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FERMENTATION AND CO FIXATION STUDIES OF STRAIN DCB—l, A UNIQUE DEHALOGENATING BACTERIUM By Todd Owen Stevens A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Microbiology and Public Health 1987 $1/‘///U ABSTRACT Fermentation and CO Fixation Studies of Strain DCB—l, A Unique Dehalogenating Bacterium By Todd Owen Stevens Strain DCB-l is a unique, strictly anaerobic bacterium that carries out the reductive dechlorination of 3-chlorobenzoate. Little is known of the physiology or taxonomy of this organism. This study attempted to elucidate the pyruvate fermentation of Strain DCB-l, and to identify the carbon substrates used by the organism in a syntrophic chlorobenzoate mineralizing consortium. The products of DCB-l's pyruvate fermentation were found to be acetate, fumarate, and succinate. Exogenous hydrogen interfered with growth by diverting carbon to unoxidized products. Fermentation products did not accumulate with thiosulfate in the medium. DCB-l's metabolism was found to be mixotrophic; up to 70% of cell carbon and 20% of acetate carbon was formed from COZ' Routes of carbon fixation by DCB-l were proposed. ACKNOWLEDGMENTS I owe thanks to a number of people for guidance and advice during my studies at MSU, especially to the members of my graduate committee, Dr. C.A. Reddy, Dr. M.J. Klug, and Dr. J.M. Tiedje. I'd also like to thank Dr. Klug for the time that I was able to spend in his laboratory at Kellogg Biological Station. Of course, Dr. Tiedje deserves special thanks for letting me do the research in the first place, and for supporting me while I did it. I'd like to thank the other inmates of our research group for their suggestions and companionship. Jan Dolfing deserves special mention, for proofreading this manuscript, as does Greg Walker of KBS, for helping with the analyses. I'd also like to thank the cows at the MSU dairy for contributing rumen fluid. List of Tables TABLE OF CONTENTS List of Figures CHAPTER 1. CHAPTER 2. CHAPTER 3. APPENDIX. Anaerobic Bacterial Communities in Carbon Cycling and Xenobiotic Degradation, and the 3-Chlorobenzoate Degrading Consortium . Pyruvate Metabolism by Strain DCB-l . Materials and methods Results Discussion . . Literature Cited The Substrate Range and Syntrophic Associations of Strain DCB-l Materials and Methods Results Discussion Literature Cited Effect of Medium Reductant on Growth and Dechlorination by Strain DCB-l Materials and Methods Results and Discussion Literature Cited iii Page 16 22 57 59 61 63 93 94 95 103 Table LIST OF TABLES Chapter 2 Mineral salts medium, from [12]. Fraction of DCB-l fermentation products derived from pyruvate and from bicarbonate, during pyruvate fermentation . . . . DCB-l fermentation balance; growth on pyruvate. Fraction of DCB-l fermentation products derived from pyruvate and from bicarbonate, during growth on pyruvate with thiosulfate DCB-l fermentation balance; growth on pyruvate with 10 mM thiosulfate. . . . . . . Fraction of DCB-l fermentation products derived from pyruvate and from bicarbonate, during growth on pyruvate with 20% C02, 80% H2 DCB-l fermentation balance; growth on pyruvate with excess hydrogen. . . . . . . Fraction of DCB-l fermentation products derived from pyruvate and from bicarbonate, during growth on pyruvate with 10 mM thiosulfate and 20% CO , 80% H 2 2 DCB—l fermentation balance; growth on pyruvate with 10 mM thiosulfate and excess hydrogen. Chapter 3 Protein determinations in cultures of strain DCB- l with Methanospirillum strain PM- 1 growing on lactate. iv page 39 40 41 42 43 44 45 46 85 LIST OF FIGURES Figure Page Chapter 2 1 Typical chromatogram of DCB-l fermentation products . . . . . . . . . . . . . . . . . . . . 23 2 Accumulation of products during growth of DCB-l on 0.2% pyruvate. Data points are means of 4 replicate cultures. . . . . . . . . . . . . . . 24 3 Accumulation of products during growth of DCB-l on 0.2% pyruvate, with headspace of 20% CO2 and 80% H . Data points are means of 4 replicages. . . . . . . . . . . . . . . . . . . 26 4 Accumulation of products during growth of DCB-l on 0.2% pyruvate with lOmM thiosulfate added. Data points are means of 4 replicates. . . . . . 29 5 Accumulation of products during growth of DCB-l on 0.2% pyruvate with 10 mM thiosulfate added and headspace containing 20% CO and 80% H . Data points are means of 4 replicates. . . . . . 31 6 Typical amounts of radioactivity in chromatographic fractions of DCB-l culture medium after 10 days growth. Radioactivity was introduced as 2- C pyruvate. . . . . . . . . . 34 7 Typical amounts of radioactivity in chromatographic fractions of DCB-l culture medium after 10 days gowth. Radioactivity was introduced as C bicarbonate. . . . . . . . . 36 8 Carbon assimilation by DCB-l with two acetyl CoA pools. . . . . . . . . . . . . . . . . . . . . . 53 Figure 9 page Carbon assimilation by DCB-l with one acetyl CoA pool. . . . . . . . . . . . . . . . . . . . . . . 55 Chapter 3 Dechlorination by membrane-divided 3-chlorobenzoate degrading consortium. SYMBOLS: circles, concentration in chamber containing strain DCB-l, squares, concentration in chamber containing strain BZ-2 and Methanospirillum strain PM-l . . . . . . . . . . . . . . . . . . . 64 Acetate production by divided 3-chlorobenzoate degrading cosortium SYMBOLS: circles, concentration in chamber containing strain DCB-l, squares, concentration in chamber containing strain BZ-2 and Methanospirillum strain PM-l . . . . . . . . . . . . . . . . . . . 67 Lactate production by divided 3-chlorobenzoate degrading consortium. SYMBOLS: circles, concentration in chamber containing strain DCB—l, squares, concentration in chamber containing strain BZ-2 and Methanospirillum strain PM-l . . . . . . . . . . . . . . . . . . . 69 Propionate production by divided 3-chlorobenzoate degrading consortium. SYMBOLS: circles, concentration in chamber containing strain DCB-l, squares, concentration in chamber containing strain BZ-Z and Methanospirillum strain PM-l . . . . . . . . . . . . . . . . . . . 71 Butyrate production by divided 3-chlorobenzoate degrading consortium. SYMBOLS: circles, concentration in chamber containing strain DCB-l, squares, concentration in chamber containing strain 82-2 and Methanospirillum strain PM-l . . . . . . . . . . . . . . . . . . . 74 Growth of strain DCB—l on lactate. Each data point is the mean of four observations. . . . . . 76 Growth of strain DCB-l on propionate. Each data point is the mean of four observations . . . 78 vi Figure 8 10 page Growth of strain DCB-l on butyrate. Each data point is the mean of four observations. . . . . . 80 Growth of strain DCB-l on acetate. Each data point is the mean of four observations. . . . . . 82 Consumption of lactate by strain DCB-l, and cocultures of strain DCB-l with Methanospirillum strain PM-l. Each data point is the mean of three observations . . . . . 86 APPENDIX Effect of medium reductant on dechlorination of 3-chlorobenzoate by strain DCB-l. Data points are means of 4 observations. Bars bars represent +/- one S.D. . . . . . . . . . . . 99 Effect of medium reductant on growth of strain DCB-l in pyruvate medium without chlorobenzoate and rumen fluid. Data points are means of 10 observations. . . . . . . . . . . . . . . . . . . 101 Chapter One Anaerobic Bacterial Communities in Carbon Cycling and Xenobiotic Degradation, and the 3-Chlorobenzoate Degrading Consortium The major role of microorganisms in biogeochemical cycling is that of decomposers, recycling organic material to inorganic precursors. Until recently, anaerobic bacteria were assumed to play a minor part in this cycle, but some estimates have shown that anaerobic processes are significant in many environments [3,16,21,33,39]. Anaerobic environments are found in waterlogged soils and in microsites in damp soils, in marine and freshwater sediments, in the intestinal tracts of animals and insects, and even within living plants. Sewage treatment plants are also important anaerobic environments. Organic matter entering into anaerobic environments includes readily metabolized compounds such as sugars and starches, as well as more recalcitrant molecules such as cellulose, hemicellulose, and lignin. Man made chemicals also enter these environments through sewage discharge, accidental spills, and agricultural application. A given anaerobic microorganism can rarely take up a complex organic molecule and mineralize it to carbon dioxide. Rather, a succession of specialized organisms modify the molecule in turn, each deriving a small amount of carbon and/or energy from the reaction [13,22,33]. For example, a typical organic polymer may be degraded into monomers by one organism, the monomers may be fermented by one or more other organisms into volatile fatty acids, and the VFA's may then be fermented to CO2 and acetate which can be used by other organisms to produce methane or CO2 [16,39]. Complex trophic webs can arise from the interactions between these organisms. Some of these interactions are so tightly coupled that associations of two organisms have been classified as a single species for years before they were discovered to be mixed cultures [6,26]. The mechanisms used by these bacterial communities to degrade organic molecules are of interest not only to increase the understanding of biogeochemical cycling, but also to explore the potential of microorganisms to degrade xenobiotic molecules [2]. This has been of increasing concern in recent years, as many man—made chemicals are found to be toxic or mutagenic. New methods must be found to dispose of these compounds when they are no longer of use, and to clean up contaminated areas. Digestion by anaerobic bacteria is an attractive possibility for disposal of synthetic chemicals because it requires less energy than other methods of waste disposal, construction costs for anaerobic treatment facilities are lower, and anaerobic digestion offers the possibility of recovering useful products, such as intermediary metabolites or methane gas [38]. In the early part of this century, the fate of these chemicals in the environment was not seen to be of particular importance. It was often assumed that the volume of the contaminants was so small, compared to the volume of the biosphere, that they would be diluted so much that they would have no biological effect [1]. Later, it was realized that such chemicals can become bioconcentrated by adsorption and active uptake into microorganisms. Once they have entered into biological systems, chemicals can be subject to biomagnification, in which higher concentrations of the compound are found in each successive step in the food pyramid [37]. For instance, high concentrations of DDT residues are found in predatory birds and in humans [11]. Long term persistance was not often