mediated Indirect Reduction of Cr(VI)

Redox potential-pH. Changes in pH and redox conditions are known to occur in

medium during growth of bacterial cultures due to various biochemical reactions and the

metabolites formed. These changes may indirectly affect the reduction of Cr(VI) in the

medium. Lower redox and pH has been shown to favor reduction of Cr(VI)(36).

Cr(VI) reduction occurs in a wide range of redox potentials. The optimum redox

potential range has not been well established as yet. Reduction of Cr(VI) has been

reported in redox conditions as high as +250mV (53). In the same culture, after 48 hours,

Cr(VI) reduction was observed even when the redox potential dropped to -500mV. A

higher rate of Cr(VI) reduction by Agrobacterium radiobacter was observed at -240mV

compared with -198mV (82). In contrary, no reduction of Cr(VI) was observed with

redox potential of -140mV for the first hour of incubation in cultures of Escherichia coli.

Fe(III)-mediated reduction of Cr(VI). Fe(III) is the most abundant electron

acceptor for anaerobic respiration in many sedimentary environments due to its ability to

act as terminal electron acceptor for many organisms. Microbial reduction of Fe(III)

significantly affects Cr(VI) biogeochemistry as reduced iron in sediments is one of the

most significant electron donors for the reduction of Cr(VI). Three equivalents of Fe(II)

are required for the reduction of one equivalent of Cr(VI).

3Fe(II) +Cr(VI)43Fe(III) + Cr(III).

Therefore, Fe(III) reducing bacteria that are unable to support their growth on

reduction of Fe(III) can indirectly reduce Cr(VI) via Fe(III) reduction (Figure 1-4).

Reduction of chromate by dissimilatory iron-reducing bacteria was reported by Wielinga

et al (169). They elucidated the reduction of Cr(VI) to Cr(III) via a closely coupled

biotic-abiotic pathway under iron-reducing conditions.









Quinone mediated reduction of Cr(VI). Humic substances are ubiquitous in the

environment. They are heterogeneous organic high-molecular-weight macromolecules

that are composed of many potentially reactive moieties. Humic substances were

considered resistant to microbial degradation until recently, when the ability of humics to

serve as electron acceptors and support bacterial growth under anaerobic conditions was

reported (90). Humics function as primary electron acceptors for iron-reducing bacteria,

and mediate transfer of electrons from humics to Fe(III) oxides, thereby stimulating the

reduction of insoluble Fe(III) oxides (Figure 1-5) (90). Quinones serve as the primary

electron-accepting moiety in the humic acids when they are reduced to hydroquinones by

accepting two electrons, as shown in Figure 1-6. Scott et al. demonstrated the higher free-

radical content of humic substances with higher electron accepting capacity with electron

spin resonance measurements by showing a proportional increase in semiquinones and

electron-accepting capacity of humic substances (137).

To date several humic reducing bacteria have been isolated from a variety of

environments (30). All iron-reducing bacteria that have been evaluated to date have

shown the ability to transfer electrons to humic substances and other extracellular

quinones.(91). Microbially reduced humics are also capable of reducing other metals,

including manganese (IV) and technetium (VII) (83). Reduction of Tc(VII) mediated by

Fe(III) was enhanced in the presence of anthraquinone di-sulfonate (AQDS), a humic

acid analog that behaved as an electron shuttle (90). Although humic-mediated Cr(VI)

reduction has not been reported so far thermodynamically, transfer of electron from

humics (E = 0.2mV) to Cr(VI) (E = 1.23mV) is plausible.









Factors Affecting Microbial Chromium Reduction

Cell density. Rate of Cr(VI) reduction has been shown to be a affected by cell

density under both aerobic and anaerobic conditions. Wang et al. (164) reported increase

in the rate of Cr(VI) reduction with increase in cell density under anaerobic conditions.

Similar observations were made in both aerobic and anaerobic cultures of Escherichia

coli. However, the rate of Cr(VI) reduction was not proportional to the increase in the cell

density, and the specific rate of Cr(VI) reduction was higher at relatively lower cell

densities (142). These observations were also documented in cultures of Enterobacter

cloacae, Agrobacterium radiobacter, Pseudomonas fluorescens LB300, Bacillus

coagulans, and Microbacterium sp.

Initial Cr concentration. Depending upon the initial concentration of Cr(VI), its

complete or incomplete reduction has been observed in Enterobacter cloacae HO1, (48,

71). Even though a decrease in cell viability was observed in the culture on addition of

Cr(VI) to the growing culture (72, 163), the initial rate of Cr(VI) reduction increased with

the increase in the initial rate of Cr(VI) in some cultures of Enterobacter cloacae (163),

E. coli (139) P. flourescens (167) and Bacillus sp.(167). Similarly, initial specific rate of

Cr(VI) reduction by cultures of E coli increased with increasing Cr(VI) concentrations.

However, an increase in time required for complete reduction was also observed (142).

Effect of other electron acceptors. Presence of oxygen does not completely

inhibit Cr(VI) reduction in some bacteria but it represses it as in the case of

Agrobacterium radiobacter EPS-916, E. coli ATCC 33456 and Pseudomonas stutzeri

CMG463 (9, 71, 82, 139, 141, 165). Microbial reduction of Cr(VI) is completely

inhibited in aerobic condition as in the case of E. cloacae HO1, even though cell growth

was observed (48). Studies with enrichment microcosms showed only 41% reduction of









Cr(VI) under aerobic conditions when compared with the 84% reduction observed in

anaerobic conditions (97).

Marsh et al. concluded that lower reducing conditions were required for Cr(VI)

reduction because reduction of Cr(VI) was inhibited by oxygen and nitrate (97).

Among other naturally occurring dominant electron acceptors, sulfate and nitrate

have little effect on the Cr(VI) reduction upto concentrations of 10 mM and 16mM,

respectively. The concentration of sulfate and nitrate to which microbial Cr(VI) reduction

is not affected varies with the bacterial species. In the case of Cr(VI) reduction by

Pseudomonas. putida Cr(VI), reduction was not affected by ImM of sulfate and 0.2mM

of nitrate. Concentrations of sulfate and nitrate, that did not affect Cr(VI) reduction, in

case of Bacillus sp., were 10 mM and 16mM respectively, in case of E. coli were 83 mM

sulfate and 129mM nitrate. Sulfate concentration as high as 50 mM did not affect the

Cr(VI) reduction by Desulfovibrio vulgaris (93). In contrast, the chromate reduction by

Enterobacter cloacae is inhibited by 32% in the presence of just 25iM of sulfate and

84% in the presence of 5mM NaNO3. Enrichment studies with alternative electron

acceptors done by Marsh et al. showed that nitrate reduction proceeded Cr(VI) reduction.

However, Fe(III) reduction and sulfate reduction always followed the Cr(VI) reduction.

They supported their results by the Gibbs energy obtained by thermodynamic reactions

(97).

Temperature and pH effects. Optimum temperature and pH conditions reported

for microbial Cr(VI) reduction strongly suggest that the reduction process is related to

growth. Cr(VI) reduction was observed in cultures of Enterobacter cloacae at pH range

of 6.0-8.5, and at pH range of 3.0 -8.0 in cultures of Escherichia coli and Bacillus









coagulans. However the maximum initial specific rate of Cr(VI) reduction by all three

bacteria was at pH 7.0, an optimal pH for most bacterial growth. Even though Cr(VI)

reduction by E. coli and Enerobacter. cloacae occurred at a wide range of temperature of

100C to 500C, optimum temperature was found to be 360C and 300C respectively. These

conditions were found to be optimal for the anaerobic growth of the bacteria. Studies

with sediments have shown temperature optima of 220C and 500C and a pH optimum of

6.8 (97).

Carbon sources. Studies have been conducted to try and establish the

relationship between the electron donors and the rate of Cr(VI) reduction. Enrichment

studies with the soils done by Marsh et al showed hydrogen to be an efficient electron

donor for the reduction of Cr(VI). Addition of electron donors that increase the

bioavailable hydrogen such as glucose, format, and hydrogen stimulated the Cr(VI)

reduction in the soils as compared with acetate and benzoate and lactate. The study also

documented the dissolved hydrogen concentration in the Cr(VI)-reducing conditions.

Based on the observation that very low hydrogen concentration was present under Cr(VI)

reducing conditions similar to that reported under nitrate- and manganese-reducing

conditions, and the observation that Cr(VI) reduction occurs before iron or sulfate

reduction it was concluded that very highly reducing conditions were not required for

Cr(VI) reduction (98).

Rege et al. reported the utilization of sucrose as a carbon source for Enterobacter

cloacae HOlfor reduction Cr(VI) (128).









Outline of Dissertation

The work presented in this dissertation was performed to get more insight into the

diversity of electron donors and acceptors utilized by the indigenous chromium reducing

bacteria (CRB). Special attention was paid to the kinetics of chromium reduction in the

presence of alternative electron acceptors Fe(III) and AQDS. An attempt was made to

explain the mechanism of Cr(VI) reduction by fermentative organisms.

Chapter 2 describes the enrichment studies with indigenous CRB capable of

utilizing various electron donors and acceptors. Difference in reduction of Cr(VI) is

determined by the organisms enriched by the various electron donors chosen to represent

the range of electron donors that naturally exist in nature. Effect of electron acceptors viz.

Fe (III) oxides and anthraquinone di-sulfonate (AQDS), an humic acid analog on

reduction of Cr(VI) is also investigated. Identification of the organisms isolated from the

enrichments is also described based on the phylogenetic studies. Chapter 3 describes the

isolation of the Cr(VI)-reducing consortium from the glucose enrichments and detailed

kinetic studies of Cr(VI)-reduction by this consortium. In chapter 4 the isolation,

identification, and detailed characterization of a novel species of chromium-reducing

fermentative organism GCAF-1 is described. Chapter 5 includes detailed kinetic studies

with fermentative isolate GCAF-1. It describes the possible mechanism adopted by

Clostridium sp. GCAF1 reduce Cr(VI). Finally, the results presented in this dissertation

are summarized and the implications of this research are discussed in Chapter 6.



















C..citrate
CSI


leaching
plant uptake
adlorpllOn/
-. precipitation


0

0
c(
p


Figure 1-1. Chromium cycle in environment (174)







20


>1.2V >1.2V

+2e +2e

0.91V 0.91V 0.91V 0.91V 0.91V
C r(V I) ...................... C r(V ) ...................... C r (IV ) -------............. C r (III)................................... C r (II) ........ C
+le +le +le +le +le

1.41 V; E(pH 7.4) 0.3-0.5 V


+3e



Figure 1-2. Reduction potential diagram for chromium. Positive E0 values favor the
reduced form. E "values for Cr(VI) and Cr(V) are dependant on the pH
because the protons are involved in the reaction (111).














CHROMIUM


+0.5-






Eh 0.0 -






-0.5-


1 3 5 7 9
pH


Figure 1-3. Eh-pH diagram for chromium-water system at standard state conditions.
Source: Dragun Figure (111)
















Surface


Aerobic


SFRB 1 Fel11)
Fe(II)l


CRB'"


---------------------0 -1C V I) MM>P C n -1111I


SHRB Hiui 'E II


SFceIIi


Anaerobic


Schematic diagram showing the possible pathways for anaerobic Cr(VI)
reduction by the three groups of bacteria, Fe(III) reducing bacteria (FRB);
Cr(VI) reducing bacteria (CRB); humics reducing bacteria (HRB). Solid lines
represent the biotic reduction of Cr(VI) and the abiotic reduction is
represented by the dashed lines.


Figure 1-4.











