fifii’ m -s i L‘ - ..,, W». .«n _ A 3...“- _.... , . . . A .. 1 . _.._. awr—I .‘ ”-0 .«o—nw '53-‘3'935 if: M... ti‘ ;.‘;‘,3‘~! ‘ V ‘ ,. .wv unw- 1......m-n wap~ w... , . .. a itoz‘ .913}, “E“:xtf-i‘: 2 {‘19 ‘. '«i {m . : I V o 3514: 41:: .. i 1 g‘ 20!} a“ u. 1....» "v. 5“ "wow—4 ..F)V~ > .' ”’3‘ ...<. .41" W .vv - ‘wuu no... -.~4 .w m FE: mm ,. '11: W.;:."-‘~ v i . if‘Ele‘J l‘fl‘) llllllllllllllllllllllllllllllllllllllllllllllllllllllllllll 31293 01812 This is to certify that the dissertation entitled Enrichment, Isolation, and Characterization of Reductively Dechlorinating Microorganisms from Coastal Marine Sediments presented by Baolin Sun has been accepted towards fulfillment of the requirements for Ph-D- degree in Mil Sciences Major professor 9/1443 MSU is an Affirmatiw Action/Equal Opportunity Institution 0-12771 *fiw ._,.—_ p v v vrr“ v-u '— LIBRARY Michigan State University PLACE IN RETURN Box to remove this checkout from your record. To AVOID FINES return on or before date due. MAY BE RECAUiD with earlier due date if requested. DATE DUE DATE DUE DATE DUE 233?. (winks 0 1 1/98 chlHCIDuaDupfi-nu ENRICHMENT, ISOLATION, AND CHARACTERIZATION OF REDUCTIVELY DECHLORINATING MICROORGANISMS FROM COASTAL MARINE SEDIMENTS By Baofin Sun A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the Degree of DOCTOR OF PHILOSOPHY Department of Crop and Soil Sciences 1999 ABSTRACT ENRICHMENT, ISOLATION, AND CHARACTERIZATION OF REDUCTIVELY DECHLORINATING MICROORGANISMS FROM COASTAL MARINE SEDIMENTS BY Baolin Sun Most of the halogenated natural chemicals are produced in marine habitats but the existence of dechlorinating microbes in this environment has not been explored. Such organisms could be important in pollutant cleanup in harbors. Seven coastal marine sediments were tested in sediment microcosms for reductive dechlorination activity on four chloroaromatic compounds, 2- chlorophenol (2—CP), 2,4,6-trichlorophenol (2,4,6-TCP), 3-chlorobenzoate (3- CB), and 3-chlorotoluene (3-CT). 2-CP and 3-CB dechlorination activities were sustained in sediment microcosms and enrichment cultures in synthetic seawater medium, whereas 2,4,6-TCP dechlorinating activity was lost. Microbial characterization of the 53-08 dechlorinating cultures by ARDRA (amplified ribosomal DNA restriction analysis) and 168 rFlNA sequence analysis showed that the patterns of the dominant clones shifted when the cultures were more highly enriched. Two anaerobic dechlorinating microorganisms, strains SF3 from San Francisco Bay sediment and DCB-M from Gulf Breeze, FL sediment, were isolated and characterized. Strain SF3 is a gram-negative, motile, short curved rod that grows by coupling reductive dechlorination of 2-CP to acetate oxidation. Strain SF3 also used fumarate, sulfate, sulfite, thiosulfate, and nitrate as electron acceptors for growth and grew at sodium chloride concentrations ranging from freshwater to seawater. Growth by halorespiration was confirmed by the growth yield of 1 g of protein per mole of 2-CP dechlorinated. Morphology, physiology, and 16S rRNA sequence analysis indicated that this organism belongs to the Desulfovibrio group of the sulfate-reducing bacteria and represents a novel genus. Strain DCB-M is a gram-negative, nonmotile, long rod with a collar girdling the cell, and is capable of growth by reductive dechlorination of 3-CB to benzoate. Strain DCB-M grew in seawater medium but was not capable of growth in the freshwater concentration of sodium chloride. The growth yield was 1.7 g of protein per mole of 3-CB dechlorinated. Strain DCB-M's 16S rRNA sequence places it in the delta proteobacteria and close to Desulfomonile tiedjei strain DCB-i. The morphology, physiology, and 168 rFlNA sequence suggest that strain DCB-M is a marine relative of Desulfomonile tiedjei strain DOB-1. TO MY PARENTS ACKNOWLEDGMENTS I would like to express my sincere thanks to my major professor, James M. Tiedje, for his guidance, encouragement, and patience during my Ph.D study. I always feel fortunate to have had the opportunity to work in his lab. I would like to give my special gratitude to James Cole, my other research supervisor, who has helped me in so many ways. Special thanks are given to my advisory committee, Michael Klug, Eldor Paul, and Michael Thomashow for their instruction, guidance, and encouragement throughout the research work, and their valuable comments and suggestions on this dissertation. Special thanks are extended to fellow lab colleagues: Frank Loeffler, Jorge Rodrigues, Robert Sanford, John Urbance, Joyce Wildenthal, Jim Champine, Klaus Nusslein, Chris Wright, Ben Griffin, Joanne Chee-Sanford, Olga Maltseva, Sabine Rech, John Davis, Steve Nold, Hector Ayala, Sherry Seston, Mike Dollhopf, Tamara Tsoi, Jim Stoddard, Guangyao Sheng, and Beicheng Xia for their kind help, support and friendship. This study was funded by the Office of Naval Research (N00014-95-1- 0115). TABLE OF CONTENTS LIST OF TABLES ....................................................................................................... .viii LIST OF FIGURES .......................................................................................................... x CHAPTER I INTRODUCTION ..................................................................................... 1 Overview ..................................................................................... 1 Background ................................................................................. 3 Reductive dehalogenation ..................................................... 3 Why marine sediments .......................................................... 4 Reductively dehalogenating microorganisms ............................. 7 References ................................................................................ 14 CHAPTER II REDUCTIVE DECHLORINATION IN MARINE SEDIMENT MICROCOSMS AND ENRICHMENT CULTURES ................................................................... 18 Abstract .................................................................................... 18 Introduction ................................................................................ 1 9 Materials and Methods ................................................................. 21 Source of sediments ........................................................... 21 Microcosms ...................................................................... 22 Formation of the synthetic seawater medium .......................... 22 Chemical analysis ............................................................. 23 ARDRA and 168 rRNA sequencing ........................................ 24 Results ...................................................................................... 25 Dechlorinating microcosms .................................................. 25 Transferred activity and enrichment cultures ........................... 27 Molecular characterization .................................................. 29 Discussion ................................................................................ 34 References ................................................................................ 40 CHAPTER III ISOLATION AND CHARACTERIZATION OF A NOVEL DESULFOVIBRIO THAT GROWS BY COUPLING OXIDATION OF ACETATE TO REDUCTIVE DECHLORINATION OF 2-CHLOROPHENOL ............................................ 44 Abstract .................................................................................... 44 Introduction ................................................................................ 45 Materials and Methods ................................................................. 47 Medium and growth conditions ............................................. 47 Isolation of 2-CP dechlorinating microorganisms ..................... 48 Microscopy ....................................................................... 48 Characterization of reductive dehalogenation ......................... 48 Growth rate and protein yield ................................................ 50 Test of antibiotic sensitivity .................................................. 51 Chemical analysis .............................................................. 51 Rep-PCR fingerprinting ....................................................... 51 168 rRNA gene sequencing and analysis .............................. 52 Results ...................................................................................... 53 Isolation of strain SF3 ......................................................... 53 Effect of salt concentrations on reductive dechlorination ............ 58 Electron donors and acceptors ............................................. 58 Induction of dechlorination reaction ....................................... 64 Protein yield coupled to reductive dechlorination ..................... 68 Antibiotic sensitivity ............................................................ 68 Evidence of culture purity .................................................... 68 Phylogeny of strain SF3 ...................................................... 68 Discussion ................................................................................ 71 References ................................................................................ 79 CHAPTER IV ISOLATION AND CHARACTERIZATION OF A 3-CHLOROBENZOIC ACID DECHLORINATING BACTERIUM FROM A COASTAL MARINE ENVIRONMENT ................................................................................... 84 Abstract .................................................................................... 84 Introduction ................................................................................ 85 Materials and Methods ................................................................. 87 Media and growth condition .................................................. 87 Isolation of a 3-CB dechlorinating bacterium ........................... 87 Microscopy ....................................................................... 88 Characterization of reductive dechlorination ........................... 88 Growth rate and protein yield ................................................. 89 Chemical analysis ............................................................... 90 16S rRNA gene sequencing and analysis ............................... 90 Results ...................................................................................... 91 Enrichment and isolation of strain DCB-M ............................... 91 Range of electron donors and acceptors used .......................... 93 Inhibition of reductive dechlorination ..................................... 93 Effect of sodium chloride on dechlorination and growth .............. 99 Protein yield coupled to reductive dechlorination ..................... 99 Phylogeny of strain DCB-M ................................................. 99 Discussion ............................................................................... 103 References ............................................................................. 106 vii LIST OF TABLES CHAPTER I Table 1.1. Characteristics of some dehalogenating isolates ............................ 8 CHAPTER II Table 2.1. Source and characteristics of coastal marine sediment samples .............................................................................................. 26 Table 2.2. Reductive dechlorination activity observed in sediment microcosms and enrichment cultures ........................................................................ 30 CHAPTER III Table 3.1. Characteristics of strain SF3. Data show general features observed in strain SF3 ........................................................................................ 57 Table 3.2. Electron donors tested for reductive dechlorination. A positive score indicated growth, which was determined by monitoring the depletion of 2-CP and the consumption of electron donors as well as observing culture turbidity ............................................................................................... 62 Table 3.3. Other growth substrates used by strain SF3. No other electron donors or electron acceptors were provided when these organic compounds were used as growth substrates ............................................................................. 