)1. ‘1‘. y V131,. .h 9 ‘ . .. l UMIW ; . i... 1!: : A... :k 10)»..5. v .n.) l).io . .§.l.;§‘f £30. .I. El.) .5127. 40 t 7. 1.4. (:31... :1; . 1| 5.. .. c . l. 29.35.; 3?. .y pv10),lppn it “ (I‘v- I 2.5.19 4 7- , , . .33 . .. .Ii :2 . 3V3 . . , . I41 . . . , ‘ . é: . . . .3 3...: .3... . . . , V , 2.... ... , . x , . . V . . . : . . . D. 21. .. . . . v ‘1,~III.L air . , . . .x .. . . . .. 2.3:...) V A v . .‘ II "AI‘ l .u , . 2 t: . :74... . .3 1.1.5-; 2.. .. . . {$31.11.}..131 l....\\v.o , . . . A13. ..\év»1)!l. 2.: (1 . . . . E... ,2|.I\.. ,Ixax....2il.uv. V ‘ y lr- -. -- - I 1 I‘ll: t “m 4 meme»: 3 UN nsmr LlBRARIES ill ll u \\\llll\\\\\lll\\\\l\ “Will 3 1293 00891 4297 L. This is to certify that the dissertation entitled PHYSIOLOGY OF DESULFOMONILE TIEDJEI, A STRICTLY ANAEROBIC BACTERIUM CAPABLE OF REDUCTIVE DEHALOGENATION presented by William W. Mohn has been accepted towards fulfillment of the requirements for Ph . D . degree in Microbiglggy Date fl AngusLlQQO__ MS U is an Affirmative Action/Equal Opportunity Institution 0-12771 ”A F LIBRARY Michigan State 1 University \.__._ *- ‘— PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINES return on or More date due. DATE DUE DATE DUE DATE DUE MSU Is An Affirmative Action/Equal Opportunity Institution cmmma-pd PHYSIOLOGY OF DESULFOMONILE TIEDJEI, A STRICTLY ANAEROBIC BACTERIUM CAPABLE OF REDUCTIVE DEHALOGENATION By William W. Mohn A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Microbiology and Public Health 1990 William W. Mohn Desulfomonile tiedjei (formerly strain DCB-l) is a strictly anaerobic bacterium which is capable of reductive dehalogenation of benzoates and ethylenes. This reaction is of possible significance for bioremediation of toxic wastes and pollutants. The organism is not closely related to any other known bacterium. General physiological studies were undertaken for the following purposes: (1) to better classify D. tiedjei, (2) to understand what physiological significance reductive dehalogenation has for this organism, (3) to devise strategies for the isolation of other dehalogenating anaerobes and (4) to better understand ecological principles which might affect reductively dehalogenating organisms in natural environments. D. tied jei was determined to be a sulfate- reducing bacterium capable of reducing sulfate and thiosulfate stoichiometrically to sulfide with the following electron donors: H2, formate, CO, lactate, pyruvate, butyrate and 3-methoxybenzoate. In the absence of an electron donor, thiosulfate was instead fermented to sulfide plus sulfate. D. tiedjei grew by a novel fermentation of pyruvate plus C02, the latter serving as an electron acceptor and being reduced to acetate. Carbon monoxide dehydrogenase activity indicated that CO2 reduction was probably via the acetyl coenzyne A pathway. Autotrophic and diazotrophic growth were possible. 3-chlorobenzoate, which was reductively dehalogenated to benzoate, also served as an electron acceptor for energy metabolism. This reaction was stoichiometrically coupled to oxidation of formate to 002. D. tiedjei was grown on formate plus 3-chlorobenzoate in William W. Mohn defined medium. Resuspended cells catalyzed dehalogenation-dependent ATP synthesis. The effects of respiratory inhibitors suggest that dehalogenation and. ATP synthesis are coupled via a chemiosmotic mechanism. The effects of an ATPase inhibitor and of imposed pH gradients suggest that a proton-driven ATPase is involved in the above chemiosmotic process. Thus, reductive dehalogenation appears to be a novel mode of anaerobic respiration. Isolation of other dehalogenating anaerobes was attempted using strategies based on the above findings. One isolate very similar to D. tiedjei was obtained, but the dehalogenating agents in the majority of enrichment cultures used were not isolated. in the hope that our curiosity (science) does more good than harm iv ACKNOWLEDGMENTS I am of course indebted to a long line of characters in my life who have guided me here. My parents, Thelma and Richard Lareau, gave me appreciation and all opportunity for education. My best friend, Marie-Claude Fortin, contributed tremendously to my personal and academic lives. Many exceptional teachers and colleagues, including Ronald W. Hoham and Ronald L. Crawford, gave me encouragement, inspiration and ideas. In this work, I have particularly benefitted from the company of Juha Apajalahti and James R. Cole. Stephen A. Boyd, John A. Breznak, Michael J. Klug, C.A. Reddy and J. Gregory Zeikus have kindly served on my guidance committee at various times. Finally, James M. Tiedje has been an excellent advisor; I have especially gained from his guidance and the environment he has created in which to learn science. I am deeply grateful to them all and I hope these pages will have some meaning for each. TABLE OF CONTENTS List of tables .................................................. viii List of figures .................................................... x Chapter 1: Reductive dehalogenation of aromatic compounds, a review.... ....................................................... 1 Introduction ................................................. 2 SCOPE ................................................... 3 Range of activity ............................................ 4 RECENTLY REPORTED SUBSTRATES ................................... 4 Undefined cultures ........................................... 8 UNIFYING PRINCIPLES ........................................ 10 ENRICHMENT ............................................... 11. ACCLIMATION .............................................. 12 ELECTRON ACCEPTORS ......................................... 13 OTHER NUTRIENTS ........................................... l 7 Tmmmummm .............................................. l8 SUBSTRATE AVAILABILITY ...................................... 18 CONCLUSION ............................................... 19 Desulfomonile tiedjei ....................................... 