considered to be a problem either. Indeed, a major selling-point of DDT in the 1940's was that it would continue to kill pests long after its initial application [15]. Chemicals introduced to the biosphere are often unstable, and undergo spontaneous degradation or are transformed by photochemical reactions [19]. Other chemicals were assumed to short-lived due to the so-called "Doctrine of Microbial Infallability." Briefly stated, the theory is that since microorganisms are ubiquitous, and since they are presented with an awesome array of different environments, they have probably evolved mechanisms to degrade almost any conceivable organic compound. As a number of chemicals were found to be recalcitrant to degradation, this theory fell out of favor [1]. Even though some compounds may be degraded by bacteria, they may persist because they partition to parts of the environment inhospitable to those bacteria, or because the metabolic state of the bacteria is not amenable to the degradation reaction. For these reasons, anaerobic environments often contain relatively high levels of ~recalcitrant organic compounds [2]. Microbially mediated reactions of xenobiotics can be of several types. The chemical may be "transformed," to another chemical which may be either more or less toxic, and more or less persistant. On the other hand, the chemical can be completely "mineralized" or degraded to one-carbon compounds. The microorganism can metabolize the compound, gaining energy and possibly cell carbon in the process, or the reaction can be due to "cometabolism" in which the transformation is due to a fortuitous property of some enzyme, and yields no benefit to the organism [14]. Efforts have been made to "manufacture" organisms to degrade specific chemicals. An organism that can degrade a similar chemical may be subjected to mutagenesis, or strong selective pressure, and variants are selected for the ability to degrade the new compound [20,29]. Other experiments have been directed at combining genes from different organisms into a single bacterium to construct new pathways for metabolism of synthetic chemicals [27,28]. Because anaerobic bacterial communities consist of a large number of different specialized organisms, such a community can be subjected to selection for degradation of a compound more readily than can a given single organism. When incubated with slowly increasing concentrations of a compound, bacteria which are able to derive carbon or energy from the compound, or its metabolites, multiply, and the population becomes enriched with higher numbers of these species. New associations of specialized anaerobes can join into syntrophic reactions to degrade the compound without major genetic mutations and rearrangements that would be required if a single species were to degrade the compound. Such enrichment experiments have been used to explore the degradation of chlorinated aromatic compounds [4,5,24,25,3l,34,35,36]. Chlorinated aromatic compounds are common constituents of pesticides, and are found in industrial waste streams. They include such serious contaminants as dioxins and polychlorinated biphenyls. Environments such as sewage treatment plants have experienced selection of haloaromatic degrading organisms. It is not entirely unreasonable to expect to find natural enzymes that can degrade these compounds, since some chlorinated aromatic compounds are found in nature, although they are probably mostly of marine origin [12,17,30]. Enzyme systems which have evolved to degrade non-chlorinated compounds may also be non-specific enough to degrade their chlorinated homologs. Enrichment experiments have produced consortia that could degrade compounds including 3-chlorobenzoate [34], 2,4-D, 2,4,5-T [24], and various chlorinated phenols [5], including pentachlorophenol [25]. One of these enrichments was adapted to a laboratory upflow bioreactor system, which continuously degraded chlorophenols for over a year [18]. Another enrichment was immobilized in an agar matrix, allowing degradation of higher concentrations of phenols [10], a system which might be adaptable to continuous culture. One model system for studying the ecology of microorganism-xenobiotic interactions is a sludge community which has been enriched on chlorobenzoates for a number of years [31]. Eight anaerobic bacteria have been isolated from this enrichment which can grow on 3-chlorobenzoate as its sole carbon and energy source [32]. After physiological characterization of these isolates, it appeared that at least three were needed to grow on the 3-chlorobenzoate substrate. By recombining these three isolates: a dechlorinating bacterium, a benzoate oxidizing bacterium, and a methanogen, the same rate of 3-chlorobenzoate metabolism was acheived as was found in the original enrichment. [8]. The dechlorinating bacterium, designated strain DCB-l is so far unique in its morphology and metabolism. This is the only obligately anaerobic bacterium isolated so far which is known to carry out reductive dechlorination. In this syntrophic association, it carries out the reaction [8,32]: - - + 3-Cl benzoate + H2 ---> benzoate + C1 + H It does not grow on benzoate or chlorobenzoate, and presumably receives some carbon from later stages in the oxidation of benzoate. Strain DCB—l is a long, rod-shaped, non-motile bacterium with a distinctive "collar" structure near one end [32]. The only substrate known to support its growth is pyruvate, although rumen fluid has been observed to stimulate its growth. Strain DCB-l is able to reduce sulfite and thiosulfate, which stimulate growth and inhibit dechlorination, but sulfate does not support growth [23]. In DNA-DNA homology experiments, DCB-l has been found to be more closely related to Desulfovibrio species than to Clostridium (J. Jansson and J.M. Tiedje, unpublished data). DCB-l can also reductively demethoxylate 3-methoxybenzoate, but this activity has been found to be unrelated to its dechlorinating ability [7]. The benzoate oxidizer, designated strain BZ-2, grows only in coculture with a hydrogen consuming bacterium such as a methanogen or a sulfidogen [32]. Benzoate is the only substrate known to support its growth, and is oxidized stoichiometrically to acetate by the reaction: Benzoate -—-> 3 acetate + 3 H2 + C02 The methanogen, designated strain PM-l has been identified as a Methanospirillum species, and is required to keep the hydrogen levels low to enable benzoate oxidation to take place [32]. The only known substrate for Methanospirillum is formate or H2/C02 [3]. In this association, it evidently carries out the reaction: 4 H2 + CO2 ---> CH4 + 2 H20 Another methanogen, a Methanobacterium sp., designated strain PM-2 was also isolated from the consortium. In addition, the consortium contained a sulfidogen, designated as Desufovibrio strain PS-l, and two butyrate oxidizing bacteria. One of the butyrate oxidizers, a spore former was designated strain SF-l, and the other, a non-spore former was designated strain NSF-2. These organisms were not required for mineralization of 3-chlorobenzoate, and their place in the food web was not established [32]. Recent experiments using the reconstructed consortium, consisting of strains DCB-l, BZ-2 and PM-l, have shown that the consortium derives energy from the reductive dechlorination, since the cell yield is greater when the consortium is grown on 3-chlorobenzoate, than when it is grown on benzoate [9]. Other experiments have shown that the association is a true trophic "web," rather than a "chain," since one third of the hydrogen from benzoate oxidation is returned to the dechlorinator for reduction of 3-chlorobenzoate [8]. Presumably, since strain DCB-l is incapable of growing on benzoate or on 3-chlorobenzoate, some form of carbon compound is also returned to it from benzoate oxidation. The further study of these organisms, and the interactions between them should lead to a better understanding of the fate of xenobiotics, as well as the basic ecology of anaerobic environments. The objectives of the experiments undertaken here were to determine how Strain DCB-l ferments pyruvate, whether it groWs chemotrophically with thiosulfate as electron acceptor, and to try to determine what carbon source DCB-l uses in the 3—chlorobenzoate degrading consortium. 10. Literature Cited Alexander, M. 1973. Nonbiodegradable and other recalcitrant molecules. Biotechnol. Bioeng. 15, 611-47. Alexander, M.A. 1979. Recalcitrant molecules, fallible micro-organisms. In J.M. Lynch and N.J. Poole (ed.), Microbial Ecology: a conceptual approach. Blackwell Scientific Publications, Oxford. Balch, W.E., G.E. Fox, L.J. Magrum, C.R. Woese, and R.S. Wolfe. 1979. Methanogens: Reevaluation of a unique biological group. Microbiol. Rev. 43:260-296. Boyd, S.A., D.R. Shelton, D. Berry, and J.M. Tiedje. 1983. Anaerobic biodegradation of phenolic compounds in digested sludge. Appl. Environ. Microbiol. 46:50-54. Boyd, S.A. and D.R. Shelton. 1984. Anaerobic biodegradation of chlorophenols in fresh and acclimated sludge. Appl. Environ. Microbiol. 47:272-277. Bryant, M.P., E.A. Wolin, M.J. Wolin, and R.W. Wolfe. 1967. Methanobacillus omelianskii, a symbiotic association of two species of bacteria. Archiv fur Mikrobiologies 59:20-31. DeWeerd, K.A., J.M. Suflita, T.G. Linkfield,J .M. Tiedje, and P.H. Pritchard. 1986. The relationship between reductive dehalogenation and other aryl substituent removal reactions catalyzed by anaerobes. FEMS Microbiol. Ecology. In press. Dolfing, J., and J.M. Tiedje. 1986. Hydrogen cycling in a three-tiered food web growing on the methanogenic conversion of 3-chlorobenzoate. FEMS Microbiol. Ecol. 38:293—298. Dolfing, J., and J.M. Tiedje. 1986. Submitted for publication. Dwyer, D.F., M.L. Krumme, S.A. Boyd, and J.M. Tiedje. 1986. Kinetics of phenol biodegradation by an immobilized methanogenic consortium. Appl. Environ. Microbiol. 52:345-351. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 11 Edwards, C.A. (ed.) 1973. Persistant Pesticides in the Environment, 2nd ed. CRC Press. Gschwend, P.M., J.K. Mac Farlane, and K.A. Newman. 1985. Volatile halogenated organic compounds released to seawater from temperate marine macroalgae. Science 227:1033—1035. Hamilton, W.A. 1979. Microbial energetics and metabolism. In J.M. Lynch and N.J. Poole (ed.), Microbial Ecology: a conceptual approach. Blackwell Scientific Publications, Oxford. Hill, I.R. 1978. Microbial Transformation of Pesticides. In Hill, I.R. and S.J.L. Wright (ed ), Pesticide Microbiology. Academic Press, London. Hill, I.R. and S.J.L. Wright (ed.). 1978. Pesticide Microbiology. Academic Press, London. Hobson, P.N., S. Bousfield, and R. Summers. 1974. Anaerobic digestion of organic matter. Crit. Rev. Environ. Control. 