63 Table 3.4. Test of halogenated aromatic compounds and other chemicals as electron acceptors by strain SF3. A positive score indicated growth, which was assessed by measuring the consumption of the electron donor and acceptor as well as observing culture turbidity ............................................................. 65 Table 3.5. Growth of SF3 on various electron donor and acceptor combinations. Growth was assessed by HPLC analysis of electron donors and acceptors viii consumed and products formed, and by the increase of visual turbidity ............................................................................................... 66 Table 3.6. Inhibition of dechlorination by potential competitive electron acceptors. 2-CP (0.25 mM) was provided in all media. Dechlorinaton was determined by monitoring the depletion of 2-CP. A positive score indicated inhibition of dechlorination, whereas a negative score indicated no inhibition of dechlorination ...................................................................................... 67 Table 3.7. Protein yield for strain SF3 cultures grown on acetate and ortho chlorophenols. Date for each replicate culture are indicated by 1, and 2 in parentheses. Protein yield was calculated after subtracting protein measured in controls ................................................................................................ 70 Table 3.8. Haloaromatic compounds as electron acceptors used by different 2- chlorophenol dechlorinating isolates. A positive score indicated that reductive dehalogenation and acetate consumption occurred over three successive feedings of the halogenated substrate. The data for strains 2CP-1, 2CP-C and ZCP-3 are from Cole et al. (1994) and Sanford (1996), respectively ........................................................................................ 74 CHAPTER IV Table 4.1. Electron donors tested for use by strain DCB-M. 3-CB served as an electron acceptor. A positive score indicated growth, which was monitored by measuring the depletion 3-CB and consumption of electron donors ............... 97 Table 4.2. Electron acceptors tested with strain DCB-M. A positive score indicated growth, which was determined by measuring the depletion of electron acceptors. Pyruvate was used as the electron donor .................................... 98 Table 4.3. Growth yield for strain DCB-M grown on 3-CB and lactate. Date for duplicate cultures are indicated by 1, and 2 in parentheses. Protein yield was calculated after subtracting protein measured in control cultures with lactate only ................................................................................................... 101 LIST OF FIGURES CHAPTER I Figure 1.1. Some naturally occurring haloaromatic compounds. Taken from King (1986) and Gribble (1992) .................................................................. 6 CHAPTER II Figure 2.1. Dechlorination activity of Gulf Breeze, FL primary microcosm and enrichment cultures at different temperatures ............................................. 28 Figure 2.2. ARDRA of the first transfer of a Gulf Breeze, FL 3-CB metabolizing culture. Each operational taxonomic unit represents a separate restriction pattern found in 16S rDNA clones ............................................................ 31 Figure 2.3. Microbial diversity characterization by ARDRA of the second transfer of a Gulf Breeze, FL 3-CB metabolizing culture .......................................... 32 Figure 2.4. Microbial diversity characterization of the third transfer (serial dilution from the second transfer) of a Gulf Breeze, FL 3-CB metabolizing culture .................................................................................................. 33 CHAPTER III Figure 3.1. Phase-contrast micrograph of strain SF3. Reference bar is 1 pm ..................................................................................................... 55 Figure 3.2. Scanning electron micrograph of strain SF3. Reference bar is 1 pm ..................................................................................................... 56 Figure 3.3. Exponential growth of strain SF3 on 2-CP plus acetate at 25°C and 30°C. Data are averaged from duplicate cultures ........................................ 59 Figure 3.4. Exponential growth of strain SF3 on fumarate plus pyruvate at 25°C. Data are averaged from duplicate cultures ............................................... 60 Figure 3.5. Growth of strain SF3 in various concentrations of sodium chloride, sucrose, and potassium chloride. Growth was measured by the rate of increase in reductive dechlorination of 2-CP. The concentration of sodium chloride in the synthetic seawater medium was 424 mM (1X). The freshwater concentration of sodium chloride (1/16X) was provided in all media when sucrose and potassium chloride were tested. Cultures were grown at 25°C ....................... 61 Figure 3.6. Induction of dechlorination. Induction of dechlorination activity was determined by comparison of two sets of cultures previously grown in the presence or absence of 2-CP. Fumarate was substituted as the electron acceptor for growth in the absence of 2-CP ................................................ 69 Figure 3.7. Maximum-likelihood phylogenetic tree based on the 16S rRNA sequences of strain SF3 and representative bacteria. Numbers at internal nodes are the percentage of 100 bootstrap samples in which the group to the right of the node was monophyletic. The scale is the expected number of substitutions per position ........................................................................ 72 CHAPTER IV Figure 4.1. Phase-contrast micrograph of strain DCB-M. Reference bar is 5 pm ..................................................................................................... 94 Figure 4.2. Scanning electron micropraph of strain DCB-M. Arrow points to collar structure of the cell. Reference bar is 1 pm ......................................... 95 Figure 4.3. Exponential growth of strain DCB-M on 3-CB plus pyruvate at 37°C. Data are averaged from duplicate cultures ................................................ 96 Figure 4.4. Effect of sodium chloride on growth of strain DCB-M and a brackish water dechlorinator, strain DCB-F. The concentration of sodium chloride in the standard synthetic seawater medium was 424 mM (1X). Growth was measured by the rate of reductive dechlorination of 3-CB .......................................... 100 Figure 4.5. Maximum-likelihood phylogenetic tree based on the 16S rRNA sequences of strain DCB-M and representative bacteria. Numbers at internal nodes are the percentage of 100 bootstrap samples in which the group to the right of the node was monophyletic. The scale is the expected number of substitutions per position ....................................................................... 102 xii CHAPTER I INTRODUCTION Overview Some microorganisms are capable of utilizing haloaromatic compounds as physiological electron acceptors in the reductive dehalogenation reaction. Anaerobic halo-respiring bacteria conserve energy by coupling organic carbon oxidation to reduction of halogenated compounds in a process analogous to the energy-yielding oxygen reduction carried out by aerobes. The ability of microorganisms to halorespire in anaerobic environments is potentially beneficial to the microorganisms carrying out reductive dehalogenation, since considerable energy is released in this reaction (Mohn and Tiedje 1991; Mohn and Tiedje 1992; Cole et al. 1994; Sanford 1996; Fetzner 1998; Holliger et al. 1999). Many halogenated aromatic compounds are common environmental contaminants and reductive dehalogenation of these compounds has been studied in freshwater systems, soils and sludges (Goshal et al. 1982; Suflita and Miller 1985; Suflita et al. 1988; Sharak Genthner et al. 1989; Sharak Genthner 1999). Little of the ecological research, however, has been done to understand the fate and behavior of halogenated aromatic compounds in marine environments. Studies have shown that some halogenated aromatic compounds are produced and can be degraded anaerobically in marine environments (King 1986; King 1988). This observation implies that a selection for reductive dehalogenation may have occurred during the bacterial evolution. Hence, It seems productive to explore the marine environments for reductive dehalogenators able to degrade halogenated aromatic compounds. The primary questions that this study addressed are as follows: 0 Do microbial populations able to degrade halogenated aromatic compounds exist in marine environments? 0 Can these dehalogenating organisms be enriched and isolated? o What Is the population structure of the degradative consortia? o What are the phylogenetic and physiological similarities between marine and freshwater dehalogenators? To study the potential for reductive dechlorination under anoxic conditions, lset up microcosms with seven different coastal marine sediments and four different chlorinated aromatic compounds in a synthetic seawater medium. Reductive dechlorination was observed in most of the sediment microcosms. Results from microcosm studies indicated that different dechlorinating populations have various responses. to substrates, temperatures, and acclimation periods. I chose two different dechlorinating enrichment cultures for further study, since they had relatively faster degradation rates. For molecular studies, I chose 3-CB dechlorinating enrichment cultures for characterization of degradative microbial communities. From these two cultures I isolated and characterized two new microorganisms, strains SF3 and DCB-M that reductively dechlorinate 2-CP and 3-CB, respectively. These marine isolates are capable of coupling growth to reductive dechlorination reaction. Background Reductive dehalogenation. Reductive dehalogenation is an important means of hazardous waste remediation since I the chemicals susceptible to the process include organochlorine pesticides, alkyl solvents, and aryl halides. These compounds include many of the most toxic and environmentally persistent pollutants. Reductive dehalogenation involves the removal of the halogen substituent(s) from a molecule with concurrent addition of electrons to the molecule. In a reductive dehalogenation reaction, the halogenated compound is not usually used as a carbon source, but as an alternate electron acceptor. Studies on reductive dehalogenation have shown that the Gibbs free energy, AG, associated with reductive dechlorination is significantly exergonic, in the range of -140 to -160 kJ/reaction for chlorinated aromatic compounds and -130 to -170 kJ/reaction for chlorinated aliphatic compounds (Dolfing and Harrison 1992; Dolfing and Janssen 1994). This energy production in reductive dechlorination is very close to the AG“ of nitrate reduction to nitrite (Sanford 1996). Many halogenated aromatic compounds have been of great environmental concern because of their polluting properties and their recalcitrance in natural environments (Mohn and Tiedje 1992; Fetzner 1998). This has led to considerable efforts to study the biodegradation of these compounds under aerobic and anaerobic conditions. Reductive dehalogenation is the only known biodegradation mechanism for certain significant pollutants including highly chlorinated polychlorinated biphenyls, perchloroethene (tetrachloroethene), and certain steps in pentachlorophenol degradation (Boyd et al. 1983; Mohn and Tiedje 1992). Reductive dehalogenation usually makes xenobiotic compounds less toxic and more readily degradable. Hence, reductive dehalogenation is of particular importance and interest because of its potential application to bioremediation of pollutants and hazardous wastes (Boyd and Shelton 1984). Also, studies and understandings of reductive dehalogenation will contribute significantly to advancing basic microbiology knowledge in the areas of microbial ecology, physiology, and diversity. Why marine sediments. Microbial ecologists and environmental chemists have provided information on the distribution and metabolism of halogenated organics (King 1986; King 1988). To date, much of the ecological research, however, has been directed toward understanding the fate and behavior of halogenated pollutants in freshwater systems, soils, and sludges (Goshal et al. 1985; Suflita and Miller 1985; Gibson and Suflita 1986; Suflita et al. 1988). Reductive dehalogenation studies have predominantly used anaerobic freshwater sediments and sludges as inocula for microcosms or enrichment cultures (Suflita et al. 1982; Boyd and Shelton 1984; Quensen et al. 1988; Zhang and Wiegel 1990; Sharak Genthner 1999). Although marine systems have been exposed to a diversity of halogenated pollutants, very little has been known from an ecological perspective such as the behavior and fate of these pollutants and their effect on marine systems (King 1988; Neilson et al. 1990; Steward et al. 1995). Actually, marine systems may have been exposed to numerous naturally occurring organohalides for a much longer time than people thought, perhaps hundreds of millions of years (King 1986; King 1988). A large number of marine biota produce a remarkable array of aliphatic and aromatic compounds containing chlorine, bromine, and iodine (Ashworth and Cornier 1967; Craigie and Gruening 1967; Gribble 1992; Gribble 1994). Some of these halogenated aromatic compounds are illustrated in Figure 1.1. The natural biogenic halogenated compounds may have provided the selection that has resulted in the current microbial mediated dehalogenation activities. Some of these natural halogenated compounds have antimicrobial activities, and play an ecological role in their environment. Studies by King (King 1986) indicated that 2,4-dibromophenol occurred at concentrations up to several hundred micromolar in marine sediment. At these levels, aerobic metabolism was selectively inhibited relative to anaerobic metabolism. 