20 MORPHOLOGY / CELL DIVISION .................................... 2 1 GENERAL PHYSIOLOGY ......................................... 2]. INDUCTION OF DEHALOCENATION ................................... 2 7 INHIBITION OF DEHALOCENATION BY SULFOXY ANIONS ..................... 2 9 Tamman' ................................................ 29 SYNTROPHY ................................................ 3 0 CONCLUSION ............................................... 3 1 Attempts to find other dehalogenators ....................... 32 SULFATE - REDUCING BACTERIA .................................... 3 2 ISOLATES ................................................ 32 Cell-free activity .......................................... 33 COPACTORS ................................................ 33 CRUDE EXTRACT OP Desulfomonile tiedjei ..................... 34 Reductive dehalogenation by aerobes ......................... 34 Conclusions ................................................. 37 References .................................................. 39 Chapter 2: Catabolic thiosulfate disproportionation and carbon dioxide reduction in strain DCB-l, a reductively dechlorinating anaerobe .......................................................... 46 Chapter 3: Strain DCB-l conserves energy for growth from reductive dechlorination coupled to formate oxidation ............. 53 vi Chapter 4: Evidence for chemiosmotic coupling of reductive dechlorination and ATP synthesis in Desulfomonile tiedjei ......... 59 Introduction ................................................ 60 Materials and methods ....................................... 61 Results ..................................................... 63 Discussion .................................................. 66 References .................................................. 73 Chapter 5: Attempts to identify or isolate dehalogenating anaerobes ......................................................... 75 Introduction ................................................ 76 Materials and methods ....................................... 76 Results and Discussion ...................................... 82 References .................................................. 88 Appendix: Involvement of a collar structure in polar growth and cell division of strain DCB-l ..................................... 89 vii LIST OF TABLES Table Page Chapter 1 1 Aromatic substrates known to be reductively dehalogenated by organisms from anaerobic environments .......................... 5 Chapter 2 1 Carbon and electron balance for strain DCB—l grown on pyruvate plus CO2 ............................................. 49 2 Use of general and putatively respiratory electron acceptors by strain DCB-l ............................................... 49 3 Metabolism of sulfoxy anions by strain DCB-l during 16 days of incubation on bicarbonate-buffered medium .................. 49 4 CODH activity in whole cells of DCB-l grown on various bicarbonate-buffered media .................................... 50 Chapter 3 1 The effect of reductive dechlorination on yield of DCB-l cultures grown on several media ............................... 55 2 Substrate consumption and product formation after 34 h incubation of resuspended DCB-l cells provided with 26.8 pmol formate with or without 5.2 pmol 3CD ................ 57 Chapter 4 1 Effects of respiratory inhibitors on dechlorination and ATP concentration in resuspended cells of Desulfomonile tiedjei... 67 viii Table Page Chapter 5 l Enrichment cultures used as sources for isolates .............. 77 2 Media used for isolations and numbers of isolates ............. 78 3 Sulfate-reducing bacteria tested for dehalogenation activity ...................................................... 80 4 Media used for growth of sulfate-reducing bacteria ............ 81 ix Figure LIST OF FIGURES Page Chapter 1 Reductive dehalogenation of the heterocyclic compound, bromacil ....................................................... 9 Aromatic substrates for reductive dehalogenation by Desulfomonile tiedjei ......................................... 22 Reductive dehalogenation of 3-chlorobenzoate coupled to formate oxidation by Desulfomonile tiedjei .................... 25 Inducers of reductive dehalogenation activity in Desulfomonile tiedjei ......................................... 28 Initial steps in aerobic degradation pathways which include reductive dehalogenation reactions ............................ 36 Chapter 2 Growth of strain DCB-l on various media ....................... 48 Consumption of a limiting amount of pyruvate with transient accumulation of acetate during growth of strain DCB-l on 5 mM sulfate as an electron acceptor .......................... SO Consumption of a limiting amount of 3-methoxybenzoate with accumulation of 3-hydroxybenzoate as an end product during growth of strain DCB-l on 5 mM sulfate as an electron acceptor ...................................................... 50 Chapter 3 Formate consumption and dechlorination by DCB-l cultures ...... S6 'Dechlorination of 3GB by resuspended DCB-l cells in the presence of various electron donors ........................... 56 Formate consumption and dechlorination of 3GB by resuspended DCB-l cells ................................................... 56 LIST OF FIGURES Figure Page Chapter 1 l Reductive dehalogenation of the heterocyclic compound, bromacil ....................................................... 9 2 Aromatic substrates for reductive dehalogenation by Desulfomonile tiedjei ......................................... 22 3 Reductive dehalogenation of 3-chlorobenzoate coupled to formate oxidation by Desulfomonile tiedjei .................... 25 4 Inducers of reductive dehalogenation activity in Desulfomonile tiedjei ......................................... 28 5 Initial steps in aerobic degradation pathways which include reductive dehalogenation reactions ............................ 36 Chapter 2 1 Growth of strain DCB-l on various media ....................... 