4:131-191. King, G.M., 1986. Inhibition of microbial activity in marine sediments by a bromophenol from a hemichordate. Nature 323:257-259. Krumme, M.L. 1986. Reductive dechlorination of chlorinated phenols in anaerobic upflow bioreactors. M.S. Thesis, Michigan State University, East Lansing, MI. Lal, R. (ed.) 1984. Insecticide Microbiology. Springer-Verlag, Berlin. Lattore, J., W. Reineke, and H.J. Knackmuss. 1984. Microbial metabolism of chloroanilines: Enhanced evolution by natural genetic exchange. Arch. Microbiol. 140 159-165. Lein, A.Y. 1984. Anaerobic consumption of organic matter in modern marine sediments. Nature 312:148-150. Lovely, D.R. and M.J. Klug. 1982. Intermediary metabolism of organic matter in the sediments of a eutrophic lake. App. Environ. Microbiol. 43:552-560. Linkfield, T.G. 1985. Anaerobic reductive dehalogenation: The lag period preceeding haloaromatic dehalogenation, enrichment of sediment activity, and the partial characterzation of a 24. 25. 26. 28. 29. 30. 31. dehalogenating organism, strain DCB-l. Ph.D. Thesis, Michigan State University, East Lansing, MI. Mikesell, M.D. and S.A. Boyd. 1985. Reductive dechlorination of the pesticides 2,4-D, 2,4,5-T and pentachlorophenol in anaerobic sludges. J. Environ. Qual. 14:337—340. Mikesell, M.D. and S.A. Boyd. 1986. Complete reductive dechlorination and mineralization of pentachlorophenol by anaerobic microorganisms. Appl. Environ. Microbiol. 52: 861-865. Postgate, J.R., 1979. The sulphate-reducing bacteria. Cambridge University Press. Cambridge. Reineke, W. and H.J. Knackmuss. 1979. Construction of haloaromatics utilizing bacteria. Nature 277:385-386. Reineke, W., D.J. Jeenes, P.A. Williams, and H.J. Knackmuss. 1982. TOL plasmid pWWO in constructed halobenzoate-degrading Pseudomonas strains: prevention of meta pathway. J. Bacteriol. 150:195-201. Rubio, M.A., K.H. Engesser, and H.J. Knackmuss. 1986. Microbial metabolism of chlorosalicylates: Accelerated evolution by natural genetic exchange. Arch. Microbiol. 145:116-122. Sato, T., M.Mukaida, Y. Ose, H. Nagase, and T. Ishikawa. 1985. Mutagenicity of chlorinated products from soil humic substances. The Science of the Total Environment, 46:229-241. Shelton, D.R. and J.M. Tiedje. 1984. General method for determining anaerobic biodegradation potential. Appl. Environ. Microbiol. 47:850-857 Shelton, D.R., and J.M. Tiedje. 1984. Isolation and partial characterization of bacteria in an anaerobic consortium that mineralizes 3-chlorobenzoic acid. Appl. Environ. Microbiol. 48:840-848. Sleat, R. and J.P. Robinson. 1984. The bacteriology of anaerobic degradation of aromatic compounds. J. Appl. Bacteriol. 57:381-394. Suflita, J.M., A. Horowitz, D.R. Shelton, and J.M. Tiedje. 1982. Dehalogenation: A novel pathway for the anaerobic 35. 36. 37. 38. 39. 13 biodegradation of haloaromatic compounds. Science 218: 1115-1117 Suflita, J.M., J.A. Robinson, and J.M. Tiedje. 1983. Kinetics of microbial dehalogenation of haloaromatic substrates in methanogenic environments. Appl. Environ. Microbiol. 45:1466-1473 Suflita, J.M., J. Stout, and J.M. Tiedje. 1984. Dechlorination of (2,4,5-Trichlorophenoxy)acetic acid by anaerobic microorganisms. J. Agric. Food Chemistry 32 218-221 Sukanya Lal. 1984. Microbial Accumulation of Insecticides. In Rup Lal (ed.), Insecticide Microbiology. Springer Verlag, Berlin. Tiedje, J.M., S.A. Boyd and B.Z. Fathepure. 1986. Anaerobic degradation of chlorinated aromatic hydrocarbons. Dev Indus. Microbiol. in press. Wolin, M.J. 1979. The rumen fermentation: A model for microbial interactions in anaerobic ecosystems. Adv. Microb. Ecol. 3:49-77. Chapter Two Pyruvate Metabolism by Strain DCB-l Since its isolation from the chlorobenzoate degrading consortium, strain DCB-l has been challenged with over 55 different substrates, of which only pyruvate was able to support good growth [6,12]. Rumen fluid was found to be stimulatory to growth, and 20% rumen fluid alone could support a small amount of growth, as could malate. In an attempt to stimulate growth on some of these substrates, exogenous hydrogen was supplied in the culture headspace, with little effect except that growth on pyruvate was inhibited [6]. Various electron acceptors were also added by Linkfield [6], to determine whether they would allow growth with different carbon sources. Again, the only carbon source that supported growth was pyruvate. Sulfate was not found to stimulate growth, although sulfite and thiosulfate did. Fumarate also resulted in some stimulation of growth. When DCB-l was transfered to a medium containing pyruvate and 3-chlorobenzoate, the addition of oxygenated sulfur anions as electron acceptors was found to inhibit dechlorination [6]. It seems unlikely that pyruvate is the natural substrate of this bacterium. Pyruvate is not a common extracellular substrate available in anaerobic environments, and was not found to be a 14 product of benzoate degradation by the consortium [M.Kane, unpublished data], or by any isolated member of it [12]. Preliminary observations of the pyruvate fermentation products of DCB-l [6] indicated that although the major product was acetate, other unidentified peaks appeared on HPLC chromatograms, suggesting that the fermentation was more complicated than simple cleavage of pyruvate to acetate and C02. Furthermore, the addition of H2 and fumarate caused accumulation of formate and succinate [6]. Although different extinction coefficients of compounds could influence peak size, the amount of product in unknown peaks appeared to be greater than the amount of pyruvate used, suggesting that CO might be fixed by DCB-l. 2 The present study was undertaken to increase understanding of the metabolism of pyruvate by this organism, in hopes that this could provide some clues on the natural trophic relationships of strain DCB-l with the rest of the chlorobenzoate degrading consortium members. Materials and Methods Bacterial Culture Conditions. All growth media were based on the basal salts medium previously described [12], which is listed in Table l. Pyruvate (0.2% w/v) was added as the carbon source. Cultures were maintained in this pyruvate medium with 10% clarified rumen fluid added. 3-Chlorobenoate (800 uM) was added to the medium to determine if dechlorination occurred. No rumen fluid was added to cultures used for fermentation experiments. Because the addition of thiosulfate or hydrogen was previously observed to alter the fermentation products [6], all fermentation experiments included four treatments: pyruvate, with and without hydrogen, and pyruvate plus 10 mM thiosulfate, with and without hydrogen. The cultures were grown in 160 ml serum bottles with butyl rubber stoppers. The medium was reduced either with 4 mmol/L Na sulfide, just before adding to culture bottles, or just before inoculation, by adding 0.1% Na dithionite, dropwise, until the indicator was decolorized (see Appendix). The headspace of the cultures, unless otherwise noted, consisted of 20% CO2 and 80% N2 added during bottling. When hydrogen was added, the bottles were repeatedly evacuated and flushed with a 20% C02, 80% H2 mixture through a sterile needle and 0.2 um filter unit, using a Hungate Table 1. Compound KH PO K fiPo: Nfi c1 Ca012.2H 0 MgCl .6H 0 FeCl .4H 0 MnCl .4H 0 H B0 2801 CaCl .6H 0 NiCl .2H 0 CuCl2 2 NaMo .2H 0 vitamin salution [15] NaHCO resazarin NNNN NNUNNN l7 Mineral salts medium [12]. Amount per liter 0.27 g 0.35 g 0.53 15 mg 20 mg 4 mg 0.5 mg 0.05 mg 0.05 mg 0.05 mg 0.05 mg 0.03 mg 0.01 mg 2.4 1.0 18 gassing manifold [3]. All cultures were incubated at 37°C, in the dark, and were not shaken. Stock cultures were maintained by simultaneous transfer of 5% inoculum into both fresh maintenence medium (described above) and dechlorination medium. Prior to transfer, each culture was checked for dechlorination activity by HPLC, and for purity by microscopy. Product curve (figures 2-5) and cell yield experiments were inoculated, using sterile syringes and needles, with 5 ml transfers of stock cultures into 50 m1 fresh medium. Each treatment included four replicates. Samples were periodically removed from cultures by sterile syringe for fermentation product analysis (see below). Cell yields were determined by measuring protein concentration by the method of Lowry, after alkaline digestion of the cells, as described in the American Society for Microbiology Manual of Methods for General Bacteriology [2]. Cell dry weight was assumed to be twice the weight of the measured protein. For all other experiments, grown cultures were transferred to sterile centrifuge bottles in an anaerobic glove box. The bottles were sealed before being removed and centrifuged at 16,000 x g for 20 min., then returned to the glove box before opening. The pellets were resuspended in 5 m1 of culture medium and injected into serum bottles of fresh medium. To determine the fermentation balance, active cultures were transferred, in triplicate, to fresh pyruvate medium, containing either sodium (140)-bicarbonate or sodium (2-14C)-pyruvate, and sampled periodically until sufficient product had accumulated for analysis. When sufficient product had built up, the labeled cultures were sampled and analyzed for organic acids and labeling pattern, as described below. The cultures were sampled as early as possible to minimize the influence of growth on the fermentation balance. Analytical Methods. Organic acids were determined by high pressure liquid chromatography, using a Biorad Aminex Ion Exclusion HPX-87H column. The mobile phase was either 0.007 or 0.008 N H2804. Analysis was carried out using an automated Shimadzu LC-6A system with UV spectrophotometric detector, coupled to a Gilson model 202 fraction collector. Detection was by UV absorption at 210 nm, with a flow rate of 0.6 mL/min and column temperature of either 35°C or 60°C. Samples to be analyzed were acidified with 2.5 N H2804, filtered through a 0.45 um pore size filter, and transferred into clean 1.5 mL autosampler vials for automated injection. Chlorobenzoate and benzoate were determined by HPLC with a Hibar LiChrosorb, 10 um RP—18 column. The mobile phase was a 70:40:13 mixture of water, methanol and acetic acid, diluted with methanol. The chromatograph was a Varian 5000 HPLC coupled to a 20 Hitachi model 100-40 spectrophotometer with an Altex 100-55 flow through cell. Detection was by UV absorption at 284 nm with flow rate of 1.5 ml/min, using a 50:50 mix of the above solution with methanol. Samples to be analyzed were filtered through a 0.45 um pore size filter before injection. Radioisotope Methods. Sodium (14C)-bicarbonate was obtained from Research Products International (Mount Prospect, IL), and diluted to achieve a solution containing 50 uCi/ml with sterile 0.1N KOH. A total of 50 uCi label was added to each culture by syringe, just prior to inoculation and after any gas additions, and neutralized with an