2,4-dibromophenol was dehalogenated under anaerobic conditions (King 1988; Steward et al. 1995). These results suggested that bacterial populations from specific marine sites may have developed enzymatic capabilities similar to or better than those of freshwater or soil bacterial populations exposed to pollutants. Hence, the best source of dehalogenating microorganisms may be marine sediment where the OH Cl OH or I C1 C1 2 ,4-dichlorophenol 2,6-dichlorophenol O H OH Br Br I Br Br 2 ,4-dibromophcnol 2 ,6-dibromophcnol OH Br I C I O : C H NH; C 1 Cl 0 H COO' 3-chloro-5-bromotyrosine 3 ,S-dichloroanisaldehydc Figure 1.1. Some naturally occurring haloaromatic compounds. Taken from King (1986) and Gribble (1992). presence of halogenated compounds and limited oxygen environment provides regular selection for dehalogenating microorganisms. In addition, coastal marine sediments (including some at Naval facilities) contain many chlorinated pollutants. Hence, marine sources may increase the chances of finding further diversity among the dehalogenating microorganisms. Reductively dehalogenating microorganisms. Evidence that some halogenated aromatic compounds can serve as growth substrates for anaerobic microcosms and enrichment cultures has been reported since the late 19805 (Dolfing and Tiedje 1987; Dolfing 1990; Mohn and Tiedje 1991; Dolfing and Harrison 1992). Only a few microorganisms capable of transforming these compounds have been isolated. These reductively dehalogenating microorganisms are summarized in Table 1.1. These isolates include both gram-negative and gram-positive bacteria. The best studied bacterial isolate capable of growth via reductive dehalogenation is Desulfomonile tiedjei strain DCB-I, an unusual anaerobic bacterium isolated from sewage sludge (Shelton and Tiedje 1984). Strain DCB-i is a sulfate-reducing bacterium and a member of the delta proteobacteria (DeWeerd et al. 1990). For many years, this organism offered the only opportunity to study anaerobic reductive dehalogenation of halogenated aromatic compounds in pure culture. The organism was able to reductively dehalogenate halobenzoates and chloroethenes. Strain DCB-1 was capable of generating energy for growth from reductive dechlorination of 3-CB (Dolfing 1990; Mohn and Tiedje 1991; Dolfing and Harrison 1992). Growth by halorespiration in strain DCB-1 was also Table 1.1. Characteristics of some dehalogenating isolates. Reference Shelton and Tiedje 1984 Madsen and Licht 1992 Species/strain Desulfomonile tiedje/DCB-I DCB-2 Source sewage sludge sewage sludge Gram stain - + Motility - 4. Cell morphology long rod slightly curved rod Spores - 4. Dechlorinated chlorobenzoates; chlorophenols chlorophenols substrate Electron donor pyruvate pyruvate Growth yield 1.9 g protein/molea NDb Closest phylogenic sulfate-reducing bacteria Clostn'dium relative Table 1.1. Characteristics of some dehalogenating isolates (continued). Reference Gram stain Motility Cell morphology Spores Dechlorinated Substrate Electron donor Growth yield Closest phylogenic relative Source mix of sewage sludge and soil Bouchard et al. 1996 Cole and Foxworthy 1993 Species/strain Desulfitobacten’um frappieri/PCP-1T rod shaped pentachlorophenol pyruvate NDb Desulfitobacten‘um dehalogenans DCB-F brackish water sediment long red 3-CB pyruvate NDb strain DOB-1 Table 1.1. Characteristics of some dehalogenating isolates (continued). Reference Species/strain Source Gram stain Motility Cell morphology Spores Dechlorinated substrate Electron donor Growth yield Closest phylogenic relative Cole et al. 1994 2CP-1 freshwater sediment rod 2-CP acetate 2.9 g protein/molea Myxobacteria Utkin et al. 1994 Desulfitobacterium dehalogenans/ . JW/I U-DC1 freshwater sediment slightly curved rod chlorophenolic compounds pyruvate NDb Desulfotomaculum- Clostridium subphylum 1O Table 1.1. Characteristics of some dehalogenating isolates (continued). Reference Sanford 1996 Sanford et al. 1996 Species/strain Anaeromyxo dehalogenans/Z-CP Desulfitobactefium chlororespirans/C023 Source soil compost soil Gram stain - + Motility + 4- Cell morphology long slender rod curved bacillus Spores + + Dechlorinated chlorophenols 3-chloro-4-hydro benzoate Substrate (3-CI-4-H A) Electron donor acetate lactate Growth yield NDb 6.9 9 cells (dry weight)/mole° Closest phylogenic Myxobacteria Desulfitobacten’um relative dehalogens a Growth yield was determined by measuring the production of protein or cells per mole of chlorinated substrates dechlorinated. b not determined. ° Gram stain negative but Gram type positive as determined by electron microscopic observations. 11 indicated by a growth yield of 1.9 g of protein per mole of 3-CB dechlorinated (Dolfing and Tiedje 1987). Studies also indicated that aryl reductive dehalogenation activity in strain DCB-1 is inducible. meta-Halobenzoates or their analogs were found to specifically induce reductive dehalogenation activity. Additionally, strain DCB-1 was capable of dechlorinating the meta position of polychlorinated chlorophenols, but it was not able to obtain energy from chlorophenol dechlorination (Mohn and Kennedy 1992). The membrane- associated 3-CB reductive dehalogenase has been purified from strain DCB-1, and a heme compound has been suggested to be the active-site prosthetic group (Ni et al. 1995). Two new isolates, DCB-O from a sewage sludge enrichment, and DCB-F from a brackish water sediment that grow by reductively dechlorinating 3-CB to benzoate, share physiological and morphological features with strain DCB-I. (Cole and Foxworthy 1993). 168 rRNA sequence analysis indicated that these two organisms are closely related to strain DCB-I. Strain DCB-2, a gram-positive anaerobic spore-forming bacterium, is capable of transforming chlorophenols by removing chlorine from the ortho and meta positions (Madsen and Licht 1992; Christiansen and Ahring 1996). This organism was not shown to benefit from the reductive dechlorination process. Another gram-positive isolate, Desulfitobacterium dehalogenans, dechlorinates a wide range of chlorophenols and related compounds from the orthc position and appears to benefit from the dechlorination reaction when grown with pyruvate and yeast extract (Utkin et al. 1994). A reductive dehalogenase has been cloned recently from Desulfitobacterium dehalogenans and studies have 12 indicated that it is a key enzyme of halorespiration in the bacterium (van de Pas et al. 1999). The purified reductive dehalogenase catalyzed the reductive removal of a halogen atom from the ortho position of chlorophenolic compounds. Desulfitobacterium frappieri strain PCP-1T, an anaerobic spore- forming bacterium isolated from a methanogenic consortium, reductively dechlorinates pentachlorophenol (Bouchard et al. 1996). 16S rRNA sequence analysis suggests that strain PCP-1T is a new species and belongs to the genus Desulfitobacterium. Another gram-positive, spore-forming bacterium, Desulfitobacterium chlororespirans strain 0023, reductively dechlorinates chlorophenols and 3-Cl-4-HBA (Sanford et al 1996). Strain 0023 is phylogenetically similar to Desulfitobacterium dehalogenans and physiologically similar to strain DCB-2 (Sanford 1996). Growth yield indicated that the organism is capable of conserving energy by chlororespiration, a respiratory process coupling reductive dechlorination of 3-CI-4-HBA to growth (Sanford et al. 1996). 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Inhibition of microbial activity in marine sediments by a bromophenol from a hemichlodate. Nature (London) 323:257-259. King, M. G. 1988. Dehalogenation in marine sediments containing natural sources of halophenols. Appl. Environ. Microbiol. 54:3079-3085. 15 Madsen, T. and Licht, D. 1992. Isolation and characterization of an anaerobic chlorophenol-transforming bacterium. Appl. Environ. Microbiol. 58:2874-2878. Mohn, W. W. and Kennedy, K. J. 1992. Reductive dehalogenation of chlorophenols by Desulfomonile tiedjei DCB-1. Appl. Environ. Microbiol. 58: 1 367-1 370. Mohn, W. W. and Tiedje, J. M. 1991. Evidence for chemicsmctic coupling of reductive dechlorination and ATP synthesis in Desulfomonile tiedjei. Arch. Microbiol. 157:1-6. Mohn, W. W. and Tiedje. J .M. 1992. Microbial reductive dehalogenation. Microbiol. Rev. 56:482-507. Neilson, A. H., Allard, A.-S., Hynning, P.-A., Remberger, M. and Viktor, T. 1990. The fate of phenolic compounds in freshwater and marine environments. Adv. Appl. Biotechnol. 4:249-265. Ni, S, Fredrickson, J. K. and Xun, L. 1995. Purification and characterization of a novel 3-chIorobenzcate-reductive dehalogenase from the cytoplasmic membrane of Desulfomonile tiedjei DCB-1. J. Bacteriol. 177:5135- 5139. Quensen, J. F., 111, Tiedje, J. M. and Boyd, S. A. 1988. Reductive dechlorination of polychlorinated biphenyls by anaerobic microorganisms from sediments. Science 242:752-754. Sanford, R. A. 1996. Characterization of microbial populations in anaerobic food webs that reductively dechlorinate chlorophenols. Ph.D. thesis. Michigan State University, East Lansing. Sanford, R. A., Cole, J. R., Loeffler, F. E. and Tiedje, J. M. 1996. Characterization of Desu/fitobacterium chlororespirans sp. nov., which grows by coupling the oxidation of lactate to the reductive dechlorination of 3-chlorc-4- hydroxybenzoate. Appl. Environ. Microbiol. 62:3800-3808. Sharak Genthner, B. R. 1999. Preliminary characterization of four 2- chlorobenzcate-degrading anaerobic bacterial consortia. Biodegradation 10:27-33. Sharak Genthner, B. R., Price 11, W. A. and Pritchard, P. H. 1989. Anaerobic degradation of chloroaromatic compounds in aquatic sediments under a variety of enrichment conditions. Appl. Environ. Microbiol. 55:1466- 1471. 16 Shelton, D. R. and Tiedje, J. M. 1984. Isolation and characterization of bacteria in an anaerobic consortium that mineralizes 3-chlorobenzoic acid. Appl. Environ. Microbiol. 48:840-848. Steward, C. C., Dixon, T. C., Chen, Y. P. and Lovell, C. R. 1995. Enrichment and isolation of a reductively debrominating bacterium from the burrow of a bromometabolite-prodiicing marine hemichordate. Can. J. Microbiol. 41:637-642. Suflita, J. M., Gibson, S. A. and Beeman, R. E. 1988. Anaerobic biotransformations of pollutant chemicals in aquifers. J. Ind. Microbiol. 3:179- 194. Suflita, J. M., Horowitz, A., Shelton, D. R. and Tiedje, J. M. 1982. Dehalogenation: a novel pathway for the anaerobic biodegradation of haloaromatic compounds. Science 218:1115-1117. Suflita, J. M. and Miller, G. I. 1985. Microbial metabolism of chlorophenolic compounds in ground water aquifers. Environ. Toxicol. Chem. 4:751-758. Utkin, I., Woese, C. and Wiegel, J. 1994. Isolation and characterization of Desulfitobacterium dehalogenans gen. nov., an anaerobic bacterium which reductively dechlorinates chlorophenolic compounds. Int. J. Syst. Bacteriol. 