48 2 Consumption of a limiting amount of pyruvate with transient accumulation of acetate during growth of strain DCB-l on 5 mM sulfate as an electron acceptor .......................... SO 3 Consumption of a limiting amount of 3-methoxybenzoate with accumulation of 3-hydroxybenzoate as an end product during growth of strain DCB—l on 5 mM sulfate as an electron acceptor ...................................................... 50 Chapter 3 l Formate consumption and dechlorination by DCB-l cultures ...... 56 2 'Dechlorination of 3GB by resuspended DCB-l cells in the presence of various electron donors ........................... 56 3 Formate consumption and dechlorination of 3GB by resuspended DCB-l cells ................................................... 56 Figure Page Chapter 4 l Desulfomonile tiedjei dependence on 3GB for growth (A) and metabolism of 3GB (B) on defined medium with formate plus 3GB as substrates ............................................. 64 2 Reductive dechlorination and consequent ATP pool increase in Desulfomonile tiedjei ......................................... 6S 3 Effect of DCCD on dechlorination rate and ATP concentration... 68 4 ATP pool increase in Desulfomonile tiedjei resulting from an imposed pH gradient ........................................... 69 Chapter 5 1 Growth on pyruvate and metabolism of 3-Chlorobenzoate by strain DCB-2 and Desulfomonile tiedjei ........................ 86 Appendix 1 Scanning electron micrographs of strain DCB-l showing the collar in various locations ................................... 9O 2 Transmission electron micrographs of strain DCB-l showing the collar in various locations and internal structures ....... 91 3 Transmission electron micrographs showing membranes of strain DCB-l .................................................. 92 4 Transmission electron micrographs showing the collar region of dividing cells of strain DCB-l ............................. 93 5 Histogram showing distribution of collar locations in a randomly selected population of 28 cells ...................... 94 6 Relative collar location as a function of cell length for the same population of cells as in Fig. 5 with nonlinear regression fit of polar growth model .......................... 94 7 Proposed mode of growth and division of strain DCB-l .......... 94 xi Chapter 1: Reductive dehalogenation of aromatic compounds, a review Introduction Reductive dehalogenation is the replacement Of a halogen substituent of a molecule with an hydrogen atom. In all known biological examples of this activity, the halogen is released as a halide anion. This process makes many xenobiotic compounds less toxic and more readily degradable, and appears to be the essential primary step in anaerobic degradation of halogenated aromatic compounds. Since it was first reported by Suflita et a1. (1982), reductive dehalogenation by anaerobic bacteria has justifiably generated great interest due to its potential application to bioremediation of hazardous wastes. The study of reductive dehalogenation has also made significant contributions to basic microbiology in the areas of ecology, physiology and phylogeny. The study of reductively dehalogenating bacteria is essential for their exploitation by humans, but is also intriguing from a more fundamental scientific perspective. Recent studies have shown that many of our most. problematic pollutants are susceptible to biological reductive dehalogenation. Anaerobic reductive dehalogenation is the only known biodegradation mechanism of certain significant environmental pollutants (e.g., highly chlorinated biphenyls , hexachlorobenzene and tetrachloroethylene). This process appears to occur within mutualistic anaerobic microbial communities. Such communities frequently are able to adapt to growth on halogenated aromatic compounds. Using undefined cultures, researchers are beginning to understand the ecological factors which influence reductive dehalogenation. However, the individual organisms which catalyze the process remain elusive, and only a single anaerobe capable of reductive dehalogenation of aromatic compounds is currently in pure culture. This organism, Desulfomonile tiedjei (formerly strain DCB-l), represents a new genus, with a morphology and mode of division singular among described bacteria, and with unusual catabolic abilities. D. tiedjei has the novel ability to gain energy for’ growth, from reductive dechlorination. The 'uniqueness of IL tiedjei and the difficulty in isolating other anaerobes capable of reductive dehalogenation suggest that continued study of this process may lead to the discovery of other novel and interesting organisms. Finally, certain aerobic bacteria are now also known to employ reductive dehalogenation steps in degradation pathways for halogenated aromatic compounds. Sam: Tiedje et a1. (1987) have reviewed literature on the subject of anaerobic reductive dedhlorination. Of aromatic compounds prior to 1986. Reductive dehalogenation has also been included as a part of general reviews on biodegradation (Reineke‘& Knackmuss, 1988; Sahm et al., 1986). The recent review by Kuhn and Suflita (1989) extensively covers anaerobic degradation, including reductive dehalogenation, of pesticides in soils and groundwater. These previous reviews have thoroughly described the range of substrates for this activity and habitats where the activity is found, and here only the most important and new information on these subjects will be summarized. The primary purpose of this review is to examine the ecological and physiological principles which are beginning to emerge from the study of reductive dehalogenation of aromatic compounds. Where possible, studies with undefined anaerobic communities are related to others using ‘pure cultures. Reductive dehalogenation of 'volatile alkyl solvents (e g., tetrachloroethylene) is not addressed, although this activity has not been proven to be distinct from reductive dehalogenation of aromatic compounds. Range of activity Microbes from a variety of anaerobic habitats