Acetate





CO2


bacteria


Humics
oxidized




Humics
reduced


Figure 1-5. Model showing reduction of Fe(III) mediated by humics (90)


benzoquinone semiquinone


hydroquinone


Figure 1-6. Quinone model compound. The semiquinone species contains an unpaired
electron (137)


Fe(II)





Fe(III)










Table 1-1. Cr(VI) reducing bacteria described in literature


Bacteria Redox Cr(VI) Gram stain Reduction Enzymatic Carbon Reference
Bacteria potential reduced conditions reduction source Reference
potential reduced_ conditions reduction source


P. putida MKI

Pseudomonas
sp. CRB5
Pseudomonas
dechromaticans


P.
chromatophila


P. fluorescent
LB300


P. ambigua G-1

P. aeruginosa


0.2mM


0.1mM


0.2mM


ND


Gram
negative
Gram
negative
Gram-
negative


Gram-
negative


ND


ND


ND


P. putida
PRS2000


0.48mM


0.4mM


0.038mM


Gram-
negative

Gram-
negative
Gram-
negative

Gram-
negative


Anaerobic ND


Aerobic and
anaerobic


Soluble
reductase


Anaerobic ND


Anaerobic ND


Aerobic and
to a lesser
extent
anaerobic
Aerobic


Membrane
associated,
NADH
dependant
NAD(P)H-
dependant


Anaerobic ND


Aerobi and
anaerobic


Soluble
protein; NADH
or NADPH


Does not
require
NADH
Peptone /
glucose
ribose/
lactate/
acetate/
succinate/
butyrate/
glycerol/
fumarate


Glucose


Nutrient broth
Acetate/
glucose

Glucose/
lactate


dependent


(117)

(101)


(130)

(79)


(22, 167)


(59)

(56)

(62)











Table 1-1. continued


Bacteria

E. coli A TCC
33456





Agrobacterium
radiobacter
EPS-916
(resting cells)
Desulfovibrio
vulgaris
Micrococcus
roses
Streptomyces
(Actinomycete)

Pantoea

SP1

Achromobacter
eurvdice


Redox
potential
ND








-200mV


Cr(VI)
reduced
0.3 mM








0.5mM under
Eh -138mV


0.1mM


ND


Gram stain

Gram-
negative






Gram-
negative


Gram-
negative
Gram-positive

Gram-positive

Gram-
negative
(facultative
anaerobe)
Gram-
negative


Reduction
conditions
Anaerobic
and aerobic;
oxygen
repressed
Cr(VI)
reduction.


Anaerobic.
Cr(VI) used
as terminal
electron
acceptor.


Enzymatic
reduction
Maj orly
Soluble
reductase little
activity by
membrane
associated.


Carbon
source
Nutrient broth


Glucose
Fructose
lactose
glutamate
succinate


Reference

(141)






(82)


(93)

(56)

(35)

(45)


NA


Lactate,
acetate,
hydrogen


(56)










Table 1-1. continued.


Bacteria
Dienococcus
radiodurans
R1
(Thermus
group)


Redox
potential


NA


Cr(VI)


Cr(VI)
reduced


0.5mM;.


Gram stain



Gram-positive


Reduction
conditions
Anaerobic
and to a
lesser extent
in aerobic
conditions


Bacillus
subtilis


Enterobacter
cloacae HOI


Rhodobacter
sphaeroides



Desulfotomacu
lum reducens


Thiobacillus
ferroxidans


ND


0.1mM to
ImM


0.5 mM


0.146 Mol h-
1(aerobic)
1.6lMol h-1



Less than
0.20 mM


ND




ND



ND


Gram-positive


Gram-
negative


Gram-
negative



Gram positive


Gram-
0.289 mM Ga
negative


Aerobic


Soluble
protein; NADH
can act as
electron donor


Membrane
Anaerobic ae
associated


Aerobic and
Anaerobic



Anaerobic


Soluble
fraction;
NADH
required.


ND


Anaerobic abiotic


(53)



Acetate/ (115)
glycerol/
glucose


Succinic acid (110)


Butyrate/
lactate/
propionate/
pyruvate/
glucose
sulfur


(152)



(125)


ND:not determined
-; no values were found in literature.


Enzymatic
reduction


Electron
donor


NA


Reference

(47)


lactate














CHAPTER 2
ENRICHMENT, ISOLATION, AND CHARACTERIZATION OF CR(VI)- REDUCING
BACTERIA

Introduction

Understanding the microbe-metal interactions in the environment has gained

considerable importance in the past decade and a half (67, 94, 160, 161, 170, 175, 177).

Among the various aspects of the interactions studied, the role of microorganisms in

remediating contaminated water, soils, and sediments is gaining much appreciation (47,

125, 158, 166, 176). Microorganisms can affect the solubility and the toxicity of metals

and provide insitu remediation of contaminated fields. Field studies, conducted to exploit

the ability of microbes to attenuate or remove contaminants from the environment by

direct or indirect means, have shown stimulation of indigenous microorganisms to be an

effective method of remediation.

Microbial reduction of soluble Cr(VI) to its insoluble Cr(III) form is a cost-

effective way to prevent the mobility of Cr(VI) beyond the compliance boundaries and to

eliminate the risk of health hazards to humans. Microbial reduction of Cr(VI) is

controlled by many factors, including cell density, initial concentration of Cr(VI), pH,

and redox potential (97, 98, 176). Among the factors that contribute significantly to

microbial reduction of Cr(VI) by influencing the activities of particular groups of soil

bacteria are electron donors and acceptors present in the soil. Number and the activity of

Cr(VI)-reducing bacteria in soil largely depend on the growth conditions and the organic

compounds that serve as electron donors present in soil.









In aerobic environments, oxygen is the most abundant electron acceptor. In the

absence of oxygen, other electron acceptors such as NO3-, and S042- can be utilized by

the microorganisms to conserve energy. Several microorganisms also have the ability to

couple the reduction of the metal oxides such as Fe(III) and Mn(IV) to the oxidation of

the carbon source. Wide diversity of Fe(III)- and Mn(IV)-reducing has been established

to date (84, 152, 177). Although few bacteria with an ability to reduce Cr(VI) have been

reported in literature, not many Cr(VI)-respiring bacteria are reported possibly due to two

reasons (a) Cr(VI) is a mutagen and is toxic for most organisms, (b) presence of other

electron acceptors that can support the growth of the organisms is much higher. Recently,

a sulfate reducing-bacteria Desulfotomaculum sp and a consortium of sulfate-reducing

bacteria with an ability to utilize Cr(VI) were reported (152). Presence of other electron

acceptors can influence the reduction of Cr(VI). For instance, it has been shown in the

past that Fe(II) abiotically reduces Cr(VI) under reducing conditions, and that soil

organic matter with a high content of humics acts as an electron donor in Cr(VI)

reduction. However, paucity in data exists regarding the importance of microorganisms in

these transformations. The comprehensive study presented here addresses various aspects

of microbial Cr(VI) reduction in soil such as the organisms involved in Cr(VI) reduction,

and the role of electron donors and acceptors in Cr(VI) reduction.

Comparative analysis of genetic sequences provides insights into the genealogical

relationships of prokaryotes. Sequencing studies have used 5S rRNA (58), cytochrome c

and ferredoxins (136) among others as possible genetic probes for phylogenetic analysis.

However, phylogenetic patterns obtained are not congruent among each other and none

of them match the branching patterns of those of 16S rRNA. The sequence of 16S rRNA









genes (rDNA) is considered to be a valuable genetic marker for establishing phylogenetic

relationships between organisms. The conserved character of these molecules together

with regions of higher variability, their ubiquitous distribution, genetic stability, and

functional constancy make them a suitable candidate for this application. The

phylogenetic trees cluster organisms based on genetic makeup. Phenotypic characters do

not have to be considered. Therefore, 16S rDNA was used in this study for identifying

the various CRBs.

Specific objectives of this study were to (i) maximize the diversity of the CRB to

be isolated (ii) evaluate the contribution of various electron donors for Cr(VI) reduction,

(iii) study the effect of alternative external electron acceptors on Cr(VI) reduction, and

(iv) perform phylogenetic analysis of the CRB isolated.

Materials and Methods

Soil was obtained from a highly Cr(VI)-contaminated Superfund site in the Upper

Peninsula of Michigan. This site is a wetland receiving Cr(VI) from effluents discharged

from an adjacent leather-tanning facility. Soil was collected in sterile containers and

immediately shipped, while being maintained below 40C, to our laboratory. Samples

were stored under 40C until the work began. The concentration of chromium in the soil

was determined to be approximately 17 g/ kg of soil. The iron content of these soil

samples was determined to be 13 mg/kg.

Enrichment media. Anaerobic enrichments were established with a variety of

electron donors and electron acceptors in different combinations. Enrichments were

prepared in bicarbonate buffered basal media composed of (per liter) KH2PO4 (0.42 g);

K2HPO4 (0.22 g); NH4C1 (0.2 g); mineral mix (10ml); vitamin mix (15ml); KC1 (0.38 g);

NaCl (0.36 g), CaC12.2H20 (0.04 g); MgSO4.7H20 (0.10 g); NaHCO3 (1.8 g); Na2CO3









(0.5 g); ImM Na2SeO4 (Iml). vitamin mix was composed of (per liter) biotin (2.0 mg);

folic acid (2.0 mg); pyridoxine HC1 (10.0 mg); riboflavin (5.0 mg); thiamine (5.0 mg);

nicotinic acid (5.0 mg); pantothenic acid (5.0 mg); vitamin B-12 (0.1 mg); p-

aminobenzoic acid (5.0 mg); thioctic acid (5.0 mg); mineral mix was composed of (per

liter) NTA (1.5g); MgSO4 (3.0g); MnSO4.H20 (0.5g); NaCl (.Og); FeSO4. 7H20 (0.lg);

CaCl2 .2H20 (0.lg); CoCl2.6H20 (0.lg); ZnCl2 (0.13g); CuSO4.5H20 (0.01g);

A1K(S04)2.12H20 (0.01g); H3BO3 (0.01g); Na2MoO4 (0.025g); NiCl2.6H20 (0.024g);

Na2WO4.2H20 (0.025g). 90 ml of this medium were dispensed in 117 ml serum bottles

under gas (C02:N2::20:80) pressure and gassed for 30 min. When required, iron was

added to the medium in the form of Fe(OH)3 a from stock solution of 0.5M before

autoclaving. After autoclaving, media were anaerobically amended with electron donors

and acceptors. Sterile stock solutions of electron donors and acceptors were prepared

separately under anaerobic conditions. Final concentrations of supplements in the

medium were Cr(VI) (0.4mM); AQDS (0.1mM); and Fe(III) (5mM). Electron donors

included 10mM each of acetate, benzoate, citrate, and glucose as required for individual

enrichments (Table 2-1).

Enrichments. Four sets of anaerobic enrichments were established with of

different electron donors as described above. Each set contained 4 microcosms, each

amended with a different combination of electron acceptors (Table2-1). Microcosms

contained the following combinations of electron acceptors. Cr(VI) only; Cr(VI) and

AQDS; Cr(VI) and Fe(III); Cr(VI), AQDS, and Fe(III). Each set also included one

control to monitor abiotic reduction of Cr(VI). Controls were supplemented with all

electron acceptors and appropriate donor inoculated with dead (autoclaved) cells.









The inoculum was prepared by adding 10 g of soil to basal medium (10 ml). Soil

in media was stirred under continuous flushing with nitrogen gas. After considerable

stirring to break all soil aggregates, 10 ml of slurry were used as inoculum for

enrichments. The enrichments were incubated at 300C in the dark without shaking. The

concentration of Cr(VI) remaining in the microcosm was monitored at various times. On

depletion of Cr(VI) from the enrichments, transfers were made with 10% inoculum to

fresh medium containing appropriate electron acceptors and donor. Rates of Cr(VI)

reduction were determined in all enrichments after the third transfer as described below.

By the third transfer, soil particles were diluted out, thereby decreasing the likelihood of

Cr(VI) reduction by chemicals other those supplemented in the medium. Initial Cr(VI)

concentrations were measured in enrichments immediately following inoculation to

assess chemical reduction of Cr(VI) by reduced components that carried over during

transfers.

Analytical methods. Concentrations of Cr(VI) were determined colorimetrically

by UV/Visible spectrophotometer (perker) equipped with 1 cm cuvettes, using the

diphenylcarbazide (DPC) assay as previously described (43, 159). 0.25ml solution,

prepared with 0.025g of 1,5-diphenylcarbazide in 10 ml of acetone, were added to 10 ml

of sample (diluted when necessary). After 15 minutes of incubation at room temperature

(250C), absorbance at 540 nm was determined. This assay has a detection limit of 2 .iM

All readings were conducted in triplicate.

Strain isolation. Bacteria were isolated by a standard roll tube method, technique

used for the isolation of the anaerobic bacteria. Roll tubes are anaerobic, sealed serum

tubes that were prepared by rolling tubes containing sterile molten agar in medium with









carbon source. The inoculum was added while the agar was still in molten form. The

tubes were rolled till the agar layer was set along the walls of the tube. Isolated colonies

appeared either embedded or on the surface of the agar layer. Each culture was passed

through roll tubes several times until colonies with uniform morphology were obtained.