44:612-619. van de Pas, B. A., Smidt, H., Hagen, W. R., van de Oost, J., Schraa, G., Stams, A. J. M. and de Vos, W. M. 1999. Purification and molecular characterization of ortho-chlorcphenol reductive dehalogenase, a key enzyme of halorespiration in Desulfitobacterium dehalogenans. J. Biol. Chem. 274:20287-20292. Zhang, X. and Wiegel. J. 1990. Sequential anaerobic degradation of 2,4- dichlorophenol in freshwater sediments. Appl. Environ. Microbiol. 56:1119- 1127. 17 CHAPTER II REDUCTIVE DECHLORINATION IN MARINE SEDIMENT MICROCOSMS AND ENRICHMENT CULTURES ABSTRACT The fate of chlorinated aromatic compounds was tested in the anaerobic marine environment. A synthetic anaerobic seawater medium with vitamins and no additional electron donors was used in marine sediment microcosms and enrichment cultures. Seven coastal marine sediments were tested in sediment microcosms for anaerobic dechlorination activity on four chloroaromatic compounds (2-CP, 2,4,6-TCP, 3-CB, and 3-CT). San Francisco Bay, CA and Gulf Breeze, FL sediment microcosms showed 2-CP and 2,4,6-TCP dechlorination activity in about three months after inception. Pearl Harbor, HI sediment microcosm showed 2-CP dechlorination activity in five months after inception. 2,4,6-TCP dechlorination activity was also observed from other sediment microcosms (King Salmon, AK; Pearl Harbor, Hl; Mayaguez, PR) in three to four months after inception. 2-CP and 3-CB dechlorination activity were sustained in sediment microcosms and enrichment cultures, whereas 2,4,6-TCP dechlorination activity was lost after feedings and transfer. Dechlorinating microorganisms were highly enriched in 3-CB and 2-CP dechlorinating cultures. Phenol and benzoate accumulated transiently in these enrichment cultures. No dechlorination activity against 3-CT was observed in any marine sediment microcosms. 3-CB dechlorination activity was only observed and 18 enriched at 30°C and 37°C, whereas no 3-CB dechlorination activity was observed when all marine sediment microcosms were cultured at 25°C. Anaerobic bacterial plate counts for the sediments on marine agar 2216 medium were similar and ranged from 2x105 to 8x10° per g of sediment (dry weight). Molecular characterization was made for the 3-CB dechlorinating cultures of Gulf Breeze, FL. ARDRA results showed that the patterns of the dominant clones shifted when the cultures were more highly enriched. 16S rRNA sequence analysis showed that the predominant pattern in the highly enriched cultures was most related to the dechlorinating microorganism strain DCB-I isolated from a freshwater environment. INTRODUCTION Many halogenated aromatic compounds have been of great environmental concern because of their polluting properties and their recalcitrance in the natural environments (Boyd et al. 1983; Boyd and Miller 1984; Mohn and Tiedje 1992; Fetzner 1998). This has led to considerable efforts to study the biodegradation of these compounds under aerobic and anaerobic conditions (Goshal et al. 1985; Suflita et al 1988; Mohn and Tiedje 1992; Fetzner and Lingens 1994). In anaerobic environments, halogenated compounds are usually transformed by reductive dehalogenation (Suflita et al. 1982; Gibson and Suflita 1986; Quensen et al. 1988; Mohn and Tiedje 1992; 19 Tiedje et al. 1993). Reductive dehalogenation usually makes xenobiotic compounds less toxic and more readily degradable. Reductive dehalogenation most readily occurs in undefined anaerobic communities, and the responsible organisms may be obligate syntrophs. Anaerobic dehalogenation communities appear to vary significantly in composition, as they respond differently to environmental factors, particularly the availability of various electron acceptors (Beeman and Suflita 1987; Mohn and Tiedje 1992). The enrichment cultures are often complex; complete degradation of the substrate may require an interdependent community food web. Very often, dehalogenating microorganisms use organic carbon as electron donors which probably originate as end products from other anaerobes. Some products formed in reductive dehalogenation reactions such as benzoate and phenol, which are toxic to microorganisms at high concentrations, may be removed by other microorganisms. So, it is not surprising that, like other anaerobic processes, reductive dehalogenation has been observed to occur widely in complex and mutualistic undefined communities (Dietrich and Winter 1990). Studies with undefined cultures clearly indicated the potential of anaerobes to catalyze reductive dehalogenation (Shelton and Tiedje 1984; Krumme and Boyd 1988; Sharak Genthner et al. 1989; Sharak Genthner 1999). The study of undefined communities can identify ecological factors affecting reductive dehalogenation. It is especially important to understand the ecological perspective of such communities in order to employ reductive dehalogenation in bioremediation processes, since undefined 20 cultures are more likely than pure cultures to behave like bacterial populations in natural habitats. In this research, seven coastal marine sediments from different geographical sources (four Pacific, three Atlantic) were used for reductive dehalogenation studies. We were interested in testing the reductive dehalogenation activity in marine sediment microcosms to determine how reductive dehalogenation activities differ between geographically isolated sites and across environments. Furthermore, one group of delta proteobacteria, the sulfate-reducing bacteria, commonly plays a much larger role in marine environments than in freshwater environments due to the high level of sulfate in saltwater. Hence, marine sources may increase the chances of finding further diversity of dehalogenating microorganisms, since some of the dehalogenating bacteria isolated from freshwater environment are sulfate reducers. MATERIALS AND METHODS Source of sediments. Sediment samples were collected from two carbonate based coastal marine environments, Pearl Harbor and Kanehoe Bay, HI, and two silicate based coastal marine environments, San Francisco Bay, CA and Gulf Breeze, FL. These samples provide both geographic distribution as well as environmental diversity. Samples from two additional carbonate sites, one from a Pacific atoll (Johnston Island, Pacific) and one from Mayaguez, Puerto Rico as well as one from an isolated silicate site, King Salmon, Alaska 21 were also obtained. Some of the sediments were dark and silty, whereas others were white and sandy. Marine Agar 2216 medium was used for plate counting under anaerobic conditions to determine the culturable number of anaerobic bacteria in these coastal marine sediment samples. Microcosms. A synthetic seawater medium with the same concentration of sodium chloride as that in natural seawater was used in the microcosms, since all these sediments were from marine sources. Replicate microcosms were constructed in all sediment-chlorinated substrate combinations. Each microcosm consisted of about 10 g of wet sediment, 100 ml of anaerobic synthetic seawater medium and chlorinated substrate. The chlorinated substrates tested were 2-CP (0.25 mM), 2,4,6-TCP (0.25 mM), 3-CB (1 mM), and 3-CT (0.25 mM). Autoclaved cultures were used as negative controls. Serum bottles were closed with Teflon lined butyl rubber stoppers and incubated at 25°C. The sediment microcosms that showed dechlorination activity were fed successively with substrates for several times. A 10% volume of the sediment microcosms was transferred into a fresh synthetic seawater medium in order to establish enrichment cultures. No additional electron donors were added to sediment microcosms and enrichment cultures. Formulation of the synthetic seawater medium. The synthetic seawater medium contained the following mineral salts (g/Iiter): NaCl, 25; MgClz, 1.4; KH2P04, 0.2; NH,CI, 0.3; KCI, 0.5; and CaCl2, 0.1. A trace element solution was added to give the following final concentration (mg/liter): MnCI26HZO, 5; H3803, 0.5; ZnClz, 0.5; CoC|26H,_O, 0.5; NiSO,6HZO, 0.5; 22 CuCl22H,_O, 0.3; and NaMoO42HZO, 0.1. In addition, the medium contained 1 ml of NaSeO3-Na2WO, solution per liter, and 10 mg of resazurin per liter. MgCI2 and CaCl2 were added from sterile anaerobic stock solution because these two chemicals would otherwise precipitate in the medium when autoclaved. Other components were boiled under N2 and cooled to room temperature under N2- CO2 (95-5). Na,S (as a reductant) and NaHCO3 were added to final concentrations of 1 and 30 mM, respectively. The pH of the medium was adjusted to 7.3-7.5 by varying the CO2 concentration in the headspace. The medium was dispensed into Nz-COZ-flushed serum bottles and sterilized by autoclaving. The sterile medium was amended with an anaerobic sterile Wolin vitamin solution (Wolin et al. 1963), with addition of thiamine, 1,4- naphthoquinone, nicotinamide, hemin, and lipoic acid at concentrations of 50, 200, 500, 50, and 50 pg/Iiter, respectively. Other components were added from sterile anaerobic stocks. Chemical analysis. Halogenated aromatic compounds were analyzed on reverse-phase high-perfonnance liquid chromatography (HPLC). A Hibar RT C18 column was used with a flow rate of 1.5 ml/min of 66:33:01 HzO-CHaCN- H3PO, and a UV detector set to 218 nm for chlorophenols, and 230 nm for 3- chlorobenzoate, respectively. 3-chlorotoluene was detected with a gas chromatograph by analyzing the headspaces of the microcosms. Appearance of products and disappearance of substrates were verified by comparison with authentic standards and zero time culture samples. 23 ARDRA and 168 rRNA sequencing. Bacterial genomic DNA was isolated from a 20 ml culture grown in the synthetic seawater medium by a method for diverse bacteria (Visuvanathan et al. 1989). 168 rRNA gene was amplified by using primers (5’AGAG'I'TTGATCCTGGCTCAG3’ and 5’AAGGAGGTGATCCAGCC3’) from F01 and R01 (Weisburg et al 1991). The polymerase chain reaction (PCR) mixture consisted of 1.5 mM MgCl2, 0.25 mM each dNTP, 0.25 pM each primer, 1x Taq polymerase buffer, 0.5 U of Taq polymerase (Sigma), and 0.1 pg of DNA in a volume of 30 pl. Amplification was conducted with a program consisting of an initial denaturation at 94°C for 5 min followed by 30 cycles of 94°C for 15 s, 55°C for 30 s, and 72°C for 2 min 10 s, and concluding with an elongation cycle at 72°C for 7 min. The 168 rRNA gene library was constructed by using a TA Cloning kit according to the manufacture's instructions (lnvitrogen, San Diego, CA). To amplify individual 16S rRNA genes, E. coli colonies were picked up and washed with 50 pl of H20 in 1.5 ml Eppendorf tubes. The cells were pelleted by centrifugation and resuspended in 10 pl of H20. The cells were then heated for 10 min in a boiling water bath and pelleted by centrifugation. The supernatant (1 pl) was then used for PCR reaction. The PCR product (10 pl) was digested with HaeIII and HinPII (New England Biolabs, Beverly, MA) in a total of 30 pl and incubated overnight at 37°C. The restriction fragments were then analyzed by metaphor agarose gel electrophoresis (3% metaphor agarose, 1 X TBE buffer, 80 voltage). The samples (20 pl) were run at 4°C for about 5 hours. The gels were then stained 24 with ethidium Bromide (0.5 pg/ml) for 15 min and then destained in TBE buffer for 15 min to visualize the bands. The PCR product amplified from individual colonies was purified by Wizard Purification System (Promega) and sequenced in both directions by automated fluorescent dye terminator sequencer. The primers used for sequencing corresponded to conserved regions of the 16S rRNA gene sequence (Woese 1987). The resulting sequence was analyzed and the phylogenetic placement was obtained with the Ribosomal Database Project (Maidak et al. 1999). RESULTS Dechlorinating microcosms. Seven coastal marine sediments were tested in sediment microcosms for anaerobic reductive dechlorination activity on four chloroaromatic substrates. These coastal marine sediments varied in both geographical source (four Pacific, three Atlantic) and in bulk composition (four carbon based, three silicate based) (Table 2.1). Although the sediments had different color and bulk composition, anaerobic bacterial plate counts for the sediment samples were similar and ranged from 2x105 to 8x106 per g of sediment (dry weight) (Table 2.1). San Francisco Bay, CA and Gulf Breeze, FL sediment microcosms showed 2-CP and 2,4,6-TCP dechlorination activity in about three months after inception. Pearl Harbor, HI sediment microcosm showed 2-CP dechlorination activity afterfive months. 