reductively dehalogenate a great variety of aromatic compounds (Table 1). Initial studies focused on relatively simple model compounds (e.g., halobenzoates and halophenols) and are described in the above reviews. ‘Recent studies have found. that the same activity can transform compounds which are considered more Significant as pollutants (e.g., polychlorinated phenols, polychlorinated benzenes and polychlorinated biphenyls). The latter compounds tend to be more complex, less water-soluble and more toxic. These findings greatly increase the significance of reductive dehalogenation from an applied perspective. RECENT-LY REPORTED SUBSTRATES Polychlorinated biphenyls (PCBs) are currently of great concern due to their recalcitrance and toxicity. Laboratory studies have now demonstrated that the more highly chlorinated PCB congeners in mixtures such as Aroclors 1242, 1248, 1254 and 1260 can be reductively dechlorinated by anaerobic microorganisms from PCB- Thus 1. Aromatic substrates known to be reductively dehalogenated by organisms from anaerobic environments Substrate Inoculum First report Benthiocarb1 Bromacil2 Bromophenol Chloroanilines Chloroanilines Chlorocatechols Chloroguaiacols Chloronitrofen3 Chlorophenols Chlorophenoxyacetates Chlorophenoxyacetates Chlorophenoxyacetates Chlororesorcinol Diuron‘ Halobenzoates Halobenzoates Hexachlrobenzene Pentachlorophenol Polybrominated biphenyls Polychlorinated bipyenyls Propanil5 Techloftham6 TPN7 Trichlorobenzene Trichlorobenzene Paddy soils Aquifer slurry Marine sediment Aquifer slurry Pond sediment Reactor column Reactor column Paddy soil Sewage sludge Sewage sludge Aquifer slurry Pond sediment Sewage sludge Pond sediment Lake sediment Sewage sludge- Sewage sludge Paddy soil River sediment River sediment Pond sediment Paddy soil Flooded soil Rat gut River sediment Moon & Kuwatsuka, 1984 Adrian & Suflita, 1990 King, 1988 Kuhn & Suflita, 1989 Strijs & Rogers, 1989 Hakulinen et al., 1982 Hakulinen et al., 1982 Yamada & Suzuki, 1983 Boyd et al., 1983 Mikesell & Boyd, 1985 Gibson & Suflita, 1986 Gibson & Suflita, 1986 Fathepure et al., 1987 Attaway et al., 1982 Suflita et al., 1982 Suflita et al., 1982 Fathepure et al., 1988 Ide et al., 1972 Quensen et al., 1990 Quensen et al., 1988 Stepp et al., 1985 Kirkpatrick et a1, 1981 Sate & Tamaka, 1987 Tsuchiya & Yamaha, 1984 Bosma et al., 1988 18-4-chlorobenzyl-N,N-diethyl thiocarbamate 2S-bromo-3-sec-butyl-6-methyl uracil 34-nitrophenyl-2,4,6-trichlorophenyl ether l'3-(3,4-dichlor0phenyl)-l,l-dimethy1 urea 5N-(3,4-dichlorophenyl) propanamide 6N-(2,3-dichlorophenyl)-3,4,5,6-thtrachlorophthalamic acid 72,4,5,6-tetrachloroisophthalonitrile contaminated river sediments (Alder et al., 1990; Quensen et al., 1988, Quensen et al., 1990a). These studies support previous evidence for in situ activity in anaerobic sediments (Brown et al., 1984, Brown et al., 1987a, Brown et al., 1987b). Reductive dehalogenation activity was nearly entirely at the meta and para positions, and the major products were mono- and dichlorobiphenyls. Significant detoxication of the Aroclors resulted since the more highly chlorinated congeners are more toxic (Safe et al., 1982). Different patterns of dechlorination were observed in various cultures (Quensen et al., 1990a), similar to patterns previously elucidated for activity in natural sediments (Brown et al., 1984, Brown et al., 1987a, Brown et al., 1987b). The observation of these patterns has led to speculation that distinct organisms may exist having individual dehalogenation activities. The products of anaerobic PCB degradation can be mineralized aerobically (Bedard et al., 1987), suggesting that a combination of anaerobic and aerobic microbial communities might mineralize all PCB congeners. Biological reductive dehalogenation of polybrominated biphenyls has also been demonstrated very recently (Quensen et al. , 1990b). Hexachlorobenzene (HCB) and other chlorobenzene congeners are also widespread pollutants of very low water solubility. Fathepure et a1. (1988) have shown reductive dechlorination of HCB to 1,3,5- trichlorobenzene and small amounts of dichlorobenzenes in stationary incubations of anaerobic sewage sludge. Dehalogenation of HCB in incubations of sediments has also been reported (Mouse and Rogers, 1990). In the latter study, different inoculum sources exhibited different dehalogenation patterns, one resembling that of the former study and another yielding penta-, 1,2,3,4- and l,2,3,5—tetra-, 1,2,3-tri- and 1,2-dichlorobenzene. Using the three trichlorobenzene isomers as substrates, Bosma et a1. (1988) showed that anaerobic river sediment in upflow columns could reductively dechlorinate all tri- and dichlorobenzene isomers, yielding di- and monochlorobenzenes. Di- and monochlorobenzenes can be mineralized aerobically (De Bont et al., 1986; Schraa et a1, 1986; Spain and Nishino, 1987; Van der Meer et al., 1987); thus, as in the case of PCBs, the proper sequence of conditions may allow biological mineralization of all chlorobenzene congeners. Recent reports indicate that Chloroanilines, which are used in industrial syntheses, can be reductively dechlorinated by organisms from aquifer material and pond sediment (Kuhn & Suflita, 1989; Strijs & Rogers, 1989). In both reports, more highly chlorinated anilines were dechlorinated, but monochloroanilines persisted. Chlororesorcinols are possible by-products Of industrial syntheses, and 4-chlororesorcinol has been shown to be reductively dechlorinated in anaerobic sewage sludge (Fathepure et al., 1987a). The resorcinol product subsequently disappeared from enrichment cultures from the sludge, suggesting that anaerobic communities may be capable of mineralizing 4-chlororesorcinol. In the first report of biological reductive dehalogenation of a heterocyclic compound, Adrian and Suflita (1990) clearly demonstrated removal of bromine from the herbicide, bromacil, by aquifer slurries (Figure 1). No further evidence concerning the fate of the debrominated product was given. Not all