equal volume of 0.1N HCl. Sodium (2-14C)-pyruvate, was obtained from Amersham Corp and dissolved in sterile phosphate buffer solution to an activity of 5 uCi/ml. A total of 5 uCi labeled pyruvate was added to each culture just prior to inoculation. Distribution of labeled substrates into fermentation products was measured by collecting 0.5 or 0.25 minute fractions of the effluent from the organic acid column in scintillation vials containing 0.1 ml 1 N KOH. To each vial, 12 m1 of Safety Solve (Research Products International Corp., Mount Prospect, IL) aqueous scintillation counting cocktail was added. The amount of radioactivity was then measured with a Beckman LSC-8100 liquid scintillation counter. Counting efficiencies of standard solutions of isotope in similar cocktail mixtures were used to 21 determine disintegrations per minute (dpm). Histograms were then constructed, showing the total dpm's in each time fraction, and compared with the original HPLC chromatograms, to determine the amount of radioactivity in each product. The amount of substrate incorporated into each product was calculated from this data and the specific activity: dpm/fraction I ? (moles product collected) specific activity (dpm/mole) The amount of label fixed in bacterial cells was determined by filtering an aliquot of a culture onto a 0.2 um pore size membrane filter, washing with two equal volumes of phosphate buffer, and placing the filters in scintillation vials containing 10 ml Filter Solv (Beckman, Fullerton, CA) liquid scintillation cocktail. The radioactivity on these filters was then counted, and the amount of substrate incorporated into cell material was calculated as shown above, substituting dry weight of cells for moles of product. Results In order to determine a fermentation balance for DCB-l growing on pyruvate, the products, as represented by the HPLC peaks from organic acid analysis (Figure l), were identified. Comparison of the retention times of unknown peaks with those of organic acid standards resulted in identification of lactate, succinate, fumarate, formate, and butyrate. Formate, butyrate, and fumarate were observed only when cells were grown with hydrogen, and lactate was increased more than ten fold under this condition. The identities of these compounds were confirmed by altering the chromatographic conditions, which caused the retention times to change, and the new retention times were again identical to the known standards. Peaks which did not change with time were assumed to be media components, and were ignored. Other peaks appeared to change with incubation time, but were identified as inorganic media components since they were present in media to which no organic substrate was added, and were not detected by gas chromatography with flame ionization detection. The concentrations of fermentation products, under the four sets of experimental conditions, were followed over time. With pyruvate alone, acetate and lactate appeared to accumulate continuously (Figure 2) while succinate accumulated only transiently. When hydrogen was added (Figure 3) more acetate and 22 23 4r‘3 l 2 {T 4 M M :— I C l I Figure 1. Typical chromatogram of DCB-l fermentation products LoNr—I> injection peak pyruvate lactate fumarate H acetate medium components inorganic medium components (see text) 24 Figure 2. Accumulation of products during growth of DCB-l on 0.2% pyruvate. Data points are means of 4 replicate cultures. 25 302.95 523583... 302.55 .N v.59... cozonao:_m>oo mu om n. o— r _ -ID 0 30:30:." I . 3300. film In .M. 3330 Ole roé ('l/IOUJLU) uononueouoo 26 Figure 3. Accumulation of products during growth of DCB-l on 0.2% pyruvate, with headspace of 20% CO and 80% H2. Data points are means of 4 rgplicates. 27 on cement»: 5:5 Bogota cos—5:250... 302:3. .n 0.52... mN L. ‘ ON cozonao:_m>oo m— o. m o _ _ _ od :md to.— 10; rod Im.N 323.5 I :o.n 3958 I . 3302 film 10 n 3380 Ole ('l/IOUJUJ) U0140J4ueouoo 28 lactate accumulated. Butyrate also accumulated, while formate accumulated transiently. When thiosulfate was present (Figure 4), all of the products appeared to accumulate only transiently, with the possible exception of lactate, which accumulated to only very low levels. The treatment with both thiosulfate and hydrogen added (Figure 5) shows a fermentation pattern similar to that of the thiosulfate alone treatment, with the exception that lactate accumulated as it did in the treatment containing pyruvate plus H2. The cell yield, in grams dry weight cells per mole pyruvate utilized, was 13.6 (S.D. 1.9) when grown with pyruvate alone. In the presence of exogenous hydrogen, the yield decreased to 4.8 (S.D. 0.5). When thiosulfate was present, the yield was 20.5 (S.D. 4.0), and when exogenous hydrogen and thiosulfate were present, the yield was 16.7 (S.D. 2.8). To determine if the organism could fix CO DCB-l cultures 2, were incubated with 14C bicarbonate. After incubation, both the cells and culture filtrate, (from which labeled bicarbonate had been removed by bubbling CO2 through it) were found to contain radioactivity (data not shown) indicating that CO2 had to be considered as a substrate, although not capable of supporting growth in the absence of pyruvate [6]. Examples of the 14C labeling patterns in fermentation products of pyruvate and bicarbonate, are shown in figures 6 and 29 Figure 4. Accumulation of products during growth of DCB-l on 0.2% pyruvate with lOmM thiosulfate added. Data points are means of 4 replicates. 3O 30:082.; 53 Eugen. cozoucoctou 30>..txn. .0 3:9... cosoaaoc_m>00 m— o— m 30595: I 305003 «In 3300. min 3330 olo Ind Io..v (1/loww) uononueouoo 31 Figure 5. Accumulation of products during growth of DCB-l on 0.2% pyruvate with 10 mM thiosulfate added and headspace containing 20% CO and 80% H2. Data points are means of 4 raplicates. 32 .5033: 0:0 30:03:: .93 30:03.". 5330250... 302:)". .n 0.53... on 00:09:05 m>00 mu om 2 o. m o _ h\-m]] - b — 0.0 1nd 10.— 10.— led InN 333.3 I .. . 33083 I o M. 305003 I . 3300. film In n 33000 To (“l/IOqu) uopnnuaouoo 33 7. Label from pyruvate was incorporated into lactate, acetate, succinate, formate, and cell material. Although only the second carbon atom in pyruvate was labeled, pyruvate found in cell material and in acetate was assumed to be incorporated in two-carbon units (via acetyl CoA). The label from bicarbonate was incorporated into succinate, acetate, formate, and cell material. Butyrate and fumarate did not accumulate to high enough levels during this short incubation to be measured. The proportions of each labeled substrate found in each product are shown in Tables 2,4,6, and 8. Both with and without thiosulfate, the proportion of CO2 derived carbon in excreted acetate carbon is about 33%, while with the addition of H2, the proportion is reduced to 10% to 15%. Whether or not hydrogen is present, CO2 derived carbon makes up about 70% of the cell carbon produced without thiosulfate, but only about 40% of the cell carbon produced with thiosulfate. This isotopic composition data, along with the chromatographic data on organic acids, was used to calculate the fermentation balance shown in Table 3. It was assumed that the formula for cell material was CH20 [3]. Although this balance accounts for the carbon fairly well, 10% (without thiosulfate) to 20% (with thiosulfate) of the added oxygen remains unaccounted for, while there is excess hydrogen found in the products. Because thiosulfate and hydrogen were observed to affect growth of DCB-l [6], additional experiments were carried out as 34 Figure 6. Typical amounts of radioactivity in chromatographic fractions of DCB-l culture medium after 10 days rowth. Radioactivity was introduced as 2- C pyruvate. 35 ON 302:3”. 02000.. .30003n. 00:35.50“. Flmoo :. 333000.03. .m 0.50.... 0E: .5520 2 OF m — Emil! — l—F—LI - - hull 0m.- ..l O 00. . noon 0 . d H W .83 u . e . D r 3 . u. - r room, My - . D . O . D... - O - xooow u .Sa H 0m0 fl 36 Figure 7. Typical amounts of radioactivity in chromatographic fractions of DCB-l culture medium after 10 da growth. Radioactivity was introduced as C bicarbonate. 37 30.89005 00.30.. m— o— :n 000 EChEECCCCCD DC: EB .02.. on :8 case O O 0 L0 coop oom— I I I I l I I I I I I l l l l I B U I looom room“ .3309... 8530050“. leoo :. 333000.03. K 050... 0:... c0320 uolioon MODS U! WdC] 38 above, with 10 mM thiosulfate in the medium, and a 20% C02, 80% N2 headspace (Tables 4 and 5); with no thiosulfate in the medium and 20% C02, 80% H2 headspace (Tables 6 and 7); and with 10 mM thiosulfate and 20% C02, 80% H2 in the headspace (Tables 8 and 9). 39 Table 2 Fraction of DCB-l fermentation products derived from pyruvate and from bicarbonate , during pyruvate fermentation mol l4C substrate % product carbon incorporated / derived from each compound mol product by HPLC substrate pyruvate CO2 pyruvate CO2 pyruvate 1.0 0.06 100 2.0 lactate 1.08 0 108 0 succinate 1.0 0.94 75 24 fumarate * * * * acetate 0.67 0.38 67 # l9 formate * * * * butyrate * * * * * not determined or not detected # assuming 2 carbons incorporated 1 contributions of substrates to individual products were determined from radiolabel measurements 40 Table 3 DCB-l fermentation balance; growth on pyruvate mmol mmol C mmol H mmol 0 Products recovered: pyr 14.26 42.78 42.78 42.78 lac ND ND ND ND succ 0.46 1.85 1.85 1.85 fum ND ND ND ND ace 1.43 2.86 4.29 2.86 for ND ND ND ND but ND ND ND ND CP* 2.54 5.07 10.14 5.07 CC* 11.83 11.83 23.66 11.83 Summary: total 64.39 82.73 64.39 added pyr 54.54 54.54 54.54 calc CO # 12.77 0.00 25.53 excess groducts -2.91 28.19 -15.68 * CP=moles pyruvate 2-C detected in cell material CC=moles COZ-C detected in cell material # calculated from % composition in table 2 ND = Not Detected 41 Table 4 Proportion of DCB-l fermentation products derived from pyruvate and from bicarbonate , during growth on pyruvate with thiosufate compound pyruvate lactate succinate fumarate acetate formate butyrate mol l4C substrate % incorporated mol product by HPLC pyruvate CO 2 1.01 0.07 .91 0 * * * * 0.71 0.33 * * * * * not determined or not detected # assuming 2 carbons incorporated product carbon derived from each substrate pyruvate C02 101 2.33 91 0 * * * * 71 # 16.5 * * * * contributions of substrates to individual products were determined from radiolabel measurements 42 Table 5 DCB-l fermentation balance; growth on pyruvate with 10 mM thiosulfate mmol mmol C mmol H mmol 0 Products recovered: pyr 12.22 36.67 36.67 36.67 lac ND ND ND ND succ 0.99 3.95 3.95 3.95 fum 1.01 4.04 2.02 4.04 ace 2.20 4.40 6.60 4.40 for ND ND ND ND but ND ND ND ND CP* 4.51 9.03 18.06 9.03 CC* 6.60 6.60 13.21 6.60 Summary total 64.71 80.53 64.71 added Pyr 54.54 54.54 54.54 calc CO # 9.33 0.00 18.95 excess Broducts 0.84 25.99 -8.78 * CP=moles pyruvate 2-C detected in cell material CC=moles COZ-C detected in cell material # calculated from % composition in table 4 ND = Not Detected 43 Table 6 Proportion of DCB—l fermentation products derived from pyruvate and from bicarbonate , during growth on pyruvate with 20% C02, 80% H 2 mol l4C substrate % product carbon incorporated / derived from each compound mol product by HPLC substrate pyruvate CO2 pyruvate CO2 pyruvate 1.00 0.04 100 1.3 lactate 1.14 0 114 0 succinate 1.0 0.9 75 22.5 fumarate * * * * acetate 0.91 0.15 91 # 7.5 formate 0 0.34 (66)@ 34 butyrate * * * * * not determined or not detected # assuming 2 carbons incorporated @ assumed from C02 data 1 contributions of substrates to individual products were determined from radiolabel measurements 44 Table 7 DCB-l fermentation balance; growth on pyruvate with excess hydrogen mmol mmol C mmol H mmol 0 Products recovered: pyr 7.04 21.11 21.11 21.11 lac 0.93 2.79 3.72 2.79 succ ND ND ND ND fum ND ND ND ND ace 1.13 2.27 3.40 2.27 for 0.42 0.42 0.42 0.83 but 0.04 0.17 0.30 0.09 CP* 3.10 6.20 12.39 6.20 CC* 12.10 12.10 24.20 12.10 Summary: total 45.04 65.54 45.37 added pyr 21.10 21.10 21.10 calc C0 # 12.60 0.00 5.05 excess groducts 11.34 44.44 19.22 * CP=moles pyruvate 2-C detected in cell material CC=moles COZ-C detected in cell material # calculated from % composition in table 6 ND = Not Detected 45 Table 8 Proportion of DCB-l fermentation products derived from pyruvate and from bicarbonate , during growth on pyruvate with 10 mM thiosulfate and 20% C02, 80% H2 mol l4C substrate % product carbon incorporated / derived from each compound mol product by HPLC substrate pyruvate C02 pyruvate CO2 pyruvate * 0.03 (100)@ 1.0 lactate * 0 (100)@ 0 succinate * 1.2 (75)@ 30 fumarate * * * * acetate * 0.10 (95)#@ 5 formate * 0.40 (60)@ 4o butyrate * * * * * not determined or not detected # assuming 2 carbons incorporated @ assumed from C02 data 1 contributions of substrates to individual products were determined from radiolabel measurements 46 Table 9 DCB-l fermentation balance; growth on pyruvate with 10 mM thiosulfate and excess hydrogen mmol mmol C mmol H mmol 0 Products recovered: pyr 12.19 36.57 36.57 36.57 lac 1.71 5.13 6.84 5.13 succ ND ND ND ND fum 0.79 3.18 1.59 3.18 ace 2.09 4.18 6.27 4.18 for 0.59 0.59 0.59 1.19 but ND ND ND ND CP* 6.28 12.57 25.14 12.57 CC* 10.85 10.85 21.69 10.85 Summary: total 73.06 98.69 73.66 added Pyr 54.54 54.54 54.54 calc CO # 12.05 0.00 24.1 excess roducts 6.47 44.15 -4.9 * CP=mmoles pyruvate 2-C detected in cell material CC=mmoles COZ-C detected in cell material # calculated from % composition in table 8 ND = Not Detected Discussion Strain DCB-l has been tentatively considered to be a sulfidogen because its growth is stimulated by thiosulfate and sulfite and because of its strictly anaerobic growth habit [6]. In addition, recent DNA-DNA hybridization data (J.Jansson and J.M. Tiedje, unpublished data) show that strain DCB-l is related to Desulfovibrio sp. but not to Clostridum, Bacteroides, or Methanospirillum species. The fermentation product data shown in Figures 2-5 support this view, since in the presence of thiosulfate, the fermentation pattern was clearly altered. In contrast to the pyruvate fermentation without thiosulfate, acetate, the major product, accumulated only transiently, and lactate accumulated to only very low levels. In addition, both succinate and fumarate accumulated transiently, and then disappeared from the growth medium. This suggests that use of these compounds was reduced after cultures were shifted to a medium containing thiosulfate. These transient products were probably generated by enzymes induced during growth of the inoculum without thiosulfate. Enzymes of the tricarboxylic acid cycle are common in anaerobic bacteria, although in most, the cycle is incomplete 47 48 [3,5]. Strain DCB-l has been shown to utilize malate [6], and although it was not detectable by the assay used here, it would provide a reasonable link between pyruvate and the other four carbon compounds found. Malic enzyme, which adds C02 directly to pyruvate, forming malate, has been found in all sulfidogens examined [8,10] and, the radioisotope data (Table 2) showed that the succinate is indeed formed from one pyruvate and one C02. Apparently then, at least when growing without oxygenated sulfur electron acceptors, one pathway of pyruvate utilization in strain DCB-l is: pyruvate + CO2 --> (malate —->) fumarate --> succinate The first step would require NADPH. ATP could theoretically be generated via electron transport phosphorylation from the reduction of fumarate to succinate. The fate of the succinate was then uncertain; succinyl CoA synthase is one of the TCA cycle enzymes not found in Desulfovibrio [5], although cyclic systems involving fumarate and succinate have been proposed in these organisms [8]. The fermentation balances calculated (Tables 3, 5, 7, and 9) are incomplete, since the oxygen added in the substrates is not all accounted for in the products, and there is more hydrogen found in the products than in the substrates. It is possible that the missing oxygen was lost to water in hydrolysis reactions, but 49 this assumption would increase the discrepancy of hydrogen. Strain DCB-l has been shown to consume H2 [6], but H2 was not detected (<10 ppm)during the short incubation periods used here. The main effect of exogenous hydrogen on the fermentation is to divert pyruvate to lactate. This is probably one reason why hydrogen inhibits growth [6], since as long as the hydrogen level is high, the lactate is unlikely to be re-oxidized to pyruvate, and thus is unavailable as a growth substrate. Another effect of hydrogen appears to be to divert more carbon to acetate, rather than to cell material, and this too could result in reduced growth. Finally, butyrate also accumulates under hydrogen, although there appears to be a lag period before it is produced. If the cell yields reported in "Results" section are recaclulated by not counting the pyruvate diverted to lactate, the cell yields become 15.1 (S.D. 2.3) without hydrogen, and 7.8 (S.D. 0.4) with hydrogen. When thiosulfate is present, these corrected yields are 21.3 (S.D. 4.2) without hydrogen, and 23.4 (S.D. 4.7) with exogenous hydrogen. The diversion of carbon to lactate then, can account for only part of the deleterious effect of exogenous hydrogen, without thiosulfate, but with thiosulfate, it can account for the entire amount. The formation of lactate, with exogenous hydrogen, showed that strain DCB-l possesses a lactate dehydrogenase enzyme, as do most sulfidogens, although why growth did not occur on lactate is 50 unclear (see Chapter 3). The mechanism of butyrate formation is unknown, but it is probably derived from acetyl 00A [3]. Because butyrate was not produced early in the fermentation, its isotopic composition was not determined in the radio-labeled experiments. The production of formate is also unexplained; since it only accumulated under excess hydrogen, it may not be a "normal" product of this organism. Formate production by sulfidogens has been reported [8], but its role is unknown. The radio-labeling experiments (Tables 2, 4, 6, and 8) show that one-third of the formate comes from C02, and presumably the other two thirds comes from pyruvate. The radioisotope experiments summarized in Table 2 show that CO2 is definitly fixed into both excreted products and into cell material, although strain DCB-l is not capable of growth utilizing CO2 and H2 alone [6]. This sort of CO2 fixation, described as "incomplete autotrophy" or "mixotrophy" is common in sulfate-reducing bacteria [1,9,13,14]. Carbon dioxide, and usually acetate, are used as carbon sources when energy comes from oxidation of some other molecule. Growth of Desulfovibrio has been described using H2 and SO 2- 4 with acetate and C02 as the source of carbon [1], although DCB-l as energy source redox partners, does not grow under these conditions. Desulfovibrio species have also been shown to grow with CO2 and acetate as carbon source while using organic compounds, such as lactate, formate or even 51 yeast extract, as hydrogen donors [9,13,14]. The suggested route of CO2 fixation was the formation of pyruvate from acetate and C02. When Desulfovibrio grows on acetate and CO as sole carbon 2 sources, 70% of cell material is found to be derived from acetate, and 30% from C02 [1]. With formate as the hydrogen donor, up to 50% of cell carbon is derived from CO2 [14]. When lactate was the hydrogen donor, approximatly one-third of cell carbon was formed from C02, one-third from acetate, and one-third from lactate [14]. Assuming that each mole of pyruvate 2-C detected in cell material represents two pyruvate carbons (i.e. acetyl CoA), when DCB-l was grown without thiosulfate, only one-third of cell carbon was derived from pyruvate, while two-thirds was derived from CO2 (Tables 3 and 7). When grown with thiosulfate, about 60% of cell carbon was derived from pyruvate, while about 40% was derived from CO2 (Tables 5 and 9). This is further evidence that different metabolic mechanisms are active when DCB-l grows with thiosulfate as an energy source. The ratio of 002 derived carbon to total carbon in cells of DCB-l is not the same as this ratio in excreted acetate, or as this ratio in total products, in any treatment. This suggests that different routes of carbon metabolism lead to cells than to products. It can be seen from Tables 2 through 9 that this ratio for cell carbon is affected by the addition of thiosulfate, but 52 not by the addition of hydrogen, while for excreted acetate, the ratio is affected by hydrogen addition, but not by thiosulfate addition. This is consistent with the idea that acetate accumulation in the thiosulfate grown treatments is due to enzymes previously induced. In any case, the evidence suggests either that the systems leading to cell carbon do not involve acetyl CoA, or that they are physically separated from those involved in energy metabolism (i.e. excreted products). This idea of physical separation has been put forth for Desulfovibrio for the same reasons [7,13]. One obvious method of CO2 fixation is through the four-carbon pathway leading to succinate. This cannot be the major route to cell material however, since there would always be more pyruvate carbon than C02 carbon. Figure 8 shows the proposed routes of assimilation of pyruvate and CO2 into strain DCB-l through two acetyl CoA pools. The major difference from mechanisms described for Desulfovibrio is the formation of acetate from C0 . The acetyl CoA pool on the 2 pathway from CO2 might be bound to an enzyme, as opposed to the free pool, or it might be in a different part of the cell. When thiosulfate plus hydrogen is used as an energy source, apparently pyruvate-derived acetyl CoA is diverted from ATP production to cell biosynthesis, resulting in a higher pyruvate carbon/cell carbon ratio. The presence of high levels of exogenous hydrogen 0.000 <00 .3000 03 300300 N: + N00 5.; 7000 a. 802.508 .688 .0 2:0: 6.030;. :00 33000 W \7 300.0. + <00 .3000 53 \7 3300. . 