2,4,6-TCP dechlorination 25 Table 2.1. Source and characteristics of coastal marine sediment samples Sample Appearance Plate Countsa (no.[g dry weight) , Carbonate-based Pearl Harbor, HI dark, silty 1.6x106 Kanehoe Bay, HI dark, silty and 7.9x106 sandy mix Johnston Island, Pacific white, sandy 1.7x105 Mayaguez, PR white, sandy 2.3x106 ili e-based SF Bay, CA dark, silty 1.0x106 Gulf Breeze, FL dark, silty and 3.9x106 sandy mix King Salmon, AK dark, silty NDb a Marine Agar 2216 was used for plate counting in anaerobic condition at 25°C. ° Not determined. 26 activity was also observed from other sediment microcosms (King Salmon, AK; Pearl Harbor HI; Mayaguez, PR) in three to four months. No 3-CB dechlorination activity was observed when the sediment microcosms had been incubated at 25°C for seven months after inception. After the cultures were transferred to 30°C, reductive dechlorination activity was observed in Gulf Breeze, FL sediment microcosms in about one month, and San Francisco Bay, CA, and Pearl Harbor, HI, in three months, respectively. Furthermore, these 3-CB dechlorinating cultures showed higher dechlorination activity when incubated at 37°C (Figure 2.1). None of the sediment microcosms had showed dechlorination activity against 3-CT after three years of incubation at different temperatures. In 2-CP and 3-CB dechlorinating microcosms, 2-CP and 3-CB were mineralized, since only small amount of phenol and benzoate transiently accumulated sometimes. In three of the active 2,4,6-TCP sediments, 2,4- dichlorophenol (2,4-DCP) produced from reductive dechlorination of 2,4,6-TCP accumulated, and in the fourth sediment (SF Bay, CA) both 2,4-DCP and 4- chlorophenol (4-CP) as products from reductive dechlorination of 2,4,6-TCP accumulated. 4-CP was the end product and was not degraded further. No reductive dechlorination activity was observed in the autoclaved control cultures. Transferred activity and enrichment cultures. After reductive dechlorination activity had been sustained in some sediment microcosms by feeding with chloroaromatic substrates , the active sediment microcosms were 27 Nw : 6939389 523% 5 3.2.3 .5852... new 53092:. .5863 .55.... .E 632m :30 .0 33:8 5.358.280 .mw 659". o.- 05:02 098 . o 84 .8» in! m Pea m 89 ( o. a =8 no... .8... .. I we Q. .7 45 I 89 o. a. =8 no... .3... 2....- Tbu first... .2... 2 r2. .. ill:- 88 9+ a... 28 transferred (10%) to a fresh synthetic seawater medium to establish enrichment cultures. Some of the dechlorination activities remained but some of the dechlorination activities were lost after transfer (Table 2.2). 2,4,6-TCP dechlorinating activity was lost in three sediment microcosms (King Salmon, AK; Pearl Harbor, Hl; Mayaguez, PR) after feedings of substrate, and in the enrichment cultures (San Francisco Bay, CA; Gulf Breeze, FL). 2-CP and 3-CB dechlorinating activities remained after transfer in all enrichment culture. Active enrichment cultures were fed successively with 2-3 mM substrates, and serial transfers (5-10%) from 10'1 to 10'7 were made from these cultures. Usually it took about six to seven weeks to recover the dechlorination activity in highly enriched cultures (10'6-10'7). Products such as phenol or benzoate accumulated in these highly enriched cultures and the dechlorination rates were higher than those in active sediment microcosms and the initial enrichment cultures. Molecular characterization. In the first transfer of a Gulf Breeze, FL 3-CB metabolizing culture, seventeen restriction patterns were found among the thirty clones analyzed. One dominant pattern (40%) was observed (Figure 2.2). ARDRA results of the second transfer showed nineteen patterns among forty clones analyzed (Figure 2.3). Several dominant patterns were observed in this culture. The dominant pattern in the first transfer was still dominant in the second transfer. In the third transfer (serial dilution 10") only six patterns were found among forty-nine clones analyzed (Figure 2.4). One predominant pattern (63%) was observed and 16S rRNA sequence analysis showed that this predominant pattern was most related to the dechlorinating microorganism 29 Table 2.2. Reductive dechlorination activity observed in sediment microcosms and enrichment cultures. Sediment LOP 2.4.6-TCP 3-CB 3-CT Pearl Harbor, HI +a + b 4. a . Kanehoe Bay, Hl - - - - Johnston Island, Pacific Mayaguez, PR - + - - SF Bay, CA +8 + b + a . Gulf Breeze, FL +8 + b + a - King Salmon, AK - + b - - ° Reductive dechlorination activity in these sediment microcosms was transferred and highly enriched. ° Dechlorination activity was lost after several additions of substrate in sediment microcosms and transferred cultures. 30 .wo:o_o <20. m? .0 m_w>_mcm c. acne. Eczma 8.8.59 2939.. m «.5359 .E: 0.69.98. EcozEcao comm. .9330 BEBE-é mo-m ._u. .335 So a .o 5.29. a... 9.. .0 <¢om< Nu 9:9“. 3E: 0.82.923 3.33.230 n.- ww mw 3 mp NF 5 ova m h m m v sauop VNCIJ S9I- 3 31 .9330 9.3.2.99: moi... 4”. .335 :30 m .o .989. 9.88 9.. .o < . :00 .902 04.0 0.0 09.0 80 so. So. .0. 0000.0 0.0 40.. 008 80 $0 80 E moo-0.0 mo. . 0.0 03. 80 $0 30 .0. 00-0 3. . 00.0 08. on... 03 08 E 00-0 Seal. 90. .50).... 02.08.... 4.5.05... 4.5.05. .0:0:0\:.0.0.n. 90.000.99.005 :.0.0.n. 00:50:00 0.0.00< 000300... .805. 00:50:00 0.9.003 0.9.005 .0_0..:00 :. 090000.: £0.90 05.09530 .90 00.0.3200 002. 0.0... :.0.0.n. 0000:2900 :. m 0:0 .. .5 00.00.05 0.0 92.00 0.00.3.0. :000 .0. 0.0m. .0_0:0...00.0_:0 05.0 0:0 0.0.000 :0 :26... 092.00 0......0. :.9.0 .0. 0.0... :.0.0.n. .50 0.00..- 70 sulfate-reducing bacteria (Figure 3.7). There are no close relatives among the described and sequenced bacteria. It is somewhat similar to a GenBank record for an organism called Desulfovibrio caledoniensis (with a similarity of 82%) isolated by a French company from oil field brines, but which has not been described in publication. DISCUSSION A novel Desulfovibrio bacterium growing via reductive dechlorination of 2-CP coupled to oxidation of acetate was enriched, isolated, and characterized. We have the following evidence to believe that the culture was pure: 1) same colony morphology in deep agarose shake cultures was observed in different media; 2) same cell morphology was observed in different liquid culture media; 3) multiple isolates from deep agarose shake cultures had dechlorination activity; 4) second round of isolation from agarose shake cultures gave the same results; 5) dechlorination activity was recovered in dechlorination medium when 1% of transfer was made from other cultures (LB complex medium, sulfate plus lactate, and fumarate plus pyruvate); 6) dechlorination activity was recovered from the colonies grown on sulfate plus acetate, and LB complex plates; 7) rep-PCR results of different cultures showed one identical pattern. As a coastal marine organism, strain SF3 grew on a wide range concentrations of sodium chloride, indicating that the organism is capable of growth at a wide range of Na+ concentrations ranging from freshwater to 71 .:0...000 .00 0:0..0...0:00 .0 .0080: 00.0000 0:. 0. 0.000 0:... .0..0_>:0o:0E 002. 000: 0:. .0 .:m.. 0:. 0. 000.0 0:. :0.:>> :. 00.0500 09.0.02. 00. .0 0m0.:00.00 0:. 90 0000: .0E0.:. .0 0.00:.02 000.00: 0>..0.:000.00. 0:0 mum E90 .0 000:00000 <2... mm. 0:. :0 00000 00.. 00050230 000:..0x..-E0E.x02 Hm 900.“. o F .o 029.000.0000 2.000.030.0380. 00 2.0.0.00 E0...0.0000...0I .IIr. 00F 2.0.00.0.000 2.0.0.5005 . 000.09. 000000905 .00. , ..00m :.9.0 I .0 .000... 955.0000 9 0.09000 .0.0000..000Q 02000000000 0.20.: 0.00m 8. 0.0 .00 .00 0.0.6.0000 00300.0: 0.00.: 0.00000. 0000< 0:0 0 _0.:0E:0..>:0 N0 0< 0:0.0 .0.:0:.:0..>:0 0.0.0.90 0.00.3 0..000Q 000m .00 00 2.0.8.0000 8 000 :.0..0 00. _ 00. . 72 seawater. This result is different than that of strain DCB-M and a brackish, 3-CB dechlorinating isolate DCB-F, which required certain concentrations of sodium chloride for optimum growth (see Chapter 4). Similar results were obtained when sodium chloride was substituted by sucrose, implying that strain SF3 was able to be tolerant to high osmotic pressure produced by sodium chloride or sucrose. Strain SF3 did not grow on high concentrations of potassium, probably due to the physiologically toxic effect of K+ on the cell. The Na“ tolerance may be a phylogenetically conserved feature. lt was particularly interesting that under the same enrichment conditions except for sodium chloride, a novel Desulfovibrio bacterium, strain SF3, was isolated from coastal marine environment while soil and freshwater environments yielded myxobacteria (Cole et al. 1994; Sanford, 1996). These myxobacteria, however, would not grow in the presence of concentrations of sodium chloride equivalent to seawater. It is a little surprising that 2-BP was not a dehalogenation substrate for strain SF3, since most of the previous 2-CP dechlorinatiing bacteria used 2-BP as a dehalogenation substrate (Table 3.8). The extremely limited range of substrates used by strain SF3 suggests that chlorophenol dechlorination activity is not a fortuitous or cometabolic reaction, but instead is a specific recognition reaction. In addition, dechlorination activity is not constitutive in strain SF3 but is induced by the presence of 2-CP, again implying specific recognition of substrate. These results seem consistent in the enriched populations, and pure cultures typically demonstrate substrate specificity for particular haloaromatic 73 Table 3.8. Haloaromatic compounds as electron acceptors used by different 2- CP dechlorinating isolates. A positive score indicated that reductive dehalogenation and acetate consumption occurred over three successive feedings of the halogenated substrate. The data for strains ZCP-1, ZCP-C and ZCP-3 are from Cole et al. (1994), and Sanford (1996), respectively. , Halogenated electron SF3 ZCP-1 2CP-C 2CP-3 acceptors 2-CP + + + + 3-CP - - - - 4-CP - - - - 2.3-DCP - - - - 2,4-DCP - +l-" + + 2,5-DCP - + + - 2,6-DCP + + + .,. 2,4,6-TCP - +l- + nd” 2-BP - + + nd a Dechlorination activity was observed but slow, and complete degradation did not occur. b not determined. 74 compounds. It is known that some halogenated aromatic compounds occur in natural environments such as marine sediments as a consequence of animal and algal activities (Ashworth and Cornier 1967; Craigie and Gruening 1967; King 1986; King 1988; Gribble 1992). Some of these naturally occurring haloaromatic compounds are not xenobiotic, and can be dehalogenated by microorganisms under anaerobic conditions (King 1988). Probably the dechlorination reaction has evolved for the use of natural halogenated aromatic compounds close to ortho-chlorophenols, and hence a specific recognition may exist between the dehalogenase and the substrate. It is assumed that oxygen inhibits reductive dehalogenation, since reductive dehalogenation typically occurs in anaerobic environments and requires a reduced, oxygen-free environment (Mohn and Tiedje 1992). Reductive dehalogenation may also be affected by other electron acceptors such as sulfate, or nitrate, which may affect the flow of electrons (Beeman and Suflita 1987). Evidence indicating that electron acceptors other than halogenated aromatic compounds affect reductive dehalogenation activity is accumulating. The relationship, however, seems to be complex. Some results showed that, sulfate, an electron acceptor used by sulfate-reducing bacteria, inhibited reductive dehalogenation (Beeman and Suflita 1987). In other two experiments, the effects of sulfate on reductive dehalogenation of monochlorobenzoates and monochlorophenols were tested, and the results showed that the chemical did not always inhibit reductive dehalogenation (Sharak Genthner et al 1989; Townsend et al 1997). King’s studies showed that 75 reductive dehalogenation of 2,4-dibromophenol in marine sediment also presumably occurred concurrently with sulfate reduction (King 1988). In our research, sulfate, nitrate, and thiosulfate did not inhibit reductive dechlorination of strain SF3. Sulfite inhibited reductive dechlorination of strain SF3 incompletely, probably due to its physiological toxicity. Perhaps these compounds compete for the electron donors such as pyruvate or lactate with 2- CP at first. This competition in strain SF3, however, does not last long due to the