haloaromatic compounds are xenobiotic. The burrows of a hemichordate inhabiting marine sediment were found to contain 2,4- dibromophenol (King, 1986). The worm apparently synthesizes the compound which inhibits growth of aerobic bacteria in the burrow. Subsequently it was shown that an anaerobic microbial community from the sediment could first debrominate and then mineralize this compound (King, 1988). These important studies are the first to examine anaerobic metabolism of numerous naturally occurring haloaromatic compounds. The existence of such compounds suggests that selective pressure for reductive dechlorination may have existed during the evolution of anaerobic bacteria. Further examination of the metabolism of such natural compounds will likely contribute greatly to our understanding of the metabolism of xenobiotic compounds. Undefined cultures Like most anaerobic processes , anaerobic reductive dehalogenation has typically been found to occur in syntrophic communities. It has proved very difficult to obtain pure cultures with the activity. Ecological understanding of these communities is thus critical for any applied use of the activity or investigation of the- individual organisms responsible for the activity. Laboratory investigations with undefined anaerobic communities have been employed in the study of reductive dehalogenation out of necessity. Br /C H H /C H \C/ \N/ \C/ \ / 2H + | | > | | + HBr /C C C C 0/ \If/ §O O/ \ITI/ F0 CHCHZCH3 ICHCHZCH3 CH3 CH3 Ffimmr l. Reductive dehalogenation of the heterocyclic compound, bromacil. 10 This approach can identify ecological factors affecting reductive dehalogenation, but is of limited value in determining the mechanisms of these effects. This approach does have predictive value, since undefined cultures are more likely than pure cultures to behave like populations in natural habitats or habitats which may be derived for bioremediation. Recent contributions to our understanding of the ecology of anaerobic reductive dehalogenation are described below. UNIPYINC PRINCIPLES Several general principles concerning anaerobic reductive dehalogenation were established by early work with chlorobenzoates and Chlorophenols (Boyd and Shelton, 1984; Boyd et al., 1983; Horowitz et al., 1983; Suflita et al., 1982), and, as will be shown, these principles have been corroborated by more recent studies. Almost all activity is biological and can be killed by autoclaving. Horowitz et a1. (1983) also showed that activity could be killed by gamma radiation, formaldehyde and oxygen. Reductive dehalogenation has always been found to be the primary step in anaerobic degradation of haloaromatic compounds with the dehalogenated product persisting or appearing as a transient intermediate. Natural samples often exhibit acclimation periods of months before dehalogenation occurs. Substrate specificity, both for the type of aromatic compound (i.e., benzoate, phenol) and for the halogen position, is often observed for acclimation. Enrichment of the activity is frequently possible, and often the haloaromatic compound can serve as a sole carbon and energy source for such a culture. The above phenomena have led to the 11 hypothesis that the reductive dehalogenation of aromatic compounds is catalyzed by individual organisms with enzymatic specificities for certain haloaromatic compounds; however, until the individual organisms are isolated, other possible explanations remain. Specificities could reside at the level of enzymes, organisms or broad physiological groups. ENRICHMENT All anaerobic communities are not equal with respect to potential for reductive dechlorination. Certain communities, often in polluted habitats, are adapted to certain xenobiotic compounds. Thus, inocula from PCB-contaminated river sediments dechlorinated PCBs while inocula from uncontaminated sediments did not during prolonged incubations (Quensen et al., 1988). Polluted river and estuary sediments had greater potential than unpolluted ones for dehalogenation of monochlorophenols and monochlorobenzoates (Sharak Genthner et al., 1989a). Sewage sludges also showed varied potentials for those activities (Shelton and Tiedje, 1984a). Additionally, by serial transfer of laboratory cultures, it has been possible to enrich for dehalogenation of a number of substrates, including halobenzoates (Sharak Genthner et al. 1989a; Shelton and Tiedje, 1984b), halophenols (Sharak Genthner et al. 1989a; Zhang and Wiegel, 1990), 4-chlororesorcinol (Fathepure et al., 1987a), dichloroanilines (Struijs and Rogers, 1989) and PCBs (unpublished data). Except for dichloroanilines and PCBs, these substrates apparently served as sole carbon and energy sources. Adaptation and enrichment are common observations in microbial cultures, but they 12 are not trivial observations for xenobiotic substrates. One would not necessarily expect such substrates to support growth, especially in cases, such as PCBs, HCB and Chloroanilines, where reductive dehalogenation is not followed by degradation Of the remaining hydrocarbon compound. In these cases, adaptation and enrichment clearly indicate selective pressure for reductive dehalogenation per se. Such selective pressure could be positive (e.g., for use as a substrate) or negative (e.g., for detoxication). Aamnumum Linkfield et al. (1989) have examined the acclimation period preceding reductive dehalogenation of halobenzoates. The periods of acclimation were reproducible for various compounds and inoculum sources. These authors identify the following possible explanations for the periods of acclimation (1) genetic change, (2) induction, (3) exhaustion of a preferred substrate (diauxy) or (4) growth of the active population from very low initial numbers. It was suggested that induction best explains the observed patterns. Genetic change was considered unlikely due to the reproducibility of the acclimation periods. The acclimation periods were determined to be too long for growth of the active population; although, this determination was based