0.0>:.>0 33000 300.0000 \7 <00 .3000 300053 \7 30.0.3 54 appears to decrease the amount of CO derived acetate excreted 2 into the medium. The regulatory mechanism however, is unclear, and will probably require careful measurements of hydrogen consumption to determine. An alternative scheme of carbon assimilation is shown in figure 9. This more conservative system, with a single acetyl CoA pool, assumes that the CO derived carbon in the free acetate pool 2 is due entirely to exchange reactions. Added hydrogen decreases these exchange reactions, presumeably by driving the pathway more strongly in a direction that does not favor the exchange. The CO2 derived carbon found in cells then, would be due to some entirely different route that did not involve the acetyl CoA pool. The exchange of acetate with CO2 could be measured using 14C tracers, to see if the extent of exchange is consistent with this hypothesis. Although thiosulfate enhances growth of DCB-l, it has been found to inhibit the reductive dechlorination of 3-chlorobenzoate, the property for which this organism was originally isolated. The dechlorination mechanism evidently lies along a pathway not induced during thiosulfate reduction, for instance the fumarate/succinate pathway, or else it competes unfavorably with thiosulfate reduction for reducing equivalents. Although strain DCB-l can apparently gain energy from the reduction of thiosulfate and sulfite, it is unable to grow at the 55 .000 <00 .3000 000 5.3 7.000 .3 0052.00.30 000000 .0 0.50.“. 3233 .0 .0 30E =00 N: + N00 3386 fl .\7 300:3 + <00 .3000 3300. . 300.0000 \7 300053 A3235 000>E>0\ f N00 56 expense of sulfate reduction [6]. This must be either because DCB-l lacks the APS sulfurylase enzyme, required for activation of the sulfate reducing system, or because growth of DCB-l under the culture conditions used to date does not supply enough energy to spare ATP for the formation of APS [ll]. Probably the only way to settle this question will be to assay for APS sulfurylase activity in cell extracts. Strain DCB-l then, is shown to be similar to the genus Desulfovibrio in that it reduces oxidized sulfur compounds for energy, contains desufoviridin and cytochrome c3 [6], does not form spores, fixes CO2 by mixotrophic reactions, apparently possesses a partial TCA cycle, has the ability to grow in the absence of sulfur electron accepters by utilizing pyruvate, and because it reacts positively to stringent DNA-DNA hybridization tests. Differences of DCB-l from Desulfovibrio include its lack of motility, unique morphology (the "collar" structure), its ability to reductively dehalogenate chlorobenzoate, formation of acetate from 002, and its apparent inability to utilize either sulfate or lactate. It seems likely that strain DCB-l will eventually be placed in a separate, but closely related genus to Desulfovibrio. 10. ll. Literature Cited Badziong, W., R.K. Thauer, and J.G. Zeikus. 1978. Isolation and characterization of Desulfovibrio growing on hydrogen plus sulfate as the sole energy source. Arch. Microbiol. 116241-49. Gerhardt, P., ed. 1981. Manual of methods for general bacteriology. American Society for Microbiology. Washington, D.C. Gottschalk, G. 1986. Bacterial Metabolism, 2nd ed. Springer-Verlag. New York. Hungate, R.E. 1968. A roll tube method for cultivation of strict anaerobes, p. 117-132. In J.R.Norris and D.W. Ribbons (ed.), Advances in microbiology, vol. 35. Academic Press, Inc., New York. Lewis, A.J. 1977. The tricarboxylic acid pathway in Desulfovibrio. Can.J. Microbiol. 23:916-921. Linkfield, T.G. 1985. Anaerobic reductive dehalogenation: the lag Period preceeding haloaromatic dehalogenation, enrichment of sediment activity, and the partial characteriazation of a dehalogenating organism, Strain DCB-l. Ph.D. Thesis, Michigan State University. Mechalas 3.9 and Rittenberg, S.C. 1960. Energy Coupling in De/su/zgovrbuo dameéwucaws'. J. Bacteriol., 80:501-507. Postgate, J.R. 1979. The sulfate reducing bacteria. Cambridge University Press, Cambridge. Rittenberg, S.C. 1969. The roles of exogenous organic matter in the physiology of chemolithotrophic bacteria. Adv. Microb. Physiol. 3 159-196. Thauer, R.K. and W. Badziong, 1980. Respiration with sulfate as electron acceptor. In Knowles, C.J. (ed.) Diversity of bacterial respiratory systems. CRC Press, Boca Raton, Florida. Thauer, R.K., K. Jungermann, and K. Decker, 1977. Energy conservation in chemotrophic anaerobic bacteria. Bact. Rev. 41:100-180. 57 12. 13. 14. 15. 58 Shelton, D.R. and J.M. Tiedje. 1984. Isolation and partial characterization of bacteria in an anaerobic consortium that mineralizes 3-chlorobenzoic acid. Appl. Environ. Microbiol. 48:840-848. Sorokin, Y.I. 1966. Sources of energy and carbon for biosynthesis in sulfate-reducing bacteria. Mikrobiologiya, 35:761-766. Sorokin, Y.I. 1966. Investigation of the structural metabolism of sulfate-reducing bacteria with C. Mikrobiologiya, 35:967-977. Wolin. E.A., M.J. Wolin, and R.S. Wolfe. 1963. Formation of methane by bacterial extracts. J. Biol. Chem. 238:2882-2886. 1'”. Chapter Three The Substrate Range and Syntrophic Associations of Strain DCB-l Although strain DCB-l has been maintained in pure culture for five years, no substrate other than pyruvate has been found to support its continued growth in defined media. Limited growth has been achieved with rumen fluid as a substrate. Growth slightly above this level has been observed in media containing rumen fluid and thiosulfate along with either D-arabinose, acetate, lactate, or glucose [5]. The organism has also been grown as part of a three membered syntrophic community, in which 3-chlorobenzoate was the only carbon source, and benzoate, acetate, and methane were the only products observed [3]. This association was described as a food web, because hydrogen from benzoate oxidation was utilized by DCB-l for reductive dechlorination. Since strain DCB—l can use neither 3-chlorobenzoate or benzoate directly as a carbon source, there must be a subsequent carbon product of benzoate metabolism that is used by DCB-l. Because lactate is readily formed from pyruvate when hydrogen is present (see chapter 2) it seems evident that DCB-l possesses all the enzymes necessary to oxidize lactate, and should be able 59 60 to utilize it as a substrate, like most other sulfidogens, when hydrogen concentrations are low. Since the organism was originally isolated from a consortium containing two other hydrogen scavenging organisms [7], it seems reasonable to assume that low hydrogen levels are a characteristic of the "normal" habitat of DCB-l. For this reason, the substrate range of the organism could be more versatile within syntrophic associations than in pure culture. The following experiments were undertaken in order to try to expand the known range of substrates for strain DCB-l, and to attempt to identify its carbon substrate in the 3-chlorobenzoate degrading consortium. Materials and Methods Bacterial strains and culture conditions. Strain DCB-l was grown in pure culture and in coculture with Methanospirillum strain PM-l, and with the benzoate oxidizing organism, strain BZ-2, described previously [7]. Cells were grown in the mineral medium described in chapter 2, with 0.2% of the appropriate carbon substrate added, either with, or without lOmM Na thiosulfate. For cultures incubated with acetate, the headspace was either 20% C02 and 80% N or 20% C0 and 80% H otherwise the headspace was 2 2 2’ 20% CO2 and 80% N2. The media were reduced by adding Na dithionite to a final concentration of 500 uM. Cells were grown and maintained in 160 ml serum bottles, with butyl rubber stoppers, as described earlier. For optical density measurements, cultures were grown in 24 ml anaerobic culture tubes with butyl rubber stoppers. All incubations were in the dark, at 37°C in static cultures. Divided consortium methodology. For the divided consortium experiment, the mineral medium with 3 mM 3-chlorobenzoate and 10% rumen fluid was used. The experiment was carried out in a two chambered membrane spinner flask (Bellco Glass, Inc., Vineland, 61 62 NJ), in which the two chambers were separated by a 0.2 um pore sized membrane filter. This flask was adapted for anaerobic use by sealing the tops with plastic tape to make an air-tight seal, and by sealing #0 rubber stoppers into the side-arms with the same tape. The apparatus was sterilized, with ventilation, then the seperatly sterilized medium was added, and 20% C02, 80% N2 gas was bubbled through the medium after passing through a sterile 0.2 uM filter and sterile needle. The cultures were then inoculated, and the experiment was incubated at 37°C in the dark on a rotary shaker at 60 rpm. Analytical methods. Samples were removed for analysis by sterile syringe, and filtered through 0.45 micron pore-size membrane filters. Samples were analyzed for fermentation products and benzoates by HPLC as described earlier (chapter 2). Growth curves were obtained by measuring the optical density of tube cultures in a Turner model 350 spectrophotometer at 660 nm. Protein determinations were by the method of Lowry, after alkaline degestion of the cells, as described in the American Society for Microbiology Manual of Methods for General Bacteriology [11. Results In an attempt to determine the nature of the carbon exchange between members of the 3-chlorobenzoate degrading consortium, its members were grown separated by a 0.2 micron pore size membrane. Strain DCB-l, the dechlorinating organism, was on one side, and the benzoate degrader and a methanogen were on the other. 3-Chlorobenzoate was degraded, as shown in Figure 1, although at a much slower rate than occured when the organisms were intimately associated (see for example, Figure l in the appendix). Cell mass did not remain constant during the incubation, but the overall rate of 33 uM 3-chlorobenzoate degraded per day can be compared to 70 uM per day, which was measured in a similar consortium incubated in a bottle. After two months, about 2 mmol of the 3-chlorobenzoate was degraded, with no detectable accumulation of benzoate. During earlier attempts to start the divided cultures, the membrane was broken, and these cultures grew at a visibly more rapid rate. This observation indicates that the medium and conditions in this vessel were adequate for more normal growth, and that the physical separation of the members of the consortium was responsible for the slow growth and dechlorination. 