Colonies selected and picked by sterile long stemmed pasteur pipettes were immediately

transferred liquid basal media while maintaining the electron donors and acceptors. This

set up was continuously maintained under nitrogen gas flow to keep it anaerobic. Isolates

were obtained from enrichment cultures in which the fastest reduction of Cr(VI) was

observed. Isolated colonies were tested for their ability to reduce Cr(VI). Isolates were

tested for facultative and obligate anaerobic growth. Strains prefixed as GCAF were

isolated from Glucose-Cr(VI) enrichment supplemented with Fe(III) and AQDS; Strains

designated as GCF were from the glucose-Cr(VI) enrichment with Fe(III); GCA isolates

were obtained from the glucose-Cr(VI) and AQDS enrichment. GC isolates were from

the Glucose-Cr(VI) enrichment with no external electron acceptors.

DNA isolation and amplification of 16S rDNA. Genomic DNA was extracted

from each of the 12 isolates (1ml culture of pure isolates). Cells were harvested and lysed

by boiling in 500 pl of sterile water. 16S rRNA gene was amplified using universal

bacterial primers 27f and 1492r (76)with Perkin Elmer thermocycler Model 240

(Norwalk, CT). Conditions for amplifications were as follows: 95C for 15 min, followed

by 35 cycles of 94C for 30 seconds, 58C for 1 min and 72C for 30 sec. The final

extension step was 7 minutes at 720C. The amplified product was purified using a

commercially available kit (Qiagen, Inc.) and sequenced by the DNA Interdisciplinary

Center for Biotechnology Research sequencing facility at the University of Florida.









Phylogenetic analyses of the isolates 16S rDNA sequences were screened using

the BLAST (ref.) program to identify organisms of highest similarity with 16S rDNA

sequences of the various isolates obtained. Sequence alignment were either performed

manually with sequences obtained from Ribosomal Database Project (96)or by using

PILEUP function of GCG (Genetics Computer Group version ). CLUSTALX was used to

view the alignment and finer adjustments were made manually using McCLADE version

3.0 (95) Phylogenetic trees were constructed using maximum parsimony analyses of the

aligned sequences by PAUP 4.0b8 (150). Bootstrap values were assigned on 100

replicates after reweighing the characters by heuristic search strategy to assess the

confidence level of various clades. The GenBank accession numbers for the sequences

shown in Figure 2-2.

Results and Discussion

Effect of Electron Donors and Acceptors on Cr(VI) Reduction

The results strongly suggested that the rate of Cr(VI) reduction by indigenous soil

microorganisms was affected by the available electron donor and acceptors. Even though

reduction of Cr(VI) was observed in all sets of enrichments, there was a difference in rate

of Cr(VI) reduction. Concentrations of Cr(VI) remained constant in treatments that were

not inoculated with cells, and insignificant amount of reduction was observed in

treatments inoculated with heat killed cells (Figure 2-1). Cr(VI) was rapidly reduced in

enrichments with glucose and citrate as electron donors. Cr(VI) concentrations fell below

detection levels in less than a week in glucose enrichments. Higher turbidity was also

observed in these enrichments. Enrichments with citrate as electron donor reduced Cr(VI)

within 10 days. Bacterial enrichments amended with acetate and benzoate also showed

loss of Cr(VI) although at a much lower rate. Cr(VI) in these enrichment cultures was









completely reduced after three weeks. The pH of cultures was monitored and remained

constant at 7.4.

Enrichments with different electron donors were established in order to maximize

the diversity of the CRB enriched. Acetate was chosen as an electron donor as it is

abundant in nature and most metal reducing organisms have the ability to couple acetate

oxidation to metal reduction (51, 109, 147). No loss of Cr(VI) and no bacterial growth

was observed in enrichments with Cr(VI) as sole electron acceptor. There are three

possible explanations; (i) toxicity of high concentrations of Cr(VI) (ii) absence of acetate

utilizing bacteria that couple their growth to Cr(VI) reduction (iii) bacterial communities

being among the 99.9% uncultivable bacteria. Benzoate and lactate were expected to

support the growth of organisms that conserve energy by oxidizing aromatic compounds

and fermentation products respectively. Bacteria with the ability to oxidize benzoate with

Cr(VI) reduction has been reported (140). Citrate was one of the chosen electron donors

for this study as it is a part of citric acid cycle that has major biosynthetic as well as

energetic functions and many organisms have the ability to utilize it an electron donor

and carbon source. Reduction of Cr(VI) in enrichment sets with the electron donors was

observed only in the presence of other electron acceptors. Variation in rate of Cr(VI)

reduction observed in different donor sets suggested diversity in organisms being

enriched and possibly different mechanisms of Cr(VI) reduction.

Slight turbidity was observed in glucose enrichment with Cr(VI) as sole electron

acceptor but there was no reduction of Cr(VI) indicating growth of Cr(VI) resistant

fermentative organisms.









Reduction of Cr(VI) was observed in all enrichment set when the media were

supplemented with additional electron acceptor AQDS or Fe(III). In presence of both

Fe(III) and AQDS, an accelerated rate of Cr(VI) reduction was observed. These results

can be explained by efficient channeling of electrons towards the reduction of Cr(VI) due

to Fe(III) and AQDS acting as electron shuttles and the amount of electrons shuttled in

these systems are much higher and therefore Cr(VI) is rapidly lost from the system.

Microbially reduced form of AQDS can act as a shuttle by transferring electrons to

insoluble Fe(III) and increasing the rate of Fe(III) reduction. Fe(II), in turn, can reduce

Cr(VI) to Cr(III). In the absence of either of the electron acceptors, effective electron

shuttle trains are broken, thereby lowering the rate of Cr(VI) reduction. Increased

reduction of Cr(VI) is also indicative of the combined contribution of the AQDS

respiring bacteria, Fe(III) reducing bacteria, and Cr(VI) reducing bacteria (Figure 1-4).

The results also suggested that the presence of Fe(III) and AQDS in combination

alleviate the toxicity of Cr(VI) to the bacteria more than when they are present alone with

Cr(VI).

Fastest rate of Cr(VI) was observed in enrichment culture with glucose as electron

donor. In addition higher turbidity was also observed. Preference of glucose by the

enriched indigenous CRB, over other electron donors used in the study was clearly

evident. Anaerobic Cr(VI)-enrichment with glucose as electron donor was expected to

enrich fermentative bacteria in addition to other CRB. In the environment fermentative

organisms form the primary level where they oxidize more complex electron donors and

form simpler metabolites that are used up by the secondary level microorganisms.

Therefore the fermentative organisms that are resistant to Cr(VI) were expected to enrich









first along with the other Cr(VI) resistant, non fermentative CRB. The fermentation

metabolites formed could then be used by other non fermentative CRB that are unable to

utilize glucose as carbon source. However, results from the phylogenetic study revealed

that the enrichment culture that showed the fastest reduction of Cr(VI) was dominated by

the fermentative gram positive bacteria. One of the possible explanation of the

dominance of these bacteria in the enrichment culture is that the major indigenous CRB

in the Cr(VI)-contaminated soil were fermentative bacteria, mainly Clostridium sp. and

Celullomonas sp. At this point, it was not clear if Clostridium sp. reduces Cr(VI) directly

or indirectly via Fe(III) and AQDS. The confirmation of this hypothesis and the

elucidation of the mechanism of Cr(VI) reduction by these organism was the objective of

the next study.

Phylogenetic Analysis of Cr(VI) Reducing Bacteria

Phylogenetic analysis of 16S rDNA sequences of the isolates obtained from

enrichments with glucose as electron donor yielded organisms that belonged mostly to

the high G+C gram positive Cellulomonas group and low G+C gram positive Clostridium

group of bacteria. Several isolates belonging to Micrococcus, Bacillus, and

Staphylococcus genera were also obtained. With exception of Bacillus and Micrococcus,

attempts to grow these isolates aerobically were unsuccessful. Previously described CRB

are capable of reducing Cr(VI) to Cr(III) and are phylogenetically diverse. Most CRB are

gram-negative and facultative anaerobes. Few gram positive bacteria capable of reducing

Cr(VI) have been reported to date. Reduction of Fe(III) by Clostridium sp has been

documented previously, but no reports of Cr(VI) reduction by Cellulomonas sp or

Clostridium sp have been published so far. Results from this study strongly suggest









significant role played by the fermentative organisms in the reduction of Cr(VI) and

perhaps other heavy metals.

As more chromium reducing bacteria will be isolated from various environments

it is likely that the diversity will continue to increase. It still remains to be seen if the

capability to reduce Cr(VI) evolved independently and specifically in some organisms or

if there are some organisms that can support their growth on the reduction of Cr(VI). For

example, numerous studies suggest that NADH dependant-reductase enzyme was

invariably involved in reduction of Cr(VI) to Cr(III) in E. coli and Pseudomonas sp. the

finding that many other bacteria can reduce Cr(VI) without any enzyme indicates that

there is more than one pathway for the reduction of Cr(VI). These studies emphasize that

much study is required before the microorganisms in various environments will be known

and before the mechanisms for Cr(VI) reduction will be understood.









Table 2-1. Combination of electron acceptors and donors supplemented in the media for
anaerobic enrichment studies


Electron donors Electron acceptors Code

Cr(VI) AC
Cr(VI) and AQDS ACA
Acetate Cr(VI) and Fe(III) ACF
Cr(VI), AQDS and Fe(III) ACAF

Cr(VI) BC
Cr(VI) and AQDS BCA
Benzoate Cr(VI) and Fe(III) BCF
Cr(VI), AQDS and Fe(III) BCAF

Cr(VI) CC
Cr(VI) and AQDS CCA
Citrate Cr(VI) and Fe(III) CCF
Cr(VI), AQDS and Fe(III) CCAF

Cr(VI) LC
Cr(VI) and AQDS LCA
Lactate Cr(VI) and Fe(III) LCF
Cr(VI), AQDS and Fe(III) LCAF

Cr(VI) GC
Cr(VI) and AQDS GCA
Glucose Cr(VI) and Fe(III) GCF
Cr(VI), AQDS and Fe(III) GCAF









Table 2-2. Accession numbers for 16S rDNA sequences used in this study
Bacterial species Acession numbers*

Clostridium cellasea X83804

Cellulomonasflavigena AF 140036

Cellulomonas persica AF064701

Cellulomonas sp. strain 1533 Y09658

Cellulomonas hominis X82598

Oerskovia turbata X79454

Clostridium acetobutylicum X81021

Clostridium beijerinckii X68179

Clostridium roseum strain DSM 51 Y18171

Clostridium sp. (C.corinoforum) X76742

Clostridium sp. (C.favososporum) X76749

Clostridium puniceumm X71857

Clostridium butyricum X68176

Clostridium paraputrificum strain M-21 AB032556

Staphylococcus sp. strain LMG-19417 AJ276810

Bacillus megaterium D16273

Bacillus macroides X70312

Bacillus macroides AF 157696

Bacillus subtilis N5 AF270793

Desulfotomaculum acetoxidans Y 11566

Pseudomonas putida AF307869

*Gen Bank accession numbers except for those species sequenced used in this study















B Benzoate enrichment


0 14 24
0 14 24


* ca
* cf
l caf
U control


100-

80-

60-

40-

20-

0-


I I I


Time(days)


0 10 20


Time (days)


C Citrate enrichment


100 -1 I


M ca
Scf
Scaf
I control


D Glucose enrichment


100.

80

60

40

20

0


0 4 10
Time(days)


0 3 6
Time (days)


Figure 2-1. Rate of reduction of Cr(VI) in enrichment cultures amended with different
electron donors and electron acceptors. A: acetate amended enrichments. B:
benzoate amended enrichments; C: citrate amended enrichments, D: glucose
amended enrichments. Each of the enrichment sets was supplemented with
additional electron acceptors. ca :Cr(VI) and AQDS, cf: Cr(VI) and Fe(III);
caf: Cr(VI),AQDS and Fe(III); control was set up with dead (autoclaved)cells
amended with Cr(VI),AQDS and Fe(III) as electron acceptors. No cell
control showed very insignificant reduction of Cr(VI).