on the questionable assumption that the specific growth rate of the dechlorinating population would be approximately that of strain the isolate, D. tiedjei, in pure culture. A diauxy response was considered unlikely because sediment stored for two years at 4°C exhibited the same acclimation period as fresh sediment. However, Kohring et al. (1989a) found that storage of sediment for two months 13 at 12°C increased acclimation time. The latter finding would also not be conSistent with a diauxy response, as storage should then decrease acclimation time, but would be consistent with an acclimation period due to growth of a dehalogenating population which decreased in viability during storage. Such an effect on viability might not occur during storage at 4°C as in the former study (viability of anaerobic cultures can be remarkably stable during storage at low temperatures). It is not clear that the acclimation periods do not have different causes in the different sediments studied or combinations of the above mentioned causes. Acclimation periods required for anaerobic reductive dehalogenation may be very long (6 me or longer) and must be allowed for in studies of this activity. Our present understanding of the acclimation periods preceding reductive dehalogenation by natural communities is highly speculative. EumanAanmnms Electron acceptors are frequently the limiting nutrient for anaerobic communities, typically being a major determinant of the structure of these communities; therefore, the presence of reductively dechlorinating organisms in a community may be affected by electron acceptors. Phrthermore, it might be expected that the availability of electron acceptors might affect the flow of electrons required for reductive dehalogenation. This effect might occur via intracellular channelling of electrons or via interspecific competition for electron donors. Accordingly, evidence is accumulating which indicates that electron acceptors do affect 14 dehalogenation activity in anaerobic communities. However, this relationship appears to be complex. The laboratory of J. M. Suflita has examined two closely located sites within an aquifer contaminated by landfill leachate (Beeman and Suflita, 1987; Gibson and Suflita, 1986; Suflita and Miller, 1985). The sites differed in being dominated by either methanogenesis or sulfate reduction. Only samples from the methanogenic site demonstrated the ability to dechlorinate and mineralize chlorobenzoates, Chlorophenols and Chlorophenoxyacetates. The potential for dehalogenation existed at both sites with sulfate apparently inhibiting dechlorination in samples of the sulfate- reducing site, since addition of sulfate inhibited dehalogenation of 2,4,5-trichlorophenoxyacetate by samples from the methanogenic site, and since depletion of sulfate by addition of acetate (as an electron donor) allowed this activity by samples from the sulfate-reducing site. The occurrence of methanogenesis did not insure the activity, since stimulation of methanogenesis in samples from the sulfate- reducing site by addition of methanol (as a substrate only used by methanogens) did not allow the activity. This does not prove that methanogenesis from other substrates would not cause dehalogenation. Additionally, amendment of aquifer slurries with nitrate or sulfate was found to inhibit debromination of the heterocyclic ring of bromacil (Adrian and Suflita, 1990). These data clearly indicate inhibition of dehalogenation by sulfate in this aquifer. However, this conclusion should be extrapolated to other habitats or xenobiotic compounds with caution. 15 Kohring et al. (1989b) examined dechlorination of phenols by samples from freshwater sediments. Addition of nitrate to samples completely inhibited dechlorination of 2,4-dichlorophenol. Sulfate increased the adaptation time, decreased the rate of the initial dechlorination at the ortho position, and prevented dechlorination of the 4-chlorophenol product. When samples were first acclimated to 4- Chlorophenol without sulfate, subsequently added sulfate was reduced to sulfide and had little effect on dechlorination of 4-chlorophenol. In this case, it appears that sulfate does not inhibit reductive dehalogenation directly, but rather, sulfate inhibits enrichment of the activity. In a broader survey, Sharak Genthner et al. (1989a and 1989b) tested the effects of sulfate and nitrate on degradation of monochlorobenzoates and monochlorophenols by samples from a variety of sediments taken from river and estuary locations. Added sulfate and nitrate, generally inhibited degradation by the samples, but there were several exceptions. An inhibitor Of‘ methanogenesis, bromoethane sulfonate (BES), also generally inhibited degradation by the samples. This finding may imply that methanogenesis is required for degradation for various possible reasons including (1) methanogens simultaneously metabolize natural substrates and the xenobiotic compounds, or (2) the dehalogenating organisms are dependent on me thanogens . However , the effect of DES on methanogenes is was not verified , and a direct effect on dehalogenat ion was not excluded . Sulfate and nitrate more consistently inhibited degradation in transfers of the original 16 samples, but there were still notable exceptions. Transfers of one sample required nitrate for degradation of 3- and 4-chlorobenzoate, another transfer was stimulated by sulfate in the degradation of 4- chlorophenol. ‘Thus, electron acceptors 'have variable effects on reductive dechlorination which are subject to biological and chemical variables. In river sediments incubated under sulfate reducing conditions, all three monochlorophenols and 2,4-dichlorophenol were degraded (Haggblom et al., 1990). In these cultures sulfate reduction appeared to be required for degradation, as an inhibitor of sulfate reduction, molybdate, inhibited degradation. In the previously mentioned study of 2,4-dibromophenol in marine sediment (King, 1988), molybdate did not affect debromination but did prevent