incomplete oxidation of acetate from pyruvate or lactate by these inorganic compounds. Hence, even in the presence of these compounds, acetate can be used as an electron donor and carbon source for reductive dechlorination by strain SF3. Although nitrate is usually thought as a favorable electron acceptor, it seemed that strain SF3 grew slower on nitrate plus lactate that sulfate plus lactate, probably due to the accumulation of nitrite which is thought to have a toxic effect on the cell. This new isolate appears to gain energy from the dechlorination reaction. lt grew in a synthetic anaerobic seawater medium amended with only vitamins, acetate, and 2-CP, producing phenol. Acetate was shown to support growth with 2-CP as an electron acceptor. But no growth occurred when only acetate was present. The coupling of oxidation of acetate to reductive dechlorination of 2-CP was only observed before in myxobacterium strains 20P-1, ZCP-Z, 20P- C, and ZCP-S (Cole et al. 1994; Sanford 1996). The protein yield was proportional to the amount of chlorine removed and was about twice as much on 2,6-DCP as on 2-CP, implying that, like strain DOB-1 strain SF3 gains 76 energy by using the chlorinated substrate as a respiratory electron acceptor. Also, the stoichiometry of chlorine removed to acetate consumed is in agreement with the theoretical maximal value of four electron pairs produced per acetate oxidized to 2C02, with the remaining reducing equivalents going to biomass. Surprisingly, the organism can grow on formate producing H2. From a thermodynamic consideration, this is almost impossible since the energy produced by this reaction is so low. Another Desulfovibrio bacterium, strain FOX 1, isolated by Sanford showed the same feature (Sanford 1996). The results from strain FOX1 indicated that the exponential growth of FOX1 continued to a AG' of -5 to -10 kJ, providing evidence for a new lower limit of free-energy that supports growth. Strain FOX1 was the first organism for which this type of growth had been demonstrated. The existence of bacterial populations like strains FOX1 and SF3 suggests a more energetically dynamic role for formate cycling in anaerobic ecosystems. It is very interesting that acetate can not be oxidized by coupling growth to sulfate, sulfite, thiosulfate, and nitrate, but can be used as an electron donor for reductive dechlorination. That the same organic compound can be used under different electron accepting conditions is a quite novel phenomenon. To our knowledge, no Desulfovibrio member of the sulfate-reducing bacteria can oxidize acetate under any electron accepting conditions. Still, strain SF3 used formate as sole carbon source for sulfate reduction, but not for reductive 77 dechlorination. Perhaps, strain SF3 employs different electron transport systems for dechlorination and sulfate reduction. Comparision of the 168 rRNA sequence of strain SF3 with a database of 168 rRNA sequences indicated that the organism is a member of the delta subdivision of proteobacteria, as are other 2-CP dechlorinating bacteria such as strains ZCP-1 and ZCP (Cole et al. 1994; Sanford 1996). The 16S rRNA sequence of strain SF3, however, does not place the organism among the myxobacteria, but instead maps it to the Desulfovibrio branch of the sulfate- reducing bacteria, which have the nutritional versatility and phylogenetic diversity (Devereux et al. 1990). The physiological features and 168 rRNA sequence of strain SF3 suggest that it represents a novel genus of the Desulfovibrio group. It is a common feature that the Desulfovibrio branch uses hydrogen as an electron donor for sulfate reduction. Although the organism can not use hydrogen as an electron donor for reductive dechlorination or sulfate reduction, anaerobic growth, motility, and sulfate reduction are all consistent with sulfate-reducing bacterial characteristics. The delta subdivision includes members capable of using a wide variety of substrates as physiological electron acceptors such as oxygen, nitrate, sulfate, fumarate, and iron (Oyaizu and Woese 1985; Lovley et al. 1993). Given the energetic benefit, it is reasonable that some members of this group are able to utilize halogenated aromatic compounds as their physiological electron acceptors. Strain SF3 has some advantageous features desired for basic studies of reductive dechlorination and for bioremediation. Since growth is selected by the 78 chlorinated substrate, the process is much easier to manage than that mediated by cometabolism. The sensitivity of strain SF3 to all the antibiotics tested and relatively faster growth compared with other chlorophenol dechlorinating isolates make strain SF3 more suitable for genetic manipulation such as transposon mutagenesis which could identify interesting genes such as the dehalogenase gene. The ability to use various electron donors for reductive dechlorination may make strain SF3 more readily survive in a polluted environment when in use for in situ bioremediation. The feature that strain SF3 is able to grow at different concentrations of sodium chloride ranging from freshwater to seawater makes the organism a very good candidate for bioremediation in river, brackish, and ocean environments. REFERENCES Ashworth, R. B. and Cornier, M. J. 1967. Isolation of 2,6-dibromophenol from the marine hemichordate, Balanoglossus biminiensis. Science 156:158- 159. Beeman, R. E. and Suflita, J. M. 1987. Microbial ecology of a shallow unconfined ground water aquifer polluted by municipal landfill leachate. Microbiol. Ecol. 14:39-54. Bouchard, B., Beaudet, R., Villemur, R., McSween, 6., Lepine, F. and Bisaillon J.-G. 1996. Isolation and characterization of Desulfitobacterium frappierisp. nov., an anaerobic bacterium which reductively dechlorinates pentechlorophenol to 3-chlorophenol. Int. J. Syst. Bacteriol. 46:1010-1015. Christiansen, N. and Ahring, B. K. 1996. Desulfitobacterium hafniense sp. nov., an anaerobic, reductively dechlorinating bacterium. Int. J. Syst. Bacteriol. 46:442-448. 79 Cole, J. R., Cascarell, A. L., Mohn, W.W. and Tiedje, J. M. 1994. Isolation and characterization of a novel bacterium growing via reductive dechlorination of 2-chlorophenol. Appl. Environ. Microbiol. 60:3536-3542. Craigie, J. S. and Gruening, D. E. 1967. Bromophenols from red algae. Science 157:1058-1059. Devereux, R., He, 8., Doyle, C. L., Orkland, S., Stahl, D. A., Legall, J. 8: Whiteman, W. G. 1990. Diversity and origin of Desulfovibrio species: phylogenetic definition of a family. J. Bacteriol. 172:3609-3619. DeWeerd, K. A., Mandelco, R. S., Tanner, R. 8., Woese, C. R. and Suflita, J. M. 1990. Desulfomonile tiedjei gen. nov. and sp. nov., a novel anaerobic, dehalogenating sulfate-reducing bacterium. Arch. Microbiol. 154:23-30. Dietrich, G. and Winter, J. 1990. Anaerobic degradation of chlorophenol by an enrichment culture. Appl. Microbiol. Biotechnol. 34:253-258. Dolfing, J. 1990. Reductive dechlorination of 3-chlorobenzoate is coupled to ATP production and growth in an anaerobic bacterium, strain DCB-1. Arch. Microbiol. 153:264-266. Dolfing, J. and Harrison, B. K. 1992. Gibbs free energy of formation of halogenated aromatic compounds and their potential role as electron acceptors in anaerobic environments. Environ. Sci. Technol. 26:2213-2218. Fetzner, S. 1998. Bacterial dehalogenation. Appl. Microbiol. Biotechnol. 50:633-657. Gibson, 8. A. and Suflita, J. M. 1986. Extrapolation of biodegrdation results to groundwater aquifers: reductive dehalogenation of aromatic compounds. Appl. Environ. Microbiol. 52:681-688. Gribble, G. W. 1992. Naturally occurring organohalogen compounds- a survey. J. Nat. Prod. 55. 1353- 1395. Hanson, R. S. and Phillips, J. A. 1981. Chemical composition. p. 328-364. In: Manual of methods for general bacteriology. Gerhardt, P., Murray, R. G., Cortilow, R. N., Nester, E. W., Wood. W. A., Krieg, N. R. & Phillips, G. B. American Society for Microbiology, Washington D. C. Holliger, C., Wohlfarth, G. and Diekert, G. 1999. Reductive dechlorination in the energy metabolism of anaerobic bacteria. FEMS Microbiol. Rev. 22:383-398. 80 King, M. G. 1986. Inhibition of microbial activity in marine sediments by a bromophenol from a hemichlodate. Nature (London) 323:257-259. King, M. G. 1988. Dehalogenation in marine sediments containing natural sources of halophenols. Appl. Environ. Microbiol. 54:3079-3085. Klomparens, K., Flegler, S. L. and Hooper, G .R. 1986. p. 146. In: Procedures for transmission and scanning electron microscopy for biological and medical sciences. Burlington, VT, Ladd Research Industries. Lovley, D. R., Giovannoni, S. J., White, D. C., Champine, J. E., Phillips, E. J. P., Gorby, Y. A. and Goodvin, S. 1993. Geobacter metallireducens gen. nov. sp. nov., a microorganism capable of coupling the complete oxidation of organic compounds to the reduction of iron and other metals. Arch. Microbiol. 159:336-344. Madsen, T. and Licht, D. 1992. Isolation and characterization of an anaerobic chlorophenol-transforming bacterium. Appl. Environ. Microbiol. 58:2874-2878. Maidak, B. L., Cole, J. R., Parker, Jr, C. T., Garrity, G. M., Larsen, M., Li, B., Lilburn, T. G., McCaughey, M. J., Olsen, G. J., Overbeek, R., Pramanik, S., Schmidt, T. M., Tiedje, J. M. and Woese. C. R. 1999. A new version of the RDP (Ribosomal Database Project). Nucleic Acids Res. 27:171-173. Mikesell, M. D. and Boyd, S. A. 1986. Complete reductive dechlorination and mineralization of pentachlorophenol by anaerobic microorganisms. Appl. Environ. Microbiol. 52:861-865. Mohn, W. W. and Tiedje, J. M. 1991. Evidence for chemicsmctic coupling of reductive dechlorination and ATP synthesis in Desulfomonile tiedjei. Arch. Microbiol. 157:1-6. Mohn, W. W. and Tiedje, J. M. 1992. Microbial reductive dehalogenation. Microbiol. Rev. 56:482-507. Oyaizu, H. and Woese C. R. 1985. Phylogenetic relationships among the sulfate respiring bacteria, myxobacteria and purple bacteria. Syst. Appl. Microbiol. 6:257-263. Rademaker, J. L. W., Louws, F. J. and de Bruijn, F. J. 1997. Characterization of the diversity of ecologically important microbes by rep-PCR genomic fingerprinting, p.1-26. In: Molecular Microbial Ecology Manual. Akkennans, A. D. L, van Elsas, J. D. and de Bruijn, F. J. Kluwer Academic Publishers, Dordrecht, Supplement 3. 81 Sanford, R. A. 1996. Characterization of microbial populations in anaerobic food webs that reductively dechlorinate chlorophenols. Ph.D. thesis. Michigan State University, East Lansing. Sanford, R. A., Cole, J. R., Loeffler, F. E. and Tiedje, J. M. 1996. Characterization of Desulfitobacterium chlororespirans sp. nov., which grows by coupling the oxidation of lactate to the reductive dechlorination of 3-chloro-4- hydroxybenzoate. Appl. Environ. Microbiol. 62:3800-3808. Sharak Genthner, B. R. 1999. Preliminary characterization of four 2- chlorobenzoate-degrading anaerobic bacterial consortia. Biodegradation 10:27-33. Sharak Genthner, B. R., Price 11, W. A. and Pritchard, P. H. 1989. Anaerobic degradation of chloroaromatic compounds in aquatic sediments under a variety of enrichments conditions. Appl. Environ. Microbiol. 55:1466- 1471. Shelton, D. R. and Tiedje, J. M. 1984. Isolation and characterization of bacteria in an anaerobic consortium that mineralizes 3-chlorobenzoic acid. Appl. Environ. Microbiol. 48:840-848. Stevens, T. 0., Linkfield, T. G. and Tiedje J. M. 1988. Physiological characterization of strain DCB-1, a unique dehalogenating sulfidogenic bacterium. Appl. Environ. Microbiol. 54:2938-2943. Steward, C. C., Dixon, T. C., Chen, Y. P. and Lovell, C. R. 1995. Enrichment and isolation of a reductively debrominating bacterium from the burrow of a bromometabolite-producing marine hemichordate. Can. J. Microbiol. 41:637-642. Suflita, J. M., Horowitz, A., Shelton, D. R. and Tiedje, J. M. 1982. Dehalogenation: a novel pathway for the anaerobic biodegradation of haloaromatic compounds. Science 218:1115-1117. Townsend, G. T., Ramanand, K. and Suflita, J. M. 1997. Reductive dehalogenation and mineralization of 3-Chlorobenzoate in the presence of sulfate by microorganisms from a methanogenic aquifer. Appl. Environ. Microbiol. 63:2785-2791. Utkin, I., Woese, C. and Wiegel, J. 1994. Isolation and characterization of Desulfitobacterium dehalogenans gen. nov., an anaerobic bacterium which reductively dechlorinates chlorophenolic compounds. Int. J. Syst. Bacteriol. 44:612-619. 