100 -r-r


Sca
Mcf
Scaf
I control


M ca
*cf
Scarf
M control


A Acetate enrichment









41






Bootstrap


93 Cellulomonas cellasea (X83804)
gcal0
Cellulomonas cellasea (AF140036)
100
-64 1 -- Cellulomonaspersica (AF064701)

100 | ----- gc9
81 89 gc8
gc7

100 73 Cellulomonas sp strain 1533 (Y09658)
Cellulomonas homins (X82598)
Oerskovia turbata (X79454)
95
--L 77 | -- gcal4
gcal3

91 Clostrzdium acetobutylicum (X81021)
100 Clostrzdium beyerincku (X68179)
Clostrzdium roseum (Y18171)
91
100 Clostrdium cormtforum (X76742)
84 73 Clostrdiumfavososporum (X76749)
Clostrdrium punceum (X71857)
ClostrIdrum butyricum (X68176)
79 gcaf7a
gcaf7

90 -- gcaf9
81 -- gcaf5

gcafla
gcf2

100 Clostrdium paraputrificum (AB032556)
75 | gca3
99 gca4
100 gca2
100 gca7

gca5

100 100 Staphylococcus sp (AJ276810)
---- gc4

100 Bacllus megaterum (D16273
100 gca8
89
.- Bacillus macroides (NCD01661) (X70312)

100 gc3
100 Bacllus macroides (AF157696)

100 (Bacllus subthls) AF270793
ccal
Desulfotomaculum acetoxidans (Y11566)
Pseudomonasputida (AF307869)











Figure 2-2. Phylogenetic tree constructed using maximum parsimony (PAUP version

4.0b8). Numbers above the branches represent the bootstrap values (100

replicates). Codes for isolates are provided in Table 2-1.














CHAPTER 3
CR(VI) REDUCTION BY A CONSORTIUM OF GRAM POSITIVE FERMENTATIVE
BACTERIA

Introduction

Cr(VI) is mainly generated as an effluent by many industries. Due to its soluble

nature it has been found in groundwater not only at the point source but also away from

its source. Mutagenic and carcinogenic nature of Cr(VI) makes its contamination a matter

of intense concern. Chemical processes that reduce Cr(VI) before it is discharged into the

environment include chemical reduction, ion exchange and electrodepositing. The use of

microbial reduction offers an inexpensive and long term alternative strategy to treat the

Cr(VI)-contaminated soils and ground water. In order to implement the bioremediation

strategy, it is important to understand microorganisms involved in Cr(VI) reduction and

the mechanisms by which Cr(VI) is reduced. Data from enrichment studies (chapter 2)

indicated that among the various carbon sources tested glucose was the preferred electron

donor by the organisms to carry out the reduction of Cr(VI). Enrichments showing the

fastest reduction of Cr(VI) had enriched gram positive bacteria belonging predominantly

to Clostridium sp and Cellulomonas sp. Presence of fermentative bacteria like

Clostridium sp. in metal contaminated sites has been described in the past, but no detailed

study has been done to elucidate their role in metal reduction. Their major role in the

metal contaminated environments has been described as being the provider for the carbon

sources for other metal reducing bacteria. The metabolic products of fermentative









bacteria act as electron donors for other microorganisms that are involved in metal

reduction.

Studies with consortium are significant as they provides a closer picture of what

happens in the field where the microorganisms do not live in pure cultures. Pure culture

studies are important to study the particular microbial activity and the consortium studies

explain how the activity may be influenced by the presence of other organisms.

Considering the above mentioned reasons the following study was done to understand the

kinetics of Cr(VI) reduction by the cosortium GCAF isolated from glucose enrichment.

The objectives of this study were to (i) monitor the kinetics of Cr(VI) reduction by the

consortium GCAF, in the presence of Fe(III) and AQDS (ii) to elucidate the mechanism

of Cr(VI) reduction by the consortium, (iii) to study the effect of Cr(VI) on the

fermentation

Materials and Methods

Culture conditions for bacterial consortium. Fermentative consortium GCAF-1

was obtained during the previously conducted enrichment study (chapter 2). Glucose

(10mM) was supplied as the electron donor. Cr(VI) (0.4 mM), Fe(III) (5 mM) and AQDS

(0.1mM) were provided as electron acceptors. For inoculum purposes, the consortium

was grown in the absence of any electron acceptor. All manipulations were made under

an atmosphere of N2-CO2 (80:20).

Metal reduction experiments. Late log phase cultures were inoculated in fresh

anaerobic medium with glucose as electron donor and incubated under 30C till early log

phase was reached. The optical density of the inoculum was 0.6 (OD at 550nm). Media

supplemented with appropriate electron donor and acceptors was inoculated with 1 %

inoculum. Experimental set up consisted of eight different treatments.The treatments









consisted of basal medium supplemented with (i) glucose and all three electron acceptors,

(ii) glucose, Cr(VI) and Fe(III), (iii) glucose and Cr(VI), (iv) glucose, Fe(III) and AQDS,

(vi) glucose and no electron acceptors, (vii) all three electron donors and no glucose and,

(viii) glucose and all three electron acceptors and no bacterial inoculation. The

concentration of glucose, Cr(VI), Fe(III) and AQDS were maintained as in the original

consortium. All the addition of electron acceptors and donors was done separately from

sterile stock solutions. After inoculation the experimental cultures were incubated at 30C

throughout the study. Samples were taken with sterile syringe and needles that were

flushed with N2 to avoid any contamination of the cultures with oxygen at appropriate

time intervals. Each test was performed in triplicates.

Determination of cell numbers. Number of cells in the cultures were determined

by direct count using acridine orange stain and the fluorescence microscope. Dilutions of

the cultures were made wherever necessary to keep the cell count within the range of 50-

100 per field area. 25% gluteraldehyde was used to fix the cells for counting. Cells were

suspended in oxalate solution prior to counting to dissolve any insoluble Fe-oxides in the

solution.

Chemical analyses. Chromium analysis was performed by colorimetric method

using UV/Vis spectrophotometer (Shimadzu) as mentioned previously (chapter 2).

Glucose analyses was done by the colorimetric method using UV/Vis spectrophotometer

at 490nm. Sample was filtered through 0.2[im filter. 2001l of 5% phenol was added to

the equal amount 2001l of sample. Immediately lml of conc. H2SO4 was added to the

mixture and gentally shaken. The reaction was kept stationary for 30 minutes to allow the

solution to cool down. The solution was gently shaken before taking the reading at









490nm. Precipitate analysis was done by EDX. Organis acids formed as he fermentation

products were measured by High pressure liquid chromatography (Waters Co.) equipped

with a UV detector (Waters, Co). Aminex HP 87H column was used as the separating

column (300 X 7.5 mm). Sulfuric acid (5mM) was used as an eluent at the flow rate of

0.6 ml/ minute.

Electron microscopy. Precipitates formed in the consortium was obtained by

using the microcentrifuge (12000X g), rinsed twice with distilled water, and air dried on

carbon coated mounts prior to viewing it via scanning electron microscopy.

Results and Discussion

Composition of Fermentative Consortium GCAF

Active Cr(VI) reducing consortium isolated from the glucose enrichment was

dominated by high G+C and low G+C gram positive bacteria. Fermentative consortium

GCAF was unable to grow or reduce Cr(VI) when Cr(VI) was added as the sole electron

acceptor to the medium. Growth of cells was inhibited by the toxicity of Cr(VI).

However, in the presence of Fe(III) and AQDS, reduction of Cr(VI) was observed

suggesting the possibility of Fe(III) alleviating the toxicity of the Cr(VI) to the cells of

Consortium GCAF (Figure 3-1).

Biotic versus Abiotic Reduction of Cr(VI)

Cr(VI) reduction was a biotic process that did not occur in medium that was not

inoculated with cells. No reduction of Cr(VI) was observed in the medium in the absence

of the electron donor suggesting that the biotic reduction of Cr(VI) required the

metabolically active cells.









Kinetics of Cr(VI) Reduction

Fermentative consortium GCAF was able to reduce Cr(VI) under anaerobic

conditions. Presence of other electron acceptors had an effect on the amount of Cr(VI)

reduced, the rate at which Cr(VI) was reduced, and the rate at which glucose was

oxidized by the consortium GCAF. Media that were supplemented with other electron

acceptors showed high turbidity indicative of bacterial growth. This was also confirmed

by observing the sample under the microscope. Cr(VI) reduction was also observed in the

active cultures. Complete reduction of Cr(VI) was observed in medium with AQDS and

Fe(III) as additional electron acceptors. However, in the absence of AQDS only 66% of

Cr(VI) was reduced. The rate of reduction of Cr(VI) was slower in the absence of AQDS.

Similar trends were observed in the oxidation of glucose. In the absence of AQDS

complete glucose (10mM) was not utilized and the rate of oxidation was much slower.

These results suggested that the electrons being generated by oxidation of glucose, by the

consortium were being transferred to Cr(VI) via Fe(III) and AQDS. In the absence of

AQDS, the electrons were shuttled from the cells to the insoluble Fe(III). Soluble Fe(II)

was then behaving as an electron shuttle and transferring the electrons to Cr(VI). Due to

the requirement of contact between bacterium and insoluble Fe(III) to transfer electrons,

the process was slow. This affected the overall reduction of Cr(VI). In presence of AQDS

there is a higher turnover of the electrons and faster reduction of Fe(III). This in turn

increases the rate of Cr(VI) reduction. AQDS is soluble unlike Fe(III) and it alleviates the

need for contact between the cells and the metal. It accepts two electrons unlike Fe(III)

that can accept only one electron at a time. Therefore higher reduction of Fe(III) occurs in

the presence of AQDS which in turn augments the rate of Cr(VI) reduction. The higher

rate of Cr(VI) reduction corresponds well to the high rate of glucose consumption in the









presence of AQDS. In the absence of Cr(VI) the rate of glucose consumption was much

higher suggesting that Cr(VI) effects the metabolic machinery of the cells.

The initial color of the medium was yellow but with the reduction of Cr(VI) it

became colorless. Insoluble amorphous Fe(III) was red in color that reduced to white

precipitate that had disc shaped crystals when seen under a SEM microscope(Figure3-6).

The X-ray diffraction analysis indicated this precipitate to be vivianite

Effect of Electron Donor on Cr(VI) Reduction

Concentration of electron donor in the medium affected the amount of Cr(VI)

reduced (Table 3-2). However the rate of reduction of Cr(VI) was not depedant on the

concentration of the electron donor. These results have implications in the field of

bioremediation. Limitation of electron donors in the environment can impede the

microbial reduction of Cr(VI) by the fermentative organisms.

Effect of Cr(VI) Reduction on Cell Growth in Consortium GCAF

The final cell concentration was lower by 47% in the medium in the presence of

Cr(VI). Although presence of AQDS in the medium with Fe(III) increased the rate of

Cr(VI) reduction, there was no significant difference in the cell numbers observed when

compared with those in the absence of AQDS (Table 3-2).

Effect of Cr(VI) on Metabolites

Since consortium GCAF comprised mainly of fermentative bacteria effect of

Cr(VI) on the fermentative products was determined. The variability in the rate of

glucose oxidation during Cr(VI) reduction in presence of different electron acceptors

suggested varied rates of product formation. Furthermore, reduction of external electron

acceptors by the fermentative bacteria suggested some change in the fermentative

metabolic products. Therefore the production of fermentation products of the consortium









were monitored with time. The results observed showed that formation of products

corresponded well with the time when oxidation of glucose started and the production

carried on till glucose was consumed (Figure 3-3, 3-4, 3-5).

The major fermentation products that were generated by the consortium GCAF

when grown in presence of glucose were acetate, butyrate and lactate. No change in the

proportions of products was observed when Fe(III) and AQDS was added to the medium.

However, there was a shift in fermentation product pattern observed when Cr(VI) was

added to the medium containing Fe(III) and AQDS as other external electron acceptors.

Oxidation of glucose by the cells in consortium resulted in significantly less amount of

butyrate, and more amount of acetate in the presence than in the absence of Cr(VI) (Table

3-3). These results suggested that the reduction of Cr(VI) effects the cells to shift the

fermentation products to more oxidative forms. During the glucose fermentation by the

fermentative cells, glucose is oxidized to pyruvate and generates two molecules of

NADH (four reducing equivalents). The oxidative decarboxylation of pyruvate generates

one molecule of format and one molecule of acetyl CoA. Acetyl CoA has two

alternative fates: it either forms acetate with a generation of ATP or can sacrifice the

generation of energy by forming a more reduced form ethanol. The other possibilities for

disposing reducing equivalents is by the formation of lactate. Lactate results in the

reoxidation of one NADH. Butyrate formation is at the expense of 4 reducing equivalents

(or 2 NADH). In the presence of Cr(VI) the drop in butyrate and lactate formation

indicated that less of NADH were being reoxidized by those pathways. Increase in

acetate could perhaps be the result of excess pyruvate being converted to acetate and

format. The energy generation step that is liked to acetate would prove beneficial to the









cell as it has to survive in the toxic environment of Cr(VI) and reduce it. The excess

NADH that were not reoxidized by lactate and butyrate were then channeled towards the

reduction of Cr(VI).Whether reduction of Cr(VI) occurs inside the cell membrane or on

the cell surface was not clear at this point. No difference in the pattern of the products

formed in the medium in the presence of Fe(III) and AQDS and in their absence indicated

that mechanism for reduction of Fe(III) and AQDS was different from the way Cr(VI)

was reduced by the cells in consortium GCAF.