degradation of the phenol product. The effect of the inhibitor on sulfate reduction was not verified, but the effect on phenol degradation. probably indicates that sulfate reduction. was blocked. It is not clear that, as the author suggests, sulfate reducers were not responsible for debromination, since the specific blockage of ATP sulfurylase by molybdate would not stop all other activities of these organisms. The evidence available suggests that nitrate and sulfate most Often inhibit dehalogenation by anaerobic communities, but the nature of this inhibition varies. Direct inhibition of the dehalogenation process by electron acceptors appears to occur in some communities; while in others, electron acceptors apparently select for a nondehalogenating population. A nondehalogenating population might l7 outcompete dechlorinators by virtue of a higher growth rate, but once established, certain dechlorinating populations apparently can compete successfully for electron donors. In a minority of cases tested, sulfate or nitrate did not inhibit dehalogenation or were even required for activity. The effects on dehalogenation of other electron acceptors such as iron, manganese and carbon dioxide have yet to be examined. OTHER NUTRIENTS In contrast to the effects of electron acceptors, the effects of other nutrients on reductive dehalogenation have not been examined extensively. In most studies of dehalogenation using undefined communities, specific electron donors and carbon sources were not added but were presumably supplied by the source material (i.e., sediment, sludge, aquifer solids) in complex and rather recalcitrant forms. When halogenated compounds were oxidized after dehalogenation, they also provided electrons and carbon. In several of the studies detailed above, enrichments were serially transferred, mineralizing haloaromatic compounds and using them as sole carbon and energy sources. Added nutrients can stimulate dehalogenation activity or enrichment of that activity as demonstrated for 4- chlororesorcinol (Fathepure et a1. , 1987a) and P088 (unpublished data). In the case of 4-chlororesorcinol dehalogenation in sewage sludge enrichments, yeast extract, trypticase, rumen fluid, glucose, sludge supernatant and resorcinol, in order of decreasing effect, stimulated dehalogenation activity. Thus, the enrichments appeared to be limited by some or all of the following: carbon, electron 18 donor, amino acids and micronutrients. Of course, certain nutrients may also select against dehalogenating populations. It would be of value to determine nutrients which limit dehalogenation; however, studies with undefined cultures are severely limited. in such determinations. TEMPERATURE Temperature might be expected to affect reductive dehalogenation by a direct effect on reaction rates and by selective pressure on populations. Both effects were indicated in a study of lake sediment samples to which 2,4-dichlorophenol was added (Kohring et al., 1989a). Temperature affected both the acclimation period and the rate of dechlorination activity. Activity was found between 5 and 50°C with distinct rate peaks at 30°C and 43°C, suggesting selection for two distinct dehalogenating populations. A direct correlation of temperature and dehalogenation rate was found only from 15 to 30°C. Thus, it may be difficult to extrapolate laboratory rate measurements at higher temperatures to natural habitats which are below 15°C. Clearly temperature severely limits dehalogenation rates in many habitats. SUBSTRATE AVAILABILITY The hydrophobicity of many haloaromatic compounds certainly affects their biological dehalogenation. The availability of the substrates to dehalogenating organisms will have a direct effect on dehalogenation rates. In addition, because of the toxicity of many of these substrates, availability may also have an inhibitory effect on dehalogenation rates. A large body of literature describes the 19 various nonbiological fates of ‘haloaromatic compounds in ‘natural habitats (e.g., Chiou, 1989; Hassett and Banwart, 1989). Here it will simply be _pointed out that fates such as sorption and volatilization will affect the aqueous concentration of these compounds. Additionally, physical surfaces may juxtapose sorbed hydrophobic compounds and microbes attached to the surfaces. Studies of the dehalogenation of very' hydrophobic aromatic compounds in liquid cultures have employed various carriers for these compounds, including a liquid organic phase (Holliger et al., 1989,) or sediments (Quensen et al., 1988). In the above studies, the carriers were probably required to enhance availability. Dispersants have been developed for delivery of hydrophobic antibiotics. These are molecules designed to be water-soluble but to have a'hydrophobic interior region ‘which may contain. a ‘hydrophobic Compound” Such compounds may have potential for use in studies of aerobic biodegradation of hydrophobic compounds. Studies of reductive dehalogenation have not yet addressed optimization of the availability of haloaromatic compounds. meumnm Studies using undefined cultures clearly indicate the potential of anaerobes to reductively dehalogenate aromatic compounds. Such studies also indicate ecological factors detailed above which significantly affect activity. Often results did not distinguish between direct effects on activity and effects on population selection. Results were not always consistent with different inocula and different haloaromatic substrates; therefore, in these cases it 20 is not yet reasonable to generalize conclusions. It seems likely that reductive dechlorination is catalyzed by physiologically diverse organisms in diverse anaerobic communities. In many cases a better understanding of ecological factors will require pure culture studies. Paradoxically, isolation of more reductively dehalogenating anaerobes probably depends on better understanding of their ecology. The majority of the studies described above involved sealing natural samples in serum bottles and