63 Figure l. 64 Dechlorination by membrane-divided 3-chlorobenzoate degrading consortium. SYMBOLS: circles, concentration in chamber containing strain DCB-l, squares, concentration in chamber containing strain BZ-2 and Methanospirillum strain PM-l 65 050000000 000_>_0l0003502 .3 00200002000 .F 0000: 00000000_m>00 no 00 mm on 0* 0* mm on ma 0N mp 0— m p _ L _ . . . . . _ . . plmoo Ola Nle film 0 00.0 r000 r000 1000 .00.. $0.. -00.. -0: .80 rag 0.00.0 .03 , [00.0 ('1/loww) eioozueg lQ—g 66 Similar experiments were attempted in apparatus consisting of dialysis bags, containing the BZ-2/PM~1 coculture, suspended in DCB-l cultures in stoppered Ehrlenmeyer flasks. These dialysis bags had a greater surface area than the membrane in the spinner flask, and it was hoped that this would allow a faster rate of growth. All attempts to keep these flasks anaerobic however, failed. The products of fermentation were measured over time on each side of the membrane. The analysis was complicated by the presence of rumen fluid, which contains all of these products in appreciable amounts, however only those compounds which changed appreciably are shown. Succinate, pyruvate, and formate were present in trace amounts initially, but quickly disappeared. Acetate accumulated continuously, as shown in Figure 2. Although over 4 mM acetate was present in the original medium, due to the rumen fluid, it increased to a final concentration of over 8 mM. A small amount of lactate was present in the initial medium, but as can be seen in Figure 3, it episodically increased and decreased througout the fermentation. The major concentration of lactate was always in the chamber containing strain DCB-l, and the net change was very small. Propionate, although present in much larger quantity, also increased and decreased throughout the incubation (Figure 4). The concentration of propionate varied greatly with time, although Figure 2. 67 Acetate production by divided 3-chlorobenzoate degrading consortium. SYMBOLS: circles, concentration in chamber containing strain DCB-l, squares, concentration in chamber containing strain BZ-2 and Methanospirillum strain PM-l. 68 0000000000 00230 >0 000000000 3300‘. .N 0002... 00300000_m>00 00 00 mm on 00 0* on on 0N 0N 0F _ _ — p h _ _ p _ _ _ 7000 ole «:3 0:0 0— _ m 0 0 1000. rI000N H.000n w000¢ 10000 H.0000 U0000 U0000 H.0000 r¢0+mp (“I/lown) 93,03,900 Figure 3. 69 Lactate production by divided 3-chlorobenzoate degrading consortium. SYMBOLS: circles, concentration in chamber containing strain DCB-l, squares, concentration in chamber containing strain BZ-2 and Methanospirillum strain PM-l. 70 500000000 00220 03 000000000 000000.. .0 000mm 00000000_m>00 mm 00 mm Om 0.0 00 on on nN 0N @— . _ 0 > _ 0 _ _ _ . 0— m NINm film. «Imoo Ole I 000 1 00¢ 1000 ("l/'own) eioiool Figure 4. Propionate production by divided 3-chlorobenzoate degrading consortium. SYMBOLS: circles, concentration in chamber containing strain DCB-l, squares, concentration in chamber containing strain BZ-2 and Methanospirillum strain PM-l. 72 0000000000 00320 >0 000000000 000020000 .0 00090 00000000_0>00 mm 00 mm 00 00 0* on on nm em mp OF 0 F _ _ _ _ — P _ _ _ _ _ Nle film Timon. Ola I [000.0. (1/|ou.m) eiouogdmd 73 there was a net increase. This product was not obviously associated with one side or the other of the membrane. Butyrate initially declined, but then increased for several days (Figure 5). After two weeks, the butyrate concentration began to decline, but there was still net production of this product. Butyrate concentration was always highest on the DCB-l side of the membrane. The four compounds observed to change in the chamber experiment -- lactate, acetate, propionate, and butyrate -- were used as substrates in separate experiments to determine if they could support growth. Monocultures of strain DCB-l, as well as cocultures of DCB-l with Methanospirillum strain PM-l were incubated with lactate, propionate, and butyrate, with and without thiosulfate. DCB-l was incubated with acetate as a monoculture with and without thiosulfate, with either 20% CO2 80% N2 or 20% CO2 80% H2 in the headspace. Very little increase in optical density was observed with either lactate (Figure 6) or propionate (Figure 7). Most of the treatments in the butyrate medium declined in optical density (Figure 8) except for the coculture with thiosulfate, which increased by about 0.015 O.D. units. Acetate appeared to support a small amount of growth in all treatments (Figure 9), although the treatments with thiosulfate appeared to initially grow slightly more. Figure 5. 74 Butyrate production by divided 3-chlorobenzoate degrading consortium. SYMBOLS: circles, concentration in chamber containing strain DCB—l, squares, concentration in chamber containing strain BZ-2 and Methanospirillum strain PM-l. 75 0000000000 00220 >0 000000000 0000300 .0 00090 00000000_0>00 no 00 mm on n... 0.0 mm on 0N 0N mp 0p m 0 7 _ _ . _ _ 0 0 . 0 _ _ _ 000 . -000 _ . q . . -000 n _ 1.. . (A .. J .. - w ., . 000 a . . ,. ., .- 000 m n a : 0 v w. .. [000. V K ( -00: Nle film. .. . 7000 I. loom. 76 Figure 6. Growth of strain DCB-l on lactate. Each data point is the mean of four observations. 77 00300.. 00 plmoo 0.0000 00 53000 .0 0000.0 00000000.0>00 on ma cu m. o. m o P p 0 P r _ 00.0 rmod 0:000 .000 01 . r000 10rd T r «1 {~00 . 13.0 30:03.5 5.; 003.0000 I .10...0 003.0000 pl20 «In . 00000 0.2.03.5 min 10.0 COEUUO O: I . r0N.0 099 eouquosqv 78 Figure 7. Growth of strain DCB-l on propionate. Each data point is the mean of four observations. 30.00.0000 :0 .Imoo 0.005 .0 530.5 K 0000.... cozon0oc.m>00 79 3 cu m. o. m o _ — p _ b 00.0 .30 -000 100.0 .000 .. [8.0 - .30 11.0 30:03.5 5.; 9.3.0000 olo wo—d 9.3.0000 Flzn. I . 00000 22.325 mum 18.0 00.3.0.3 0... Ole . lewd ogg aouquosqv 80 Figure 8. Growth of strain DCB—l on butyrate. Each data point is the mean of four observations. 81 30030m :0 —lmoo Eobm .0 530.0 .m 0000.“. 00:0000c_m>00 on nN ON 0 P O F n O — — — — — — $0.0 l no.0 I O F .O n. l N _. .0 fi ”Haw—amom-Lu £33 0.5323030 I l¢ Foo ”Law—3000 F'Il I 32.325 50, 010 . cozmuvo CC I 099 eouquosqv 82 Figure 9. Growth of strain DCB-l on acetate. Each data point is the mean of four observations. 83 00300.0 .00 plmoo 0.00% 00 £30.00 .m 0000.... cozon0oc.m>00 ON ON mu.— 0. m 00000 30:03.5 000 0000000... I 00000 0000000... I 00000 300.03.... film 000.000 0: Ole I Inpd 099 aouquosqv 84 Since strain DCB-l has been shown to produce lactate under excess hydrogen, (chapter 2) and to apparently consume it again, (Figure 3) another attempt was made to grow the organism on lactate. Strain DCB-l was inoculated, in triplicate. into lactate medium with and without 10 mM thiosulfate. Cocultures of strain DCB-l and Methanospirillum strain PM-l were also inoculated into these two media, in triplicate. Protein determinations were made at the begining of incubation, and after 30 days (Table 1). Some amount of growth occurred in all treatments, but growth was much greater in the presence of thiosulfate. Microscopic examination showed that in cocultures, Methanospirillum cells greatly outnumbered DCB-l cells. In both monocultures and cocultures containing thiosulfate, the concentration of lactate initially dropped by 5 mM, as shown in Figure 10, but then increased. The concentration in cocultures eventually began to decline again. In monocultures without thiosulfate, there was no appreciable change in lactate concentration. In cocultures without thiosulfate, lactate concentrations did not change for 20 days, but then began to decline, eventually using the greatest amount of lactate. 85 Table 1 Protein determinations in cultures of strain DCB-l and cocultures of strain DCB-l with Methanospirillum strain PM-l growing on lactate Protein in ug/ml (S.D.) Medium Culture Day 1 Day 30 lactate DCB-l 67 (12) 135 (28) lactate DCB-l + PM-l 156 (38) 261 (30) lactat§_ + $203 DCB-l 91 (6) 211 (24) lactat§_ + s o DCB-l + PM-l 183 (26) 403 (5) 2 3 86 Figure 10. Consumption of lactate by strain DCB-l, and cocultures of Strain DCB-l with Methanospirillum strain PM-l. Each data point is the mean of three observations. 87 7000 seam B 238.. 00 000300: .o. 2:90 00300000_m>00 on ON ON 003 0— ~40 p . 300.000.50.421... ole 300.0821 010 p21... I 000.000 00 I Imp row 1mm 3 0101001 se|ouJuJ bulugowe Discussion The object of the divided consortium experiment was to identify carbon compounds passing between the members of the sytrophic association. The products of each organism's metabolism should be present in the highest concentrations in the chamber containing that organism, and lower in the other chamber. By monitoring the concentration gradient (albeit at only two points with this apparatus) the organism responsible for each product should be identified. The apparatus was not wholly satisfactory for this purpose, since benzoate, presumably formed in the rate-limiting step of 3-chlorobenzoate metabolism, was never observed. The introduction of a third chamber, between the other two might provide a better defined gradient, however the growth rate of the consortium may be too slow to produce an observable gradient. Acetate, known to be the main product of benzoate oxidation by strain BZ-2 [3,7], accumulated to a similar extent on both sides of the membrane (Figure 2). A total production of 4 mmol acetate from 2 mmol 3-chlorobenzoate (Figure l) was observed, and is less than the 3 mol acetate per mol benzoate oxidized, which 88 89 has been previously observed [3,7], indicating that up to 1 mmol of acetate may have been consumed. Butyrate and lactate were transiently produced, and their concentrations were highest on the DCB-l side of the membrane, indicating that they were probably produced (as shown in chapter 2) by the dechlorinator. There was a net accumulation of 300 umol of propionate, although it was difficult to assign its production to one side or the other of the membrane. It appears that these three products may have been consumed sequentially, on the DCB-l side of the membrane, in the order butyrate, lactate, propionate. It is possible that the propionate represents an intermediate between benzoate and acetate, and was produced by BZ-2. Spot checks of BZ-2 cocultures (with Methanospirillum) showed that propionate was present (data not shown), but the levels were not measured over the course of complete benzoate oxidation, and propionate was a minor component of the growth medium used. Methane was detected on the BZ-2 side, as expected, but not on the DCB-l side. Quantification of methane production was not possible, because the apparatus developed a slow leak when pressure initially built up on the BZ-2 side. In summary, 3-chlorobenzoate was reductively dehalogenated by strain DCB-l to benzoate. Strain BZ-2 oxidized benzoate to acetate, COZ, and hydrogen, as previously described [3,7], and possibly to propionate as well. Methanospirillum strain PM-l then 90 converted CO2 and H2 to methane. Initially, strain DCB-l may have grown on components