So far there is no evidence that energy conservation by fermenting bacteria during

reduction of Cr(VI). Although, reduction of Cr(VI) results in increased formation of

acetate which is energetically favorable for fermenting bacteria.

Mechanism of Cr(VI) reduction by a pure strain isolated from this consortium

GCAF has been explained in chapter 5. Identification and characterization studies of the

isolate have been conducted in order to explain the phylogenetic importance of the strain.

GCAF may be a good candidate for the bioremediation of heavy metal laden waters and

sediments.













...-A----A... .. .... A --A ............. A
Q- 9:-:*:R:-- 2::--. :; :. ..-.-.'... 6


... ---- GC
-- NA
-. A---- NC
A. A... NG
-M- S


0 50 100
Time (hr


150 200 250


Figure 3-1. Cr(VI) reduction and removal from the solution as an insoluble precipitate.
Glucose (10mM) was supplied as an electron donor. Fe(III) and AQDS were
supplied as extra electron acceptors. Reduced insoluble Cr(VI) was removed
from the solution by centrifugation prior to analysis. GC: glucose, Cr(VI);
NA. Glucose, Cr(VI), Fe(III); NC. Glucose, Cr(VI), Fe(III), AQDS with no
inoculation of cells; NG: Cr(VI), Fe(III), AQDS; S: glucose, Cr(VI), Fe(III)
and AQDS.


: .::'. ::.............. 0
.-. A


---.... ...---- GC
- E NA
--- -. -- NC
-- --- NCr
------ NEA
-U-- S


0 50 100 150 200 250


Time (hrs)


Figure 3-2. Glucose consumption by consortium GCAF-1 during reduction of Cr(VI)
(0.4mM) in the presence of Fe(III) (5mM) and AQDS.(0.1 mM). GC:
glucose, Cr(VI); NA. Glucose, Cr(VI), Fe(III); NC. Glucose, Cr(VI), Fe(III),
AQDS with no inoculation of cells; NCr; glucose, Fe(III) AQDS; NEA:
glucose; S: glucose, Cr(VI), Fe(III) and AQDS.







51




S--B-- NA
4.5
4 NCr
3.5 NEA
E 3 -i-
S2.5
2
S1.5 -
1 -
0.5
01 0
0 i i*^--------------
0 50 100 150 200 250
time (hrs)







5-
I--5 0 -NA
4.5 Mil
4 -
3.5 A NEA
E 3 A NCr
a 2.5 -
S2-
S1.5 D A
1 -
0.5 -

0 2 4 6 8 10 12
Glucose (mM)



Figure 3-3. Acetate produced by oxidation of glucose by consortium GCAF.during the
reduction of Cr(VI) in presence of added electron acceptors. (A) Acetate
produced with respect to time. All treatments were set up in triplicates with
eeror bars representing the standard error. (B) Acetate produced per mole of
glucose consumed.NA. Glucose, Cr(VI), Fe(III); AQDS with no inoculation
of cells; NCr; glucose, Fe(III) AQDS; NEA: glucose; S: glucose, Cr(VI),
Fe(III) and AQDS.







52








7 NEA
E 6 ---S
5 3a
4
3
2-
1 1

0 50 100 150 200 250
time (hrs)





10
9 A NA
9

7 A NEA
E 6 ANCr
W 5
m

2 3
0O AO

1
0 II m..
0 2 4 6 8 10 12
Glucose (mM)




Figure 3-4. Butyrate produced by oxidation of glucose by consortium GCAF during the
reduction of Cr(VI) in presence of added electron acceptors. (A) Butyrate
produced with respect to time. All treatments were set up in triplicates with
eeror bars representing the standard error. (B) Butyrate produced per mole of
glucose consumed.NA. Glucose, Cr(VI), Fe(III); AQDS with no inoculation
of cells; NCr; glucose, Fe(III) AQDS; NEA: glucose; S: glucose, Cr(VI),
Fe(III) and AQDS.







53





2.5
-B- NA
2 -- NCr
S-- NEA
E 1.5 -- S

1

0.5



0 50 100 150 200 250
time (hrs)



2.25
2 A DNA
1.75 A A S
1.5 A & A NEA
1.5
E 1.25 A NC
A 1A M A6 A
S0.75

0.25 -


0 2 4 6 8 10 12
Glucose (mM)

Figure 3-5. Lactate produced by oxidation of glucose by consortium GCAF during
reduction of Cr(VI) in presence of added electron acceptors. (A) Lactate
produced with respect to time. All treatments were set up in triplicates with
eeror bars representing the standard error. (B) Lactate produced per mole of
glucose consumed. NA. Glucose, Cr(VI), Fe(III); AQDS with no inoculation
of cells; NCr; glucose, Fe(III) AQDS; NEA: glucose; S: glucose, Cr(VI),
Fe(III) and AQDS.

















































Figure 3-6. SEMs showing the insoluble precipitates formed by consortium GCAF-1
during the reduction of Cr(VI) via Fe(II)- and AQDS-mediated mechanisms










IMAGE. 255


I -


BSE. 255 I


I --, -


Figure 3-7. EDX of precipitate formed by consortium GCAF-1 showing the distribution
of Cr in the precipitate formed during Cr(VI) reduction.









Table 3-1. Cell growth in the presence of different electron acceptors. Cells were grown
in Basal Media supplemented with glucose as electron donor and Fe(III) as
external electron acceptor. Cr(VI) and AQDS were added as described in the
table. Cell counts were estimated by Acridine Orange Direct Count method
using a fluorescence microscope. All treatments were set up in triplicates.

Cell Reduction in
Treatment Fe(III) AQDS Cr(VI) numbers/ ml Cell number
( x 10) (%)

I + + 80.6 4.6a



II + + + 50.3+ 5.3 b 37.61


III + + 43.4 1.6b 47.18


Table 3-2. Cr(VI) reduction by consortium GCAF
of electron donor.


Glucose
utilized
(mM)


in presence of varying concentrations


Rate of Cr(VI)
reduction
(mM/day)


Amount of
Cr(VI) reduced
(mM)


5.73 5.62 0.066 0.19

10.92 9.05 0.066 0.34

17.96 8.42 0.066 0.37


Glucose
Initial
(mM)






57


Table 3-3. Effect of Cr(VI) on the on the pattern of products of glucose fermentation by
consortium GCAF.

Concentration
of substrate Products formed (mM)
AQDS Fe(III) Cr(VI) consumed
consumed
(mM) Acetate Butyrate Lactate
A 10 1.7 7.9 1.9

B + + 10 1.9 8.4 1.1

C + + + 10 4.1 5.5 0.9














CHAPTER 4
IDENTIFICATION AND CHARACTERIZATION OF THE CHROMIUM REDUCING
ISOLATE CLOSTRIDIUM SP. GCAF1

Introduction

In the last few decades, microbial metal reduction has been identified as an

important process for mineralization of organic compounds (92) and for detoxification

and remediation of soils contaminated with toxic metals (6, 88, 89). Microbial Cr(VI)

reduction was first demonstrated by Romanenko (129), following which a wide diversity

of CRB have been isolated. Cr(VI) reduction by organisms belonging to the genera

Bacillus, Escherichia, Pseudomonas, and Pantoea, among others.. This dissertation

describes the involvement of Clostridium and Cellulomonas species (Chapter 2) in

Cr(VI) reduction. The presence of fermentative bacteria in Cr(VI)-contaminated soils and

sediments have been reported previously, no detailed studies on the direct involvement of

these bacteria in Cr(VI) reduction have been documented to date.

The genus Clostridium forms one of the largest gram-positive taxa and has

significance in several fields. Many toxin producing pathogens (Clostridium perfringens,

C. botulinum, C. tetani and C. difficile) and industrially important solvent producing

fermentors (C. acetobutylicum, C. butyricum, C. aceticum) belong to this group (69, 145,

146, 172).

This study reports the discovery of a new spore forming bacterium, Clostridium

sp. GCAF-1. This strain has an ability to reduce Cr(VI) directly and indirectly via Fe(III-

reduction). Detailed results of extensive analysis of 16S rRNA gene sequences, DNA-









DNA homology and G+C content analyses are described here. Additional studies

supporting the designation of GCAF-1 as a novel species of genus Clostridium are also

reported.

Material and Methods

Source of sample and organism. Soil used as inocula for the enrichments

originated from the Cr(VI) contaminated wetlands in Michigan. Tranfers were made

several times into the fresh media to dissolve the soil particles out. transferred to Strain

GCAF-1 was isolated from Cr(VI)-reducing anaerobic enrichment that was provided with

glucose as electron donor and Fe(III) and AQDS as additional electron acceptors. The

enrichments were amended with Fe(III) and AQDS as additional electron acceptors.

Media and growth conditions. Standard anaerobic culture techniques were used

during the preparation of the medium (11). A bicarbonate buffered mineral medium, as

described in chapter 2, was used for the growth of the strain GCAF-1. The final pH was

adjusted with HC1 ca 7.0 and N2/CO2 was bubbled through it to remove oxygen. The

medium was dispensed into anaerobic pressure tubes or serum bottles under N2/CO2,

sealed with thick butyl rubber stoppers and then sterilized by autoclaving. Electron donor,

glucose (10mM) and appropriate electron acceptor [Cr(VI) (400 M); Fe(OH)3 (5mM);

AQDS (100M)] was added later from sterile anaerobic stocks. All incubations were

done in the dark under 30C without any shaking.

Isolation of strain GCAF-1. Strain GCAF-1 was isolated from the consortium

enriched under glucose-Cr-Fe(III) and -AQDS conditions. All procedures were carried

out under the anoxic conditions in a glove box equipped with charcoal filter and with

N2/C02/H2 (v/v) as gas phase. Traces of oxygen were removed by circulating the gases

over the palladium catalyst and desiccant.









Biochemical analyses. The biochemical features of the strain GCAF-1 were

determined to establish its species status. Production of riboflavin (yellow pigment) by

GCAF-1, under anaerobic conditions, was tested by inoculating sterile milk under

anaerobic conditions and incubating at 30C for 24 hours (70). Sensitivity to antibiotic

rifampicin was tested and turbidity was monitored to determine the resistant and sensitive

nature of the strain GCAF-1(70).

Fatty acid analysis. Methyl esters of cellular fatty acids (FAME) were prepared

according Metcalfe et al (105) and were analyzed using Microbial Identification System

(MIDI, Inc., Newark, DE, USA). Fatty Acids were identified by comparison of retention

times with those of commercial standards (Sigma Co., USA).

Determination of G+C content of the DNA. The base composition of the DNA

was determined by Deutsche Sammlung von Mikrooganismen und Zellkulturen (DSMZ,

Germany). DNA was isolated by cell disruption with French pressure cell. DNA was

purified on hydroxylapatite columns (26). The DNA was further hydrolyzed with P1

nuclease and nucleotides were dephosphorlized with bovine alkaline phosphatase (104).

The resulting deoxiribonucleosides were analyzed by HPLC using column SelectaPore

90M, C18, 5 [t m (250 x 4.6mm) equipped with guard column 201gd54H (Vydac,

Hesperia, CA 92345, USA). Chromatography conditions were as follows: temperature

45C, 10 al sample, solvent used was 0.3 M (NH4) H2P04 / acetonitrile, 40:1 (v/v), pH

4.4, flow rate 1.3 ml / min (adapted from Tamaoka and Komagata) (151). Non-

methylated lambda DNA (sigma) with GC content of 49.85 mol% (104) was taken as

standard. GC ratio calculated from the ratio of deoxyguanosine (dG) and thymidine(dT)

according to Mesbah (1989) (104).