82 Visuvanathan, 8., Moss, M. T., Stanford, J. L., Hermon-Taylor, J. and McFadden, J. 1989. Simple enzymic method for isolation of DNA from diverse bacteria. J. Microbiol. Methods 10:59-64. Weisburg, W. W., Barns, S. M., Pelletier, D. A. and Lane, D. J. 1991. 16S ribosomal DNA amplification for phylogenetic study. J. Bacteriol. 173:697- 703. Woese, C. R. 1987. Bacterial evolution. Microbiol. Rev. 51:221-271. Wolin, E. A., Wolin, M. J. and Wolfe, R. S. 1963. Formation of methane by bacterial extracts. J. Biol. Chem. 238:2882-2886. 83 CHAPTER IV ISOLATION AND CHARACTERIZATION OF A 3-CHLOROBENZOIC ACID DECHLORINATING BACTERIUM FROM A COASTAL MARINE ENVIRONMENT ABSTRACT Strain DCB-M was isolated from an anaerobic 3-CB mineralizing culture enriched from Gulf Breeze, FL marine sediment. Strain DCB-M is a large, gram- negative rod with a collar girdling the cell. The isolate is capable of reductive dechlorination of 3-CB to benzoate and the generation time is 3.5 days. Strain DCB-M used a variety of electron donors for reductive dechlorination and growth. The organism fermented pyruvate to support growth, producing acetate. Among the chlorinated aromatic compounds tested, only the meta position of chlorobenzoates was dechlorinated, and substitutions at the para position blocked meta dechlorination. Strain DCB-M also used fumarate, sulfite, thiosulfate, sulfate and nitrate as physiological electron acceptors for growth, but grew poorly on sulfate and nitrate. Reductive dechlorination was completely inhibited by sulfite and thiosulfate but not sulfate. Strain DCB-M was not capable of growth in the freshwater concentration of sodium chloride. lt grew well at seawater salt concentrations, however, the optimum growth rate was at the salt concentration of half seawater. Growth by chlororespiration was indicated by a growth yield of 1.7 g of protein per mole of 3-CB dechlorinated. The 16S rRNA sequence places strain DCB-M in the delta proteobacteria and 84 much closely related to Desulfomonile tiedjei strain DCB-1. lts morphology, physiology, and 163 rRNA sequence suggest that it is a marine relative of strain DCB-I. INTRODUCTION Understanding the fate of natural and man-made halogenated aromatic compounds in the environment such as aquifer ecosystems has become more and more important since many of the most toxic and environmentally persistent pollutants are in this chemical class. Reductive dehalogenation is the only known biodegradation mechanism for certain significant pollutants including the highly chlorinated polychlorinated biphenyls, perchloroethene, and pentachlorophenol (Boyd et al. 1983; Mikesell and Boyd 1986; Mohn and Tiedje 1992). Reductive dehalogenation usually makes xenobiotic compounds less toxic and more readily degradable. Since groundwaters and sediment microenvironments are frequently oxygen-limited, reductive dehalogenation is a key initial biological step to achieve the biodegradation of highly chlorinated compounds in these environments (Lee et al. 1998). To further understand and optimize reductive dehalogenation, major efforts have been made to isolate anaerobic dechlorinating microorganisms. These organisms have been very difficult and tedious to isolate. Several, however, have been isolated and characterized in the last fifteen years (Shelton and Tiedje 1984; Madsen and Licht 1992; Cole et al. 1994; Utkin et al. 1994; 85 Steward et al. 1995; Christiansen and Ahring 1996; Sanford et al. 1996; Bouchard et al. 1996). Strain DCB-1, a sulfate-reducing bacterium isolated from sewage sludge was the first isolate and has been the best studied of these reductively dehalogenating microorganisms. Strain DCB-1 is a large, gram- negative, obligate anaerobic rod with a unique morphological feature: a collar surrounding the cell. The organism is capable of reductive dehalogenation of halobenzoates, chloroethenes. and pentachlorophenol. The halogenated aromatic compound is not used as a carbon source, but as an electron acceptor (Shelton and Tiedje 1984; Stevens et al. 1988; Mohn and Tiedje 1992), although the dechlorinated benzoate ring can also serve as a reductant (Cole, personal communication). The organism conserves energy for growth from reductive dechlorination of 3-CB (Dolfing 1990; Mohn and Tiedje 1991; Dolfing and Harrison 1992). 168 rRNA sequence of 003-1 indicates that the organism is a member of the delta subdivision of the class Proteobacteria. and represents a new genus among the sulfate-reducing bacteria (DeWeerd et al. 1990). Although studies of strain DCB-1 and several other dechlorinating microorganisms have greatly aided our understandings of aryl reductive dehalogenation, these isolates do not represent the diversity of dehalogenating microorganisms in nature. In particular, no isolates except for that reported in Chapter 3, are from the marine environment, yet the marine environment produces most of the natural haloaromatic compounds. Furthermore, marine coastal environments receive many of society's haloaromatic pollutants. 86 Previously, 3-CB dechlorination activity in Gulf Breeze, FL marine sediment microcosms and enrichment cultures was found. In this chapter, the isolation and characterization of a 3-CB dechlorinating bacterium from this sediment sample are described. MATERIALS AND METHODS Media and growth conditions. The synthetic seawater medium as described in Chapter 2 was used for enrichment culture and isolation of dehalogenating organisms. Wolin vitamins (Wolin et al. 1963) and other components were provided from sterile anaerobic stock solutions. Cultures were grown at 37°C in 160-ml serum bottles with 50 or 100 ml of boiled degassed medium or in 30-ml anaerobic culture tubes with 20 ml of medium and closed with butyl rubber stoppers. Synthetic seawater medium amended- with 3-CB to the final concentration of 1 mM was used for enrichment cultures. Colonies were isolated in deep agarose shake cultures containing 10 ml of synthetic seawater medium solidified with 1% low-gelling-temperature agarose and supplemented with 3-CB and pyruvate or lactate to final concentrations of 1, and 5 mM, respectively. Isolation of a 3-CB dechlorinating bacterium. After the fifth serial transfer, a culture diluted to 10'7 that dechlorinated about 4 mM 3-CB was chosen for isolation of 3-CB dechlorinators using deep agarose shake cultures incubated at 37°C. Bacterial colonies were picked from deep agarose cultures 87 that had been diluted 10*3 and 10'7 fold and transferred to the same liquid medium. Reductive dechlorination activity was checked by measuring the depletion of 3-CB and appearance of benzoate at different intervals. Microscopy. Phase-contrast photomicrography was done using a Leitz Orthoplan 2 microscope. Microbial cells from different cultures were centrifuged, then suspended in 10 pl of phosphate buffer (pH 7.2), and spread on dry agarose coated slides. Cells were observed under oil-immersion with a 60 X phase-contrast objective lens. Photographs were taken using TMAX 100 black and white film. Scanning electron microscopy was done according to the procedures described by Klomparens et al. (1986). Characterization of reductive dechlorination. To test the ability of strain DCB-M to reductively dechlorinate 3-CB by using different electron donors, duplicate 20-ml cultures of synthetic seawater medium was amended with 3-CB and different electron donors to final concentrations of 1 and 2.5 mM, respectively. The cultures were inoculated with a 1% transfer from an active dehalogenating culture. Acetate, benzoate, butyrate, forrnate, fumarate, hydrogen, lactate, propionate, and pyruvate were tested as potential electron donors for reductive dechlorination. Growth was determined by measuring the depletion of 3-CB, the production of benzoate, and the increase of visual culture turbidity over three successive feedings. To determine the range of electron acceptors, the following chlorinated aromatic compounds were tested in the synthetic seawater medium with 88 pyruvate as an electron donor. They were 2-chlorobenzoate (2-CB) (1 mM), 3- Cl-4-HBA (1 mM), 2-CP (0.25 mM), 3-CP (0.25 mM), 2,3-DCP (0.25 mM), 2,3- dichlorobenzoate (2,3-DCB) (1mM), 2,5-dichlorobenzoate (2,5-DCB) (1mM), 3,4-dichlorobenzoate (3,4-DCB) (1mM), 3,5-dichlorobenzoate (3,5-DCB) (1mM), 2,3,5-trichlorobenzoate (2,3,5-TCB) (1mM), and 2,4,6-trichlorobenzoate (2,4,6-TCB) (1mM). Fumarate, sulfate, sulfite, thiosulfate, and nitrate were also tested as potential electron acceptors at 5 mM with lactate as an electron donor. The cultures were periodically monitored by HPLC To test the relationship between reductive rechlorination and potential competitive electron acceptors in strain DCB-M, inorganic compounds such as sulfate, sulfite, thiosulfate, and nitrate at 5 mM were added to the synthetic seawater dechlorination medium containing 1 mM 3-CB and 5 mM pyruvate. Dechlorination activity was determined by measuring the depletion of 3-CB and appearance of benzoate by HPLC. The effect of sodium chloride on reductive dechlorination was tested by including various concentrations of sodium chloride ranging from 13.25 mM to 848 mM in dechlorination medium. Growth was measured by monitoring the depletion of 3-CB and appearance of benzoate. Growth rate and protein yield. The rate of growth by reductive dechlorination was measured by monitoring the production of benzoate from reductive dechlorination of 3-CB. Samples were taken from duplicate cultures every twelve hours and analyzed by HPLC. 89 To measure protein yield, replicate cultures were grown in serum bottles in the synthetic seawater medium (100 ml) amended with 5 mM lactate and with or without 1 mM of 3-CB. After about 1 mM of 3-CB was consumed, cultures were harvested and analyzed for substrate transformation and protein yield. Chemical analysis. Benzoate and chlorinated aromatic compounds were analyzed by reverse-phase high-performance liquid chromatography (HPLC). A Hibar RT C,8 column was used with a flow rate of 1.5 ml/min of 66:33:0.1 HZO-CHaCN-HsPO4 and a UV detector set to 230 nm. Appearance of products and disappearance of substrates were verified by comparison with authentic standards and time zero culture samples. Organic acids such as acetate, fumarate, pyruvate, and succinate were analyzed by ion-exclusion HPLC. H2 was analyzed by gas chromatography. To measure protein yield, cultures were harvested by centrifugation, washed, and then analyzed by the method of Lowry after alkaline hydrolysis (Hanson and Phillips 1981). 168 rRNA gene sequencing and analysis. Total DNA was extracted from a 50-ml culture grown in synthetic seawater medium by a method developed for diverse bacteria (Visuvanathan et al. 1989). 168 rRNA gene was amplified by using primers (5’AGAGTTTGATCCTGGCTCAG3’ and 5’AAGGAGGTGATCCAGCC3’) from FD1 and RD1 (Weisburg et al 1991). The PCR mixture consisted of 1.5 mM MgCl2, 0.25 mM each dNTP, 0.25 (M each primer, 1x Taq polymerase buffer, 0.5 U of Taq polymerase (Sigma), and 0.1 pg 90 of DNA in a volume of 30 pl. Amplification was conducted with a program consisting of an initial denaturation at 94°C for 5 min followed by 30 cycles of 94°C for 15 s, 55°C for 30 s, and 72°C for 2 min 10 s, and concluding with an elongation cycle at 72°C for 7 min. The PCR product was purified using the Wizard Purification System (Promega) and sequenced in both directions by automated fluorescent dye terminator sequencing. The primers used for sequencing corresponded to conserved regions of the 168 rRNA gene sequence (Woese 1987). The resulting sequence was analyzed and the phylogenetic placement was obtained with the Ribosomal Database Project (Maidak et al. 1999). A maximum likelihood phylogenetic tree was created with the program fastDNAml. RESULTS Enrichment and isolation of strain DCB-M. The coastal marine sediment sample from Gulf Breeze, FL was placed in a 160-ml serum bottle with 100 ml of synthetic seawater medium supplemented with 1 mM of 3-CB and incubated at 25°C for seven months. No dechlorination activity was observed during that period. Dechlorination occurred after these cultures were transferred to 30°C for one month. 