of the rumen fluid, including butyrate and lactate, but appears to have consumed acetate (and possibly propionate) from the benzoate oxidation as well. It is difficult to draw definitive conclusions from this experiment, especially since it was performed without replicates. The data is suggestive though, and the experiment should probably be repeated with several modifications. A third, intermediate chamber could be introduced to better define the gradients. Gas traps should be added to each chamber to relieve any pressure buildup, and to quantify methane. Finally, to help clarify the interpretation, the experiment should be attempted in media which do not contain rumen fluid. In growth studies of DCB-l using the substrates identified above, only butyrate (in the presence of a methanogen and thiosulfate) and acetate appeared to be able to support growth (Figures 8 and 9). Butyrate consumers were present in the original 3-chlorobenzoate degrading consortium that these organisms were isolated from [7]. In that environment, DCB-l could have supplied butyrate, which might then have been consumed by the butyrate degraders, rather than being reconsumed, as observed here. 91 It is possible that strain DCB-l can consume these substrates in a mixotrophic reaction [6,8], while obtaining energy from the metabolism of some other substrate. That lactate did not support growth of strain DCB-l either in pure culture or in coculture with Methanospirillum (Figure 6) is surprising, since DCB-l evidently has all the enzymes needed for its use (see chapter 2), and was apparently able to consume the lactate in the divided consortium (Figure 3). It is possible that hydrogen was produced from the culture medium in higher quantities than expected, thus blocking lactate oxidation, or that the methanogen competed with DCB—l for some other compound required for growth, such as C02. The latter possibility is supported by the fact that some growth did occur in similar cultures in 160 ml serum bottles, which had a larger headspace (Table 1). Even in this later experiment however, lactate was not utilized (Figure 10) until very late in the incubation. It is possible that this time lag was required for the methanogen to reduce hydrogen levels sufficiently. These experiments suggest that strain DCB-l can utilize lactate for growth, at least in sytrophic associations, which are probably the rule, rather than the exception in natural environments [2]. The inability of DCB-l to utilize lactate in pure culture however, remains a puzzle. 92 It is worth noting that since this organism has been continuously grown in laboratory culture for five years, it has been trained to exploit the laboratory conditions. The efficiency of the dechlorination reaction has increased from an initial apparent Km of 67 UN to less than 1 uM [9]. Initially, cultures of DCB-l grown on pyruvate rarely reached a density of 0.1 O.D. units [5,7], but recent cultures (see for example, Figure 2 in the appendix) have reached more than twice that density. This selection must be taken into account when attempting to extrapolate the results of laboratory experiments to define the function of the organism in different environments. Literature Cited Balch, W.E., Fox, G.E., Magrum, L.J., Woese, C.R., and Wolfe, R.S. 1979. Methanogens: reevaluation of a unique biological group. Microbiol. Rev. 43:260-296. Bull, A.T. and Slater, H.J. 1982. Microbial interactions and communities. Academic Press, London. Dolfing, J., and J.M. Tiedje. 1986. Hydrogen cycling in a three-tiered food web growing on the methanogenic conversion of 3-chlorobenzoate. FEMS Microbiol. Ecolo. 38:293-298. Gerhardt, P., ed. 1981. Manual of methods for general bacteriology. American Society for Microbiology. Washington, DC. Linkfield, T.G. 1985. Anaerobic reductive dehalogenation: the lag period preceeding haloaromatic dehalogenation, enrichment of sediment activity, and the partioal characterization of a dehalogenating organism, strain DCB—l. Ph.D. thesis, Michigan State University, East Lansing, MI. Rittenberg, S.C. 1969. The roles of exogenous organic matter in the physiology of chemolithotrophic bacteria. Adf. Microb. Physiol. 3:159—196. Shelton, D.R. and J.M. Tiedje. 1984. Isolation and partial characterization of bacteria in an anerobic consortium that mineralizees 3-chlorobenzoic acid. Appl. Environ. Microbiol., 48:840-848. Sorokin, Y.I. 1966. Sources of energy and carbon for biosynthesis in sulfate-reducing bacteria. Mikrobiologiya, 35:761-766. Suflita, J.M., T. Linkfield, J.M. Tiedje, and P.H. Pritchard. 1987. The relationship between reductive dehalogenation and other aryl substituent removal reactions catalyzed by anaerobes. FEMS Microbio. Ecology. in press. 93 APPENDIX APPENDIX Effect of Medium Reductant on Growth and Dechlorination by Strain DCB-l Introduction Strictly anaerobic bacteria, such as strain DCB-l, are commonly propagated in media containing sulfide or cysteine as reducing agents, in order to maintain anaerobiosis [3,6]. Because cysteine is an organic compound, and could possibly support growth [5], it was eliminated from the media used for fermentation studies (chapter 2). Cultures grown with sulfide alone appeared to grow more slowly. The following experiments were performed in order to determine whether sulfide was indeed toxic to this organism, and to select a medium reductant for further studies. 94 Materials and Methods Culture conditions. Cells of DCB-l were grown in the mineral medium previously described (chapter 2) with 0.2% pyruvate. For dechlorination determinations, the medium also contained 800 uM 3—Chlorobenzoate and 10% rumen fluid. Dechlorination was monitored by growing DCB-l in 50 ml of the above medium in 160 ml serum bottles with butyl rubber stoppers at 37 C, in the dark. Growth of DCB-l was measured in 15 m1 of the pyruvate medium, in 25 ml anaerobic culture tubes with butyl rubber stoppers, at 37°C in the dark. Media reduction. Sulfide was added to growth media, after cooling and just prior to bottling, to a final concentration of 5 mM. Dithionite was added by syringe through a sterile 0.2 um pore size filter, just prior to inoculation, to a final concentration of 500 uM. The membrane fraction of E. coli was added just prior to inoculation by injecting 1 ml of the sterile preparation, described below, with a syringe. Preparation of E. coli membrane fraction. This was prepared by modifying the procedure of Alder et. al. [1]. Cultures of E. coli were prepared by inoculating 2 L of nutrient broth (Difco Laboratories) and growing this culture at 37 C on a shaker for 24 95 96 hours. Cells were harvested by centrifugation and washed with cold HEPES buffer (N-2 hydroxyethylpiperazine N'-2 ethanesulfonic acid), at pH 7.5. The pellet was resuspended in 25 ml of HEPES buffer, then passed through a French pressure cell three times at 20,000 lb/in2. Magnesium chloride was added to a concentration of 0.16 mM, and the suspension was centrifuged at 12,000 x g for 15 min. The pellet was discarded, and the supernatant was filtered through a 0.45 um filter to remove any remaining whole cells. This preparation was then frozen until use at -20°C. Analytical methods. Samples were removed periodically from the dechlorinating cultures, filtered through 0.2 uM pore size membrane filters, and frozen until analysis. Samples were then thawed, and analyzed for benzoate and 3-ch10robenzoate by HPLC, as described earlier (chapter 2). For growth curves, tubes were periodically removed from the incubator and optical density was measured with a Turner model 350 spectrophotometer. Results and Discussion The E. coli. membrane fraction has been suggested to be an efficient, non-toxic reductant for use in isolating and propagating anaerobic bacteria [2]. It was selected for these experiments because it was reasoned to be the least toxic reductant possible. As shown in Figure 1, this treatment dramatically increased the rate of dechlorination by strain DCB-l, as compared to the medium reduced with sulfide. The rate of dechlorination was also greater with dithionite as the reductant, and was not significantly different than that achieved with the E. coli membrane fraction. It is possible that this increased rate is due in part to the much lower amount of dithionite required to attain the same level of reduction as sulfide. Not only did sulfide interfere with dechlorination (Figure 1), but with growth of DCB-l as well (Figure 2.) This bacterium has been tentatively considered as a sulfidogen [4], but since no oxygenated sulfur electron acceptor was added in these experiments, the effect of sulfide cannot be considered to be due to inhibition of sulfate reduction by high levels of its product. Because of this apparent toxicity to strain DCB-l, sulfide was not used in subsequent experiments. Since dithionite does not 97 98 require extensive preparation, this method of media reduction was selected for use in chapter 2 and 3 experiments. '5 99 Figure 1. Effect of media reductant on dechlorination of 3-chlorobenzoate by strain DCB-l. Data points are means of 4 observations. Bars represent +/- one S.D. 100 00.000.002.000 0— 0 00 0030000”. 0.002 00 .0000... .— 0009.”. 00000000.m>0o 0 hi!) 00.0.2... To 0 2.8.55 010 no. 0600050: :00.“ I I o * 3 01002009 (Q—g 1000.109 I o co Bumgowa Ll 101 Figure 2. Effect of media reductant on growth of strain DCB-l in pyruvate medium (without chlorobenzoate and rumen fluid.) Data points are means of 10 observations. 102 .108 sasm .0 £380 8 0038000 £2000 .0 08.00 .N 2:00 00.000000. «>00 nNF o.N m.— 0.. m o Mariam Ole oo o u....zo....._..o .ula r I006 102... In To loud and r Iond 099 Kusuap loondo Literature Cited Adler, H.I., A. Carrasco, W. Crow, and J.S. Gill. 1981. Cytoplasmic membrane fraction that promotes septation in an Escherichia 0011 Jon mutant. J. Bact. 147:326-332. Crow, W.D., R. Machanoff, and H.I. Adler. 1985. Isolation of anaerobes using an oxygen reducing membrane fraction: experiments with acetone butanol producing organisms. J. Microbiol. Methods 4 133-139. Holdeman, L.V., and W.E.C. Moore (ed.). 1972. Anaerobe Laboratory Manual, 2nd ed. Virginia Polytechnic Institute and State University, Blacksburg, Virginia. Linkfield, T.G. 1985. Anaerobic reductive dehalogenation: the lag period preceeding haloaromatic dehalogenation, enrichment of sediment activity, and the partial characterization of a dehalogenating organism, strain DCB-l. Ph.D. thesis, Michigan State University, East Lansing, MI. Postgate, J.R. 1979. The sulphate reducing bacteria. Cambridge University Press, Cambridge. Shelton, D.R. and J.M. Tiedje. 1984. General method for determining anaerobic biodegradation potential. Appl. Environ. Microbiol. 47 850-857. 103 IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII [1W]IIHIHHHIIIH[ll/[JIM][I] 03264 31