16S rRNA gene sequencing and phylogenetic analysis..Cells from actively

growing culture of GCAF-1 were harvested by centrifuging at 3000xg. The pellet was

washed three times and resuspended with a phosphate buffer saline. Suspended culture

was lysed by boiling for fifteen minutes. The 16S rRNA gene was amplified by using the

universal bacterial primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492 R

(5'-TACGGTTACCTTGTTACGACTT- 3') (76). Purified PCR products were cloned

using TOPO TA Cloning kit (Invitrogen, Life Technologies). 14 clones forming white

colonies were chosen for further analysis. Insert DNA was amplified using M13 forward

and reverse primers to under conditions described in appendix B. The insert was digested

with Hae III and Alu I separately. Digestion reaction was set at 370C for 14 hours. The

digestion pattern of the clones was compared on a 2% agar electrophoresis gel. Based on

the results obtained, 16S rDNA amplification products of two clones were sent for

sequencing. Sequencing was done at ICBR core sequencing facility at University of

Florida with an automated sequencer.

Sequences obtained were compared with those available from public databases

using BLAST search and were aligned with type strains showing 98% or greater

similarity using GCG. Phylogenetic tree was constructed based on 1400 bp 16S rRNA

sequences using PAUP version 4.0.

DNA-DNA hybridization. These studies were performed at the DSMZ with

strains showing 98% (or higher 16S rDNA similarity) by thermal denaturation method

described by DeLey et al (ref) with Gilford System 2600 spectrophotometer equipped

with a Gilford 2527-R thermoprogrammer. DNA was isolated by chromatography on

hydoxylapatite as described by Cashion et al. (26).









Results and Discussion

Cell morphology. GCAF-1 is a spore forming obligate anaerobe. Cells of strain

GCAF-1 were rod shaped (4-7[t long and 1-1.5 [t in diameter), occurring singly or in

chains of 4-7cells (Figure 4-1). Terminal and subterminal endospores were observed

under scanning electron microscope (SEM). Spores were oval in shape (1-1.5 [t by 0.5 t )

(Figure 4-2). Spores constitute the dormant form of the bacterial cell that are produced

during the advent of starvation. The spores are resistant to adverse conditions including

high temperatures and organic solvents. Gram staining of GCAF-1 resulted in faint blue

color that was regarded as positive. Cells were motile and motility could be demonstrated

under electron microscopy when a freshly withdrawn sample was studied immediately

Negative staining of GCAF-1 revealed the presence of peritrichous flagella and pili.

Flagella represent the locomotory organelles of the cell. They are embedded in the cell

membrane and extend through the cell envelope and project as a long strand. Flagella

consist of many proteins including flagellin. Pili (synonym fimbriae) are hair like

projections of the cell that may be involved in the sexual conjugation or may allow

adhesion to the surfaces. Electron micrograph of the ultra thin section of the Cr(VI)

reducing Clostridium sp GCAF-1 showed the presence of the S-layer. S-layers are the

outer most component of the cell wall of several bacteria and archaea. They confer

stability to the cell structure and protects cell from lytic enzymes.

Biochemical characteristics. The Cr(VI) reducing strain GCAF-1 reduced higher

concentrations of Cr(VI) (400[tM) completely in the presence of AQDS and Fe(III). In

cultures with Cr(VI) as sole electron acceptor, complete reduction of Cr(VI) was

observed only when Cr(VI) was present in low concentrations such as 20 aM. Reduction

of Cr(VI) occurred simultaneously with the growth of the organism. The generation time









of this culture in the absence any electron acceptor was 2.04 Hrs. In the presence of

15[tM of Cr(VI) the time increased to 2.5 hrs. However, when the concentration of

Cr(VI) was further increased to 35[ and 50 gM the generation time also increased to 4.0

and 4.5 hrs respectively (Figure 4-3). Other substrates that were tested for the chromium

reduction but were not utilized were lactate, acetate, butyrate, format and citrate. GCAF-

1 was found to be resistant to rifampicin. It did not curdle the milk in 24 hrs and yellow

pigment, riboflavin formation was not observed. This strain seemed closer to the C.

beijerinckii than to C. acetobultylicum based on the few physiological properties tested.

Chemotaxonomic Data.

Fatty acid analysis. Cellular fatty acids profile of the isolate GCAF-1 is given in

Table 4-1. The most prevalent fatty acids were 16 and 18 carbon atoms. Hexadecanoic

acid was most abundant. Dimethylacetals (DMA) were found in the esterified

preparation. DMA's are the esterification products of plasmalogens, unique lipids found

in anaerobes (118).

DNA base composition. The G+C content of DNA from strain GCAF-1 was 30.7

mol %. The characteristics determined for strain GCAF-1 are summarized in Table 4-2

Phylogeny of Strain GCAF-1

Analyses of the 16S rRNA gene sequence. Comparative sequence searches of

EMBL and Genbank databases revealed that 16S rDNA sequence of strain GCAF-1, was

related to those of low G+C genus Clostridium of the gram-positive bacteria. Strain

GCAF-1 clusters with group I organisms of genus Clostridium. Within this subphylum

the highest sequence identity (98%) was obtained with 16S rRNA gene sequences of type

strains of, C. beijerinckii, C. saccharybutylicum, C. saccharoperbutylacetonicum, C.

butyricum, C. roseum (Table 4-3) (Figure 4-4).









Clostridium sp. GCAF-1 contains at least two 16S rRNA genes. Restriction

patterns obtained with HaeIII were identical for all DNA fragments obtained from 14

clones selected. However, two kinds of restriction patterns obtained with Alu I indicated

the presence of more than one kind of 16S rRNA gene operon. Sequencing of the selected

clones revealed the difference of 5 base pairs, one being at the site of Alu I.

The results obtained from the cloned 16S rDNA of isolate GCAF-1 suggests the

possibility of at least two types of 16S r RNA gene operons, but may be greater. A

difference of 5 bases (0.36%) can result from PCR- introduced errors (Figure 4-6).

Alternatively there is a possibility of sequence heterogeneity between 16S rRNA gene

operons as previously described in E. coli and Clostridium paradoxum (25, 107).

Presence of multiple 16S rRNA genes with heterogeneous intervening sequences has

been described in Clostridium paradoxum (127).

Such results may have an implication on the microbial ecology studies, wherein

the group of highly related environmental 16S rDNA clone sequences obtained from

many environments may represent not a group of separate, phylogenetically highly

related strain but rather the sequence heterogeneity of the 16S rDNA contained within

one strain.

DNA -DNA hybridization. As described previously, the 16S rRNA gene of 5

Clostridial species showed similarity of greater than 97% with that of GCAF-1. DNA-

DNA hybridization was carried out as with these 5 type strains of genus Clostridium

(Table 4-3). Determination of genomic similarities revealed that strain GCAF-1 had only

23% reassociation values with Clostridium beijerinckii.









Based on the general conclusion that strains with more than 97% 16S rRNA gene-

sequence similarities that do not exhibit DNA-DNA homologies of 70% of more are

accepted as being representatives of a single species (66, 168). GCAF-1 is a novel species

in the cluster I of Clostridium. The 16S rDNA sequence is being submitted tot he

Genbank.

In summary, isolate GCAF-1, obtained from a glucose-oxidizing Cr(VI) reducing

enrichment was identified as a novel species belonging to genus Clostridium. The name

Clostridium chromoreductans sp. nov is proposed. Other biochemical and

chemotaxonomic data further describes the isolate GCAF-1. The Cr(VI) reducing ability

of the organism is investigated in chapter 5 in detail. This strain can be used as a model

organism to provide an insight to the role of fermentative bacteria in metal reduction














































Figure 4-1. Scanning electron micrograph of isolate GCAF-1.A. Rod shaped cells. Scale
bar, 10tM. B. Cells occur in chains. Scale bar, 5Mm.




















































Figure 4-2. Micrograph of spores of isolate GCAF-1. A. Differential Interference image
of sporulating cells with subterminal spores. B. Scanning electron microscopy
of GCAF-1 spores. Scale bar 5Cim.















1 4








Ylg .e





. . .F

do An It
1
'l a' 'P "


Re


FlIgella


I,


)4


;.. -.-'


* .
-. ',


4- Puf
4/


r

ri

~ r
;r *b~ .rI ,

41


p '


, *"
11 -"
.


Negatively stained preparations of Cr(VI) reducing Clostridium sp. GCAF-1
showing peritrichous flagella. A. Bacterium showing peritrichous flagella. B.
Dividing cells still maintain the flagella.


Figure 4-3.


W
r


\ .*f *


S'4k






69















CM

CW


Spore
























Figure 4-4. Electron micrograph of an ultra thin section of Clostridium sp. GCAF-1
showing the S-layer. Due to slight plasmolysis, the protoplast has drawn
away from the cell wall. CM, cell membrane; CW, cell wall.

































Figure 4-5. Electron micrographs of Cr(VI) reducing Clostridium sp. GCAF-1 showing
the dividing cells containing terminal spores and glycogen inclusions in the
cells.


tj













Bootstrap
100 Clostndmm cellulose (L09177)



100
-- Clostndmm leptum (AJ305238)

100- Clostndmm cellobloparum (X71856)

100 Clostndum cellulolyticum (X71847)

Clostndmum aldnchn (X71846)

-100 Clostndum kluyven (M59092)
99
Clostndum ljungdahin (M59097)

Clostndmum histolytcum (M59094)
100
S Clostndum hmosum (M59096)
85
Clostndum proteolytcum (X73448)
100
SClostndmum roseum (Y18171)
98
69 Clostndmum beyjennckn (X68179)

GCAF1

98
8 -Clostndmm saccharobutyhcum (U16147)
56
Clostndmum saccaroperbutyacetom cum (U16122)
70
Clostndmumfavososporum (X76749)
85 95
890 -- Clostndum connoforum (X76742)

100 Clostndrumpumceum (X71857)

Clostndum butyncum (X68176)
98
Clostndmum paraputrificum (X73445)
94
Clostndmm acetobutyhcum (X78071)

Clostndmm tetam (X74770)

Lactobacillus case (D16551)

Enterobacter aerogenes (AF395913)

Figure 4-6. Phylogenetic tree based on 16S rDNA comparisons showing the relative
position of strain GCAF-1 among other representative species of genus
Clostridium. The branching pattern was generated by the neighbour joining
method and the bootstrap values, shown at the nodes were calculated from
100 replicates. Bootstrap values less than 50% are omitted from the figure.
Bar, 0.01 substitutions per nucleotide position. The gen bank acession
numbers for the 16S r RNA sequences are given after the strain names.













10




E
0 1




0
, 0.1




0.01
0.01


0 5 10 15 20


Time (Hrs)


Anaerobic growth curve of GCAF-1 in under various Cr(VI) concentrations.
o--in absence of Cr(VI); --m-- in presence of 0.015 mM Cr(VI); --A-- 0.032
mM Cr(VI);---e--0.050 mM Cr(VI); ---A-- 0.070 mM Cr(VI). aValues are
represented by the average of the duplicates. bGrowth rate was determined in
cultures kept at 300C in dark without shaking.


Figure 4-7.










Figure 4-8. Comparison of two 16S rRNA gene sequences from Clostridium sp. GCAF-1.
Two sequences are represented by CLS-1 and CLS-2. o highlight the different
bases.