3-CB dechlorinating populations from these cultures were further enriched at 37°C, since comparison of dechlorination rates at 30°C and 37°C showed that reductive dechlorination of 3-CB was faster at 37°C. The 91 enrichment cultures were fed with 3-CB for several times. A second transfer was made by transferring the active culture (10%) into a fresh synthetic seawater medium with 3-CB and no additional electron donors. After approximate 4 mM 3-CB was metabolized in the second transfer, a series of cultures (5% inoculum, diluted 10'1 to 10") was made in order to highly enrich dechlorinating microorganisms. Dechlorination activity was obtained in six weeks in the cultures diluted 10'7 fold and was sustained by successive feedings with 3-CB. Benzoate accumulated in these high enrichment cultures, and this accumulation appeared to be proportional to the degree of enrichment. Dechlorinating microorganisms were isolated from the fifth serial transfer (10'7 dilution) by using deep agarose shake cultures in anaerobic synthetic seawater medium at 37°C. After about two weeks, small white colonies became visible in the deep agarose shake cultures. Eight individual bacterial colonies were picked from these deep agarose shake cultures of 100 and 10'7 dilutions, transferred to homologous liquid medium under anaerobic conditions and tested for dechlorination activity at intervals. After 4 months, 3-CB had disappeared in three of the eight cultures with isolated colonies, with the concomitant appearance of approximately equal amounts of benzoate. To further establish culture purity, a second round of deep agarose shake cultures was made and dechlorination activity was obtained from the isolates tested. The isolated organism was named strain DCB-M indicating a dechlorinating bacterium from a marine environment. 92 Strain DCB-M is a gram-negative, nonmotile, long rod with a collar girdling the cell (Figure 4.1; Figure 4.2). Strain DCB-M is a strict anaerobe since no growth was observed when oxygen was introduced into the culture. The organism has a generation time of about 3.5 days in synthetic seawater medium amended with pyruvate and 3-CB at 37°C (Figure 4.3). Range of electron donors and acceptors used. Strain DCB-M was capable of using a wide range of electron donors for reductive dechlorination of 3-CB (Table 4.1). In addition to pyruvate, this organism oxidized formate, hydrogen, lactate, butyrate, benzoate, and propionate. Comparison of dechlorination rates indicated that pyruvate and lactate are more favorable than other electron donors. Pyruvate also supported growth in the absence of an electron acceptor and was stoichiometrically fermented to acetate. The evaluation of substrate range for reductive dechlorination showed that only the meta position of chlorobenzoates was dechlorinated. Substitutions by chlorine or hydroxyl at the para position blocked meta dechlorination (Table 4.2). Strain DCB-M was capable of using fumarate as an electron acceptor, producing succinate. Although sulfate and nitrate were reduced by strain DCB- M, they were much less desirable electron acceptors than was sulfite or thiosulfate. Inhibition of reductive dechlorination. When inorganic compounds such as sulfate, sulfite, and thiosulfate as potential competitive electron acceptors were added in the dechlorination medium (5 mM), sulfate did not show any inhibition of reductive dechlorination. Nitrate partially inhibited 93 Il Figure 4.1. Phase-contrast micrograph of strain DCB-M. Reference bar is 5 pm. 94 w x . «“I‘Wufl-“uw‘tnvx‘fi H V Figure 4.2. Scanning electron micrograph of strain DCB-M. Arrow points to collar structure of the cell. Reference bar is 1 pm. 95 1 200 1000 . 900- 800 - 1000 - 700 - 600 - 500- 400 - Doubling time = 3.5 days I 144 168 I I I I 192 216 240 264 E 3 '0 0 0 8 g 600 - 300 0 .9 G 0 N r: 0 m I I I I I O 48 96 144 1 92 240 288 336 Hours Figure 4.3. Exponential growth of strain DCB-M on 3-CB plus pyruvate at 37°C. Data are averaged from duplicate cultures. 96 Table 4.1. Electron donors tested for use by strain DCB-M. 3-CB served as an electron acceptor. A positive score indicated growth, which was monitored by measuring the depletion of 3-CB and consumption of electron donors. Electron donor Growth Acetate - Benzoate + Butyrate + Formate + Fumarate - Hydrogen + Lactate + Propionate + Pyruvate + 97 Table 4.2. Electron acceptors tested with strain DCB-M. A positive score indicated growth, which was determined by measuring the depletion of electron acceptors. Pyruvate was used as the electron donor. Electron acceptor Growth Produc_t(§_) 2-CB - 3-CB + benzoate 3-Cl-4-HBA - 2-CP - 3-CP - 2,3-DCP - 2,3-DCB + 2-CB 2,5-DCB + 2-CB 3,4-DCB - 3,5-DCB + 3-CB; benzoate 2,3,5-TCB + 2,3-DCB; 2,5-DCB; 2-CB 2,4,6-TCB - Fumarate + succinate Nitrate + nda Sulfate + nd‘il Sulfite + nda Thiosulfate + nda a Not determined. 98 dechlorination. Sulfite and thiosulfate almost completely inhibited reductive dechlorination at this concentration. Effect of sodium chloride on dechlorination and growth. Strain DCB-M was enriched and isolated in a synthetic seawater medium with sodium chloride of 420 mM. Reductive dechlorination and growth of strain DCB-M occurred at concentrations of sodium chloride ranging from 53 mM to 424 mM. The concentration of sodium chloride at 212 mM gave the best growth rate (Figure 4.4). The profile of the effect of sodium chloride on growth for strain DCB-M was very similar to that of strain DCB-F, a brackish water isolate capable of reductive dechlorination of 3-CB (Figure 4.4). The growth rate of strain DCB-M, however, was faster. Growth yield coupled to reductive dechlorination. Protein yield assay indicated that about 1.7 g of protein was produced per mole of 3-CB reductively dechlorinated (Table 4.3). Phylogeny of strain DCB-M. Comparison of 16S rRNA sequence of strain DCB-M to available 168 rRNA sequences indicated that strain DCB-M is a sulfate-reducing bacterium and a member of the delta proteobacteria, as is Desulfomonile tiedjei strain DCB-1. lts closest relative is strain DCB-1, with a similarity of 93%. The 168 rRNA sequences of strains DCB-M, DCB-1, DCB-F, and some representative bacteria were included in the construction of a maximum likelihood phylogenetic tree (Figure 4. 5). 99 0.4 Q DCB-M 0.3 - 0.2 - Growth rate (doublings per day) 0.1 — 00‘ 'l'l'l T 1I32X 1/16X 1I8X 1I4X 1I2X 1X 2X NaCI concentration relative to seawater Figure 4.4. Effect of sodium chloride on growth of strain DCB-M and a brackish water dechlorinator, strain DCB-F. The concentration of sodium chloride in the standard synthetic seawater medium was 424 mM (1X). Growth was measured by the rate of reductive dechlorination of 3—CB. Table 4.3. Growth yield for strain DCB-M grown on 3-CB plus lactate. Data for duplicate cultures are indicated by 1 and 2 in parentheses. Protein yield was calculated after subtracting protein measured in control cultures with lactate onl . EI’ectron acceptor 3-CB consumed Benzoate procficed Protein Protein/benzoate (pmonQ (omonl) (pglml) (glmol) 3-CB (1) 988 976 3300 1 .91 3-CB (2) 994 983 2790 1.4 None“ 1390 aAverage of data from duplicate control cultures. 101 005000 .00 32.0.0800 .0 00:5: 00.0000 0:. 0_ 2000 0:.. .0_.0.>:00:0E 00B 000: 0:. .0 .:0_. 0:. 0. 000.0 0:. :03? :_ 00_0:.00 00.0.00: 00. .0 000.:00.00 0:. 0.0 0000: _0:.0.:_ .0 0.00E0z 000.00: 0>_.0.:000.00. 0:0 Swmoo £000 .0 000:00000 :0 000:__0x__-:.0:._x0_2 .0... 0.00E o_..o 0.0.0000 00....00m 00000000000 0.20.: 000000. _ 000006.000 000059000000. 7000 ...0 .000... 0...:0E0..000Q w00 _ _0o. <00mI 0:0_0 _0.:0:.:0._>:0 _. _ 0-000 ...0 .00 0...:0E0..000Q |_ _00. 2-000 .00 .00 00005000000 .52 , _00. 102 DISCUSSION Strain DCB-M along with the previously isolated strains DCB-1, DCB-O, and DCB-F represent a coherent group of anaerobic 3-CB dechlorinating sulfidogenic bacteria (see Chapter 1). All these strains are long rods with a unique morphological feature: a collar girdling the cell. Studies showed that the collar structure of strain DCB-1 was involved in polar growth and cell division (Mohn et al. 1990). This unique morphology along with common physiology distinguishes these strains from other prokaryotes, and represent a novel group of sulfate-reducing bacteria. Strain DCB-M is capable of utilizing a wide range of electron donors for reductive dechlorination and growth. The range of dechlorinated electron acceptors used by strain DCB-M for reductive dechlorination, however, seems to be narrow. Only the meta position of chlorobenzoates was dechlorinated, and substitutions by chlorines or hydroxyl at the para position blocked meta dechlorination. Perhaps the substituents affect not only the chemical reactivity of the substrates but also their uptake into the cells and their affinity to the active enzyme. The specificity that strain DCB-M exhibits for removing meta-substitued chlorines from chlorobenzoates and the coupling to respiratory growth suggests that this capability has evolved for some period of time. Perhaps natural halogenated compounds are ubiquitous and abundant enough to select for and support novel anaerobic microbial populations. Many haloaromatic compounds occur in natural environments as a consequence of animal, fungal, and algal 103 ll activities (Ashworth and Cornier 1967; Craigie and Gruening 1967; King 1986; Gribble 1988; de long et al. 1994) and some of them can be dehalogenated by microorganisms under anaerobic conditions (King 1988). The reductive dechlorination reaction may have evolved for the use of naturally occurring haloaromatic compounds structurally similar to meta-chlorobenzoates. Given an ecological niche, reductive dehalogenation may be inhibited by other competitive electron acceptors. The relationship between reductive dehalogenation and potential electron acceptors seems to be complex. Inhibition tests in strain DCB-M indicated that sulfite, thiosulfate, and nitrate inhibited reduction dechlorination but sulfate did not. Inhibition may be due to the direct competition for electron flow or the accumulation of toxic products produced from nitrate or sulfate reduction. Studies on strain DCB-1 showed that the dehalogenation activity was membrane bound and was inhibited by sulfite and thiosulfate but not sulfate (DeWeerd and Suflita 1990). Sulfite reduction and reductive dehalogenation were inhibited by the same respiratory inhibitors, suggesting that reduction of sulfite and dehalogenation have some common electron carriers and use parts of the same electron transport chain (DeWeerd et al. 1991). This interpretation is reasonable but remains to be directly established. In fact, studies by Stevens showed that sulfate was a much less desirable electron acceptor than was sulfite or thiosulfate for strain DOB-1 (Stevens et al. 1988). Perhaps strain DCB-M shares the same or similar mechanisms with strain DCB-1 in the relationship between reductive dehalogenation and potential electron acceptors. 104 HI Strain DCB-M was not capable of growth on the freshwater concentration of sodium chloride. The seawater salt concentration, however, was not optimum for growth. This result is very similar to that of a brackish water isolate, strain DCB-F. Although the salt effect was not tested on all 3-CB dechlorinating strains, the Na+ requirement may be a conserved feature. Growth of strains DCB-M and DCB-F at 37°C, however, is not typical at least of open ocean marine bacteria. Perhaps these strains are adapted to harbor or near shore environments. Strain DCB-M seems to gain energy from the reductive dechlorination reaction and hence is a halorespirer. This was shown by continued serial transfer with 3-CB as the only electron acceptor and by a growth yield of 1.7 g of protein per mole of 3-CB dechlorinated. This compares favorably with values of 1.9 g of protein per mole of 3-CB dechlorinated in strain DCB-1 (Dolfing and Tiedje 1997), 1 g of protein per mole of 2-CP dechlorinated in strain SF3 (see Chapter 3), and 2.9 g of protein per mole of 2-CP dechlorinated in strain ZCP-1 (Cole et al. 1994). 168 rRNA sequence analysis indicated that strain DCB-M is a sulfate- reducing bacterium of the delta ptoteobacteria, and is much closely related to strain DCB-1, with a similarity of 93%. Although phylogenetically, morphologically, and physiologically similar to strain DCB-1, strain DCB-M has several features that delineate it from strain DOB-1. Strain DCB-1 was capable of growth in the freshwater concentration of sodium chloride but strain DCB-M was not. Propionate was an electron donor used by strain DCB-M but was not 105 by strain DCB-1. 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