74



Identities = 1378/1383 (99%)
Strand = Plus / Plus

CLS-1: 13 gttccttcgggaacggattagcggcggacgggtgaggggcacgtgggtaacctgcctcat 72

CLS-2: 1 gttccttcgggaacggattagcggcggacgggtgagtaacacgtgggtaacctgcctcat 60


CLS-1: 73 agaggggaatagcctttcgaaaggaagattaataccgcataagattgtagttt gcatga 132

CLS-2: 61 agaggggaatagcctttcgaaaggaagattaataccgcataagattgtagttt gcatga 120


CLS-1: 133 aacagcaattaaaggagtaatccgctatgagatggacccgcgtcgcattagctagttggt 192

CLS-2: 121 aacagcaattaaaggagtaatccgctatgagatggacccgcgtcgcattagctagttggt 180


CLS-1: 193 gaggtaacggctcaccaaggcgacgatgcgtagccgacctgagagggtgatcggccacat 252

CLS-2: 181 gaggtaacggctcaccaaggcgacgatgcgtagccgacctgagagggtgatcggccacat 240


CLS-1: 253 tgggactgagacacggcccagactcctacgggaggcagcagtggggaatattgcacaatg 312

CLS-2: 241 tgggactgagacacggcccagactcctacgggaggcagcagtggggaatattgcacaatg 300


CLS-1: 313 ggggaaaccctgatgcagcaacgccgcgtgagtgatgacggtcttcggattgtaaaactc 372

CLS-2: 301 ggggaaaccctgatgcagcaacgccgcgtgagtgatgacggtcttcggattgtaaaactc 360


CLS-1: 373 tgtctttggggacgataatgacggtacccaaggaggaagccacggctaactacgtgccag 432

CLS-2: 361 tgtctttggggacgataatgacggtacccaaggaggaagccacggctaactacgtgccag 420


CLS-1: 433 cagccgcggtaatacgtaggtagcaagcgttgtccggatttactgggcgtaaagggagcg 492
ii111111111111 II I111iiiiiiiiii ii ii IIIiiiiii 1 1 III
CLS-2: 421 cagccgcggtaatacgtaggtggcaagcgttgtccggatttactgggcgtaaagggagcg 480


CLS-1: 493 taggtggatatttaagtgggatgtgaaatactcgggcttaacctgagtgctgcattccaa 552

CLS-2: 481 taggtggatatttaagtgggatgtgaaatactcgggcttaacctgagtgctgcattccaa 540


CLS-1: 553 actggatatctagagtgcaggagaggaaagtagaattcctagtgtagcggtgaaatgcgt 612

CLS-2: 541 actggatatctagagtgcaggagaggaaagtagaattcctagtgtagcggtgaaatgcgt 600

CLS-1: 613 agagattaggaagaataccagtggcgaaggcgactttctggactgtaactgacactgagg 672

CLS-2: 601 agagattaggaagaataccagtggcgaaggcgactttctggactgtaactgacactgagg 660


CLS-1: 673 ctcgaaagcgtggggagcaaacaggattagataccctggtagtccacgccgtaaacgatg 732

CLS-2: 661 ctcgaaagcgtggggagcaaacaggattagataccctggtagtccacgccgtaaacgatg 720








75


CLS-1: 733 aatactaggtgtaggggttgtcatgacctctgtgccgccgctaacgcattaagtattccg 792

CLS-2: 721 aatactaggtgtaggggttgtcatgacctctgtgccgccgctaacgcattaagtattccg 780


CLS-1: 793 cctggggagtacggtcgcaagattaaaactcaaaggaattgacgggggcccgcacaagca 852

CLS-2: 781 cctggggagtacggtcgcaagattaaaactcaaaggaattgacgggggcccgcacaagca 840


CLS-1: 853 gcggagcatgtggtttaattcgaagcaacgcgaagaaccttacctagacttgacatctcc 912

CLS-2: 841 gcggagcatgtggtttaattcgaagcaacgcgaagaaccttacctagacttgacatctcc 900


CLS-1: 913 tgaattacccttaatcggggaagcccttcggggcaggaagacaggtggtgcatggttgtc 972

CLS-2: 901 tgaattacccttaatcggggaagcccttcggggcaggaagacaggtggtgcatggttgtc 960


CLS-1: 973 gtcagctcgtgtcgtgagatgttgggttaagtcccgcaacgagcgcaacccttattgtta 1032

CLS-2: 961 gtcagctcgtgtcgtgagatgttgggttaagtcccgcaacgagcgcaacccttattgtta 1020


CLS-1: 1033 gtFgctaccatttagttgagcactctagcgagactgcccgggttaaccgggaggaaggtg 1092
II IIIIIIii IIII iiii i 1i1i i1111111 1 i i 111 111 i i1
CLS-2: 1021 gtcgctaccatttagttgagcactctagcgagactgcccgggttaaccgggaggaaggtg 1080


CLS-1: 1093 gggatgacgtcaaatcatcatgccccttatgtctagggctacacacgtgctacaatggct 1152
II1 1ii iiiiII iii 11 11 11 11 III 1 IIIIIIIIIIIIIII
CLS-2: 1081 gggatgacgtcaaatcatcatgccccttatgtctagggc acacacgtgctacaatggct 1140


CLS-1: 1153 ggtacagagagatgctaaaccgcgaggtggagccaaacttcaaaaccagtctcagttcgg 1212

CLS-2: 1141 ggtacagagagatgctaaaccgcgaggtggagccaaacttcaaaaccagtctcagttcgg 1200


CLS-1: 1213 attgtaggctgaaactcgcctacatgaagctggagttgctagtaatcgcgaatcagaatg 1272

CLS-2: 1201 attgtaggctgaaactcgcctacatgaagctggagttgctagtaatcgcgaatcagaatg 1260


CLS-1: 1273 tcgcggtgaatacgttcccgggccttgtacacaccgcccgtcacaccatgagagttggca 1332

CLS-2: 1261 tcgcggtgaatacgttcccgggccttgtacacaccgcccgtcacaccatgagagttggca 1320


CLS-1: 1333 atacccaaagttcgtgagctaaccg aaggaggcagcgacctaaggtagggtcagcgatt 1392
I I I I I I I I I I I I I I I I I I I I I I I I I Ii i i i i i i i i i l i l l I
CLS-2: 1321 atacccaaagttcgtgagctaaccgraaggaggcagcgacctaaggtagggtcagcgatt 1380


CLS-1: 1393 ggg 1395
CLS-2:1381 ggg 1383
CLS-2: 1381 ggg 1383









Table 4-1. Cellular fatty acid composition of GCAF-1 grown with 10mM glucose.

Component Fatty acids in profile (area%)
14:0 FAME 5.44
16:0 ALDE 1.14
16:1 CIS 7 FAME 4.25
16:1 CIS 9 FAME 6.73
16:0 FAME 29.46
16:1 CIS 9 DMA 2.37
16:0 DMA 5.44
18:1 CIS 9 FAME 22.58
18:1 CIS 13 FAME 0.57
18:0 FAME 2.97
18:1 CIS 9 DMA 3.42
18:1 CIS 11 DMA 1.73
20:1 CIS 11 FAME 0.52
ECL Unknown 14:762 (15:2 FAME, 15:1 CIS 7) 1.61
15:0 ANTEI 3 OH FAME, 16:1 CIS 7 DMA 2.08
ECL Unknown 16:760 (17:2 FAME, 17:1 CIS 8 FAME) 0.96
ECL Unknown 16:801 (17:1 CIS 9 FAME, 17:2 FAME) 0.56
ECL Unknown 17:834 (18:1 cl 1/t9/t6/FAME) 5.84
ECL Unknown 18:622 (19:0 ISO FAME) 0.63
The cellular fatty acid compositions were analyzed by Microbial ID, Inc.
In fatty acid designation the first and second numbers indicate the number of carbon
atoms, and the number of double bonds, respectively.
Peaks lower than 0.5% in area are not represented.
aFAME, fatty acid methyl ester;
bDMA, dimethyl acetyl;
'ISO, iso;
dCIS, cis form double bond;
ec, cis form;
fECL, Equivalent chain length;
gALDE, aldehyde;
hANTEI, anteiso;
'xOH indicates the position of hydoxylation;
it, trans from double bond.









Table 4-2. Characteristics of GCAF-1

Organism GCAF-1
Genus Clostridium cluster I
G+C Mol content 30:7%
Carbon sources not utilized Acetate, Lactate, butyrate, Lactate
Resistance to Rifampicin +
Riboflavin formation in milk
Facultative growth
Spore forming +
Spore size 1-1.5 g by 0.5g
Length of rods 4.0-7.0 g y 1.5[
MIC of Cr(VI) > 0.1 mM
Major fermentation products Acetate, butyrate, lactatea
Motility motile
a Fermentation products as determined by HPLC equipped by UV /vis detector:









Table 4-3. Sequence similarity between 16S rRNA gene of isolate GCAF-1 and type
strains of the genus Clostridium showing closest similarity.


Species


C. acetobutylicum



C. butyricum


C. beijerinckii


C. paraputrificum


C. puniceum


C. roseum


C. saccharobutylicum


C. sacchaoperbutylicum


Strain


aATCC 824,
bDSM 792,
CNCIB, 8052
ATCC 19398
DSM 10702,
DSM 552
ATCC 25752,
DSM, 791,
NCIB 9362
ATCC 25780,
DSM 2630,
NCIB 10671
DSM 2619,
NCIB 11596
ATCC 17797,
DSM 2619,
NCIB 11596
ATCC BAA-
117, DSM 13864

ATCC 27021,
DSM 14923


Sequence
similarity of
16S rDNA to
isolate (%)


DNA DNA
hybridization
with the
isolate (%)


ND


23.5


ND


32.9


33.6


eND


36.3


Similarity higher than 97% is indicative of sequences belonging to same genus.
aATCC American Type Culture Collection, Manassas, VA, USA
bDSM: Cultures from Deutsche Sammlung von Mikroorganismen und Zelkulturen,
Braunschweig, Germany
cNCIMB: National Collections of Industrial, Food and Marine Bacteria, Abeerdeen, UK.
dND: Not determined
eND: Not determined. Biochemical test conducted.


Reference


Mc Coy
Emend
Keis
Prazmows
ki
Donker
Emend
Keis
Bienstock
snyder


Lund


Mc Coy
Cato


Keis


keis














CHAPTER 5
ELECTRON SHUTTLE-MEDIATED CHROMIUM REDUCTION BY CLOSTRIDIUM
sp GCAF-1

Introduction

Strictly anaerobic fermentative organisms, although ubiquitous in the

environment, have not been studied extensively in terms of heavy metal remediation.

Direct metal reduction by fermentative organisms is not generally considered to be

significant, however, there are a few reports documenting the ability of these obligate

anaerobes to reduce metals such as iron and selenium (39, 118). The currently accepted

hypothesis regarding the role of fermentative bacteria in metal reduction is that complete

oxidation of fermentable compounds to carbon dioxide is coupled to Fe(III) reduction by

the cooperative activity of fermentative and Fe(III) respiring bacteria (92) that utilize the

fermentative products as electron donors. This does not consider that fermentative

organisms may play a significant role in direct reduction of metals. Thereby it disregards

any ecologically significant role of fermentative bacteria in direct reduction of metals.

Many bacterial strains have been shown to mediate reduction of Cr(VI) to Cr(III)

both aerobically (22, 59, 75) and anaerobically (28, 93, 152, 163). Most of these

organisms belong to the gram-negative group of bacteria. Cr(VI) reducing gram-positive

bacteria reported to date, belong to the genera Bacillus (122), Staphylococcus (134) and

Micrococcus (9). Although studies based on the preliminary screening of organisms

isolated from Cr(VI) contaminated soil have reported the presence of gram-positive









fermentative bacteria in metal contaminated sites, there is no report documenting the

active participation of fermentative organisms in Cr(VI) reduction.

Results from our enrichment studies (Chapter 2) show that the most rapid rate of

Cr(VI) reduction was observed in enrichments amended with glucose as electron donor.

Even though acetate is considered to be one of the most preferred electron donors for

most metal reducing bacteria, our data indicated efficient chromium reducers to be

among glucose utilizers. Isolation and identification studies revealed that the predominant

enriched microorganisms belonged to the genus Clostridium. Cr(VI) reducing isolate

GCAF-1 was obtained from this enriched culture. In order to understand the mechanisms

adopted by GCAF-1 to reduce Cr(VI), detailed kinetic studies were undertaken. In doing

so, direct and Fe-dependant indirect pathways for Cr(VI) reduction were identified.

These results suggest that the selective advantage of strain GCAF-1 in Cr(VI)

contaminated environments, due to its ability to grow in the presence of typically toxic

concentrations of Cr(VI) and to reduce high concentrations of Cr(VI) in presence of

Fe(III) and humics, is of potential environmental relevance. This study not only adds to

the growing list of organisms involved in the Cr(VI) reduction, but also suggests the

significant role played by fermentative organisms in the reduction of Cr(VI).

Materials and Methods

Source of organism and isolation. Strain GCAF-1 was isolated from anaerobic

enrichment cultures that were initiated with the soil sample from a Cr(VI) contaminated

wetland in Michigan. Enrichment cultures were supplemented with glucose as electron

donor and Cr(VI), AQDS and Fe(III) as electron acceptors. Strain GCAF-1 was isolated

by using standard anaerobic technique of roll tube.









Cultivation of strain GCAF-1. Strict anaerobic techniques were used throughout

the course of study as described previously (87). All incubations were in dark at 30C

unless specified. Medium was prepared in serum bottles and bubbled with (N2/ CO2 :: 80:

20) to remove the dissoled oxygen. These bottles were then capped with blue butyl

rubber stoppers and aluminum crimp seals under (N2/ CO2 :: 80: 20). Media was

sterilized by autoclaving it for 30 minutes. Appropriate electron donor and acceptors

were added from sterile stock solutions. For routine maintenance of cultures, 10 ml of

medium were dispensed into anaerobic pressure tubes (Bellco glass, Inc., Vineland, N.J)

and sparged with appropriate gas mixture for 10 minutes before sterilizing the medium by

autoclaving.

Cell growth and kinetics study. Growth of cells was monitored during the

kinetic study by cell counts with acridine orange direct count. For the kinetics study, 48

ml of basal medium was dispensed into 120 ml serum bottles and sparged for 30 minutes

with appropriate gas mixture to remove traces of oxygen from the medium. Medium was

sterilized by autoclaving it for 30 minutes. Glucose was added as electron donor for all

treatments except in controls that were set up with no electron donor. To determine the

effect of Fe(III) and AQDS on Cr(VI) reduction, several treatments were set up with

different combinations of electron acceptors: (i) Cr(VI) with Fe(III)and AQDS (ii) Cr(VI)

with Fe(III), (iii) Cr(VI) with AQDS, and (iv) Cr(VI). In order to account for the effect of

Fe(III) and AQDS on the metabolism of strain GCAF-1 another treatment with Fe(III)

and AQDS in the absence of Cr(VI) was set up as a control. Fe(OH)3 was synthesized by

titrating a solution of FeCl3.6H20 with 10% NaOH to pH of 9.0. Cr(VI) was provided in

the form of K2Cr207. Anthraquinone di-sulfonate (AQDS), humic acid analog (Sigma)









was used to mimic the effect of humic acids on Cr(VI) reduction. Appropriate addition of

electron donor and acceptors were made to the bottles to get a final concentration of

glucose (10 mM), Cr(VI) (0.4 mM), Fe(III) (5 mM), and AQDS (0.1mM) in the media.

Samples were taken from serum bottles with a syringe.

Analytical techniques. Cell enumeration was done by staining the cells with

acridine orange and counting the cells under epifluorescence microscope as previously

described (87). 0.1 ml of cell culture was taken from the serum bottle and fixed with 0.1

ml of 25% gluteraldehyde for 10 minutes. 0.8ml of PBS was added to make up the

volume to 1 ml. Samples were diluted in oxalate solution in order to dissolve the

particulate forms of iron. Sample was passed through 0.2 [m millipore filter and stained

with the acridine orange (5 drops of 0.1 M). After washing off the excess stain with

filtersterlized distilled water, the filter was placed on the glass slide and viewed under the

microscope. Cr(VI) concentrations were measured by colorimetric methods using UV

spectrophotometer as described in chapter 2 (159). Glucose concentrations were

determined spectrophotometrically at 450nm. Fermentation products were analyzed with

high pressure liquid chromatography (HPLC, Waters) equipped with an Aminex HPX-

87H column (7.8 by 300 mm column) (Biorad) and UV detector (Waters) at 210 nm.

Sulfuric acid (5mM) was used as the eluent buffer. Flow rate was maintained at 0.6 ml
-1
min

Results and Discussion

Strain GCAF-1 was isolated from a Cr(VI) reducing enrichment culture that was

supplemented with glucose as electron donor and Fe(III) and AQDS as additional

electron acceptors. Earlier studies with Cr(VI) reduction have reported Fe(II) to be one of

its ecologically significant reductant in soils (13, 24, 54, 138). Also the ability of









Clostridium beijerinckii and C. butyricum (118) to reduce Fe(III) to Fe(II) made Fe(III) a

potential candidate to study the effect of Fe(III) on Cr(VI) reduction.

The Biphasic Mechanisms for Cr(VI) Reduction by Clostridium sp GCAF-1.

Strain GCAF1 was capable of direct and Fe-dependant indirect reduction of

Cr(VI). It reduced 400 tiM of Cr(VI) in the presence of Fe(III) and AQDS. In the absence

of these additional electron acceptors strain GCAF-1 was able to reduce up to 100M of

Cr(VI). Cell growth in GCAF-1 cultures supplemented with Cr(VI) as sole electron

acceptors, with concentrations higher than 100M, was negligible. Toxicity of the heavy

metal at higher concentrations may have prevented the cell proliferation. Growth rate of

the cells of strain GCAF-1 decreased as the initial Cr(VI) concentration in the medium

increased (Figure4-?). The fermentative growth rate of GCAF-1 was similar to that when

16tM of Cr(VI) was present in the medium (Figure 5-1). However, slight increase in lag

phase is observed indicating that cells need to get acclimatized to Cr(VI) before they

enter the logarithmic growth phase. Cr(VI) was completely reduced during growth. No

growth was observed in medium with Cr(VI) concentration of 200[iM. GCAF-1 was a

fermentative spore-forming organism and formation of spores was observed in the

fermentative culture in 24 hours. In presence of Cr(VI) delayed sporulation was observed

and cells exhibited altered morphology. The cells appeared longer and much thinner than

those grown in absence of Cr(VI) suggesting the effect of Cr(VI) on cell division of the

cells as documented by Theodotou et al. (153). Negligible Cr(VI) reduction was noted in

the absence of electron donor or in the absence of cells. These results indicated that

reduction of Cr(VI) was biotic and was carried out by metabolically active cells.









In the presence of Fe(III) and AQDS, strain GCAF-1 was able to reduce higher

concentrations of Cr(VI). An interesting observation was that reduction of Cr(VI) in

medium containing both AQDS and Fe(III) (GCAF medium) was faster than the Cr(VI)

reduction in the medium containing only Fe(III) (GCF medium). These results suggested

that Fe(III) acted as an electron shuttle for the reduction of Cr(VI). In the presence of

Fe(III), AQDS also formed a part of the train of shuttles transferring electrons to Cr(VI).

Besides acting as electron shuttle, Fe(III) also played an important role in alleviating the

Cr(VI) toxicity to the cells of GCAF-1. This was evident by the absence of cell growth

and negligible reduction of Cr(VI) in culture medium supplemented with AQDS only. It

must be noted that since strain GCAF-1 is fermentative it is capable of growing in the

absence of electron acceptor and the therefore, the absence of Fe(III) should not have

been the limiting factor for its growth. Toxicity of Cr(VI) concentration used in this study

however, has been established earlier. Reduction of Cr(VI) corresponded well to the

oxidation of glucose by strain GCAF-1 (Figure 5-2). There was negligible change in the

glucose concentrations in the absence of cells. No oxidation of glucose was observed in

cultures that showed no reduction of Cr(VI). Glucose oxidation by strain GCAF-1 was

observed during its fermentative growth in the absence of any external electron acceptor.

When the rate of glucose oxidation in GCF and GCAF media was compared there was no

significant difference observed. The cell numbers in GCAF cultures were slightly higher

when compared with those in GCF cultures (Figure 5-3). Cell growth in both the cultures

ceased with the complete oxidation of glucose. However, complete reduction of Cr(VI)

was only observed in GCAF medium. Incomplete and slower rate of Cr(VI) reduction

was noted in the GCF medium. This indicated that electrons generated during glucose









oxidation by GCAF-1 are channeled more efficiently towards Cr(VI) reduction in the

presence AQDS and Fe(II). This can be explained as follows. Reduction of Cr(VI) in the

presence of Fe(III) depended on the reduction of Fe(III) by the cells. Fe(III) in this case

acted as an electron shuttle. Under the prevailing pH conditions Fe(III) oxide is insoluble

and the microbial reduction of Fe(III) requires the cells to be in contact with the metal

oxide as described previously (ref). Presence of AQDS in the medium stimulates the

reduction of Fe(III) by alleviating the need for the contact by the cells. AQDS is soluble

and is capable of shuttling electrons from the cells to Fe(III) oxide. Reduced AQDS is

oxidized once the electrons are transferred to metal oxide and it is ready to accept

electrons again from the cells. Fe(II) generated directly or indirectly by microbial activity

then reduces Cr(VI). Therefore the rate of Cr(VI) reduction was dependant on the

reduction of Fe(III). Cr(VI) reduction by strain GCAF-1 was possible by four different

pathways: (i) by Fe(II) that is directly generated by the cells, (ii) by Fe(II) that is reduced

by reduced AQDS (iii) by reduced form of AQDS (iv) directly by the cells.

Effect of Cr(VI) on Production of Metabolic Products of Strain GCAF-1

In order to determine the effect of Cr(VI) on the metabolism of strain GCAF-1,

the metabolites in different media were compared. Formation of metabolic products

(Figure 5-4, 5-5, 5-6) corresponded well with the oxidation of glucose (Figure 5-2) and

the reduction of Cr(VI) (Figure 5-1). Metabolites were detected in the spent growth

medium of cells. As expected, absence of fermentation products was observed in media

that did not show any oxidation of glucose. Oxidation of glucose by strain GCAF-1 in the

absence of any external electron acceptor yielded acetate, butyrate and lactate as the three

major fermentative products (Table 5-1). In the absence of Cr(VI), no change in the

fermentation products was observed in growth media supplemented with Fe(III) and









AQDS. However, the effect of Cr(VI) on the metabolic products was apparent by the

change in concentrations of acetate, butyrate, and lactate formed per 10 mM of glucose in

the presence of Cr(VI) in the medium (Table 5-1). There was an increase in the acetate

concentrations noted along with a decrease in the butyrate concentrations (Figure 5-4, 5-

5) in GCAF in GCF cultures. This indicated the possibility that (i) Cr(VI) was directly

reduced by strain GCAF-1, and (ii) Fe(III) and AQDS were reduced by strain GCAF-1 by

a pathway that was different from that by which Cr(VI) was reduced.

Proposed Mechanism of Cr(VI) Reduction by Strain GCAF-1.

Based on the results presented above a hypothesis elucidating the pathway for

Cr(VI) reduction by fermentative strain GCAF-1 is proposed. Generally, in glycolytic

pathways the oxidation of one mole of glucose by Embden-Meyerhof-Parnas pathway

yields two moles of pyruvate. This pathway also produces two NADH, plus two ATP

molecules. The pyruvate is further decarboxylated to acetyl CoA, C02, and h2 using

pyruvate-ferredoxin oxidoreductase and hydrogenase. The acetyl CoA has two fates.

Some of it is condensed to form acetoacetyl-CoA, which is reduced to P-hydroxybutyryl-

CoA using one of the two NADHs. This product is reduced to butyryl-CoA using the

second NADH. CoASH is displaced by inorganic phosphate and butyryl phosphate

donates phosphoryl group to ADP and forms ATP and butyrate. Some aetylCoA is also

converted to acetate via acetyl -P in a reaction that yields an additional ATP. Twice as

much ATP is generated per acetate produced as opposed to butyrate. Mass balance of

fermentation products formed (within 10% error) by Clostridium sp. GCAF-1 during

oxidation of 10mM of glucose in the absence of Cr(VI) showed that 20 NADH produced

by the oxidation of 10 mM of glucose, were used as follows: 15 NADH for butyrate, 1

NADH for lactate and 2 NADH used for NADH:ferredoxin oxidoreductase. In presence









of Cr(VI), 11 NADH were used for the formation of butyrate, 1 NADH was utilized for

lactate formation and 1.2 for Cr(VI) reduction. 5.2 NADH was utilized for the reduction

of Fe(III) and AQDS. In presence of Cr(VI), decrease in concentration of butyrate would

result due to the channeling of electrons from NADH towards reduction of Cr(VI) and

more ATP formation per acetyl CoA. Reduction of Cr(VI) was perhaps one of the

defense mechanism adopted by the cell to detoxify the environment for its survival.

Therefore, NADH was perhaps acting as electron source for the reduction of Cr(VI).

This speculation is further supported by several reports of microbial NADH

dependant enzymatic reduction of Cr(VI) (10, 44, 52, 117, 149). The electrons from

NADH are transferred through the electron transport chain and transported via a

cytochrome to Cr(VI). Presence of cytochromes has been reported in some members of

Clostridium sp. Decrease in formation of butyrate content was coupled with the increase

in acetate concentrations. Increase in acetate concentrations are advantageous for the cell

as it requires to make more energy to compensate for the energy expended for activating

its defense mechanisms against the toxic Cr(VI) molecules. Whether Cr(VI) is used as an

electron acceptor by the strain GCAF-1 is currently unclear. Location of Cr(VI) reduction

by the cell is also not clear

Environmental Relevance of Cr(VI) Reduction by Gram Positive Spore-forming
Fermentative Species

Presence of fermentative organisms in soil is ubiquitous. These organisms

generally form the primary group of bacteria that oxidize the complex carbon sources

into simple carbon forms that are utilized as electron donors by the known metal reducing

bacteria. In anaerobic Cr(VI)- contaminated environments like the wetlands fermentative

bacteria such as Clostridium sp GCAF-1 are active participants in reduction of Cr(VI) as