incubating them in the laboratory. These cultures thus differed from in situ conditions in input of soluble nutrients, removal of soluble products, temperature and other aspects. Such experiments have logistical advantages and adequately address certain questions, -but empirical knowledge of predictive value for natural and derived habitats requires experiments which more closely approximate those habitats. The latter experiments have yet to be undertaken. Desulfomonile tiedjei While reductive dehalogenation appears to be 'most favored in syntrophic communities, the above studies using undefined cultures indicate that certain aspects of the process will only be understood when the responsible organisms are studied in pure culture. Pure culture studies may answer essential questions including (1) which organisms have dehalogenation activity? (2) which enzymes and cofactors are responsible for the activity? (3) what is the chemical mechanism involved in activity? (4) how do various factors directly affect activity? and (5) how does the activity benefit (or harm) the responsible organisms? In addition to satisfying basic scientific 21 interest, answers to such questions are Of obvious importance in applications of reductive dehalogenation. At present D. tiedjei (formerly strain DCB-l) represents the only opportunity to study anaerobic reductive dehalogenation of aromatic compounds in pure culture. This organism is able to dehalogenate benzoate or certain of its analogs, with activity preferentially directed to the meta position (Figure 2; J.R. Cole, 1990, personal communication; DeWeerd et al., 1986; Shelton and Tiedje, 1984b). D. tiedjei also dechlorinates tetrachlorethylene (Fathepure et al., 1987b). In addition to dehalogenation activity, this organism has several unique morphological and metabolic characteristics which are of more general microbiological interest. The uniqueness of D. tiedjei suggests that further study of reductive dehalogenation will involve the challenges as well as the rewards of studying novel organisms. MORPHOLOGY/CELL DIVISION A unique morphological feature of D. tiedjei is a collar which girdles each cell (see Appendix, Figures 1-4). This collar consists of a region where the cell wall folds over itself (Shelton and Tiedje, 1984b). The collar is the origin of polar cell growth and cell division (Mohn et a1. , 1990). GENERAL PHYSIOLOGY Initially D. tied jei could only be cultured on an undefined medium including rumen fluid, and pyruvate was the only substrate found to significantly support growth (Shelton and Tiedje, 1984b). 22 Substrates: COOH, CONH2 (not OH, NH,, cnon ) Br, I (not F, C1) Cl, Br, I (not F, CH3) Br, I (not F, C1) Ihcmm 2. Aromatic substrates for reductive dehalogenation by Desulfomonile tiedjei: one of the substituents shown in the one position is required, and the halogen substituents shown can be removed. 23 Thiosulfate was latter found to stimulate growth, suggesting that the organism might be a sulfate-reducing bacterium (Stevens et al., 1988). Growth on pyruvate was found to be mixotrophic, involving C02 fixation (Stevens and Tiedje, 1988). Identification of vitamins stimulatory to D. tiedjei (Apajalahti et al., 1989; DeWeerd et al., 1990a) has permitted studies using defined media which have greatly improved knowledge of the metabolic characteristics of this organism. D. tiedjei exploits several catabolic electron acceptors. The organism is a bona fide sulfate-reducing bacterium (Mohn and Tiedje, 1990b), having the usual ability to reduce sulfate or thiosulfate to sulfide (Equations 1 and 2) as well as the ability, in the absence of a suitable electron donor, to gain energy from the disproportionation of thiosulfate to sulfide plus sulfate (Equation 3). Growth by the latter lithotrophic fermentation is apparently possible for only a few of the sulfate reducers presently in pure culture (Kramer & Cypionka, 1989). D. tiedjei, like Desulfotomaculum spp. is more sensitive to sulfide than other sulfate reducers. Sulfide typically limits growth of D. tiedjei on sulfur compounds in batch cultures and sulfide production is usually less than 3'mM (Mohn & Tiedje, 1990b). This sensitivity' would select against D. tiedjei during routine enrichment and isolation of sulfate reducers and may contribute to the lack of similar isolates. 4 HCOOH + soa- + H+ » a 002 + H8' + 4 H20 (1) 4 HCOOH + 8203' ~ 4 002 + 2 HS“ + 3 H20 (2) 3203- + H20 » HS' + 304' + H+ (3) 24 D. tiedjei also grows by an unusual fermentation of pyruvate plus C02 (Mohn & Tiedje, 1990b). The fermentation involves the oxidation of pyruvate to acetate plus C02 (Equation 4) and the reduction of C02 to acetate (Equation 5), with the oxidative and reductive processes balancing one another (Equation 6). In many respects this resembles the terminal steps in homoacetogenic fermentation of sugars; however, D. tiedjei is unable to use sugars. Like homoacetogens, D. tied jei has carbon monoxide dehydrogenase activity and is believed to employ the acetyl-COA pathway for CO2 reduction. Unlike many homoacetogens, D. tiedjei cannot grow on H2 plus C02 or formate plus 002. 4 011300000}! + 4 H20 -» a cu3coou + 4 (:02 + 8 H (4) 8 H + 2 co2 - 033000}: + 2 H20 (5) a cu3cocoon + 2 H20 .. 5 cu3coon + 2 (:02 (6) Reductive dehalogenation is yet another reaction exploited by D. tiedjei for energy metabolism. Dolfing and Tiedje (1986) constructed a defined consortium including D. tiedjei which used 3- chlorobenzoate (3GB) as a sole substrate. Growth of the consortium was stimulated by dechlorination (Dolfing and Tiedje, 1987). Subsequently, dechlorination was found to stimulate growth of D. tiedjei in pure culture (Dolfing, 1990; Mohn and Tiedje, 1990a). Dechlorination was coupled to formate oxidation (Figure 3), or, probably, H2 oxidation (Mohn and Tiedje, 1990a). If other dehalogenating anaerobes can also employ this novel form of chemotrophy, it may support the previously mentioned enrichment of 25 COOH COOH HCOOH + (:()2 'f 'F {{(31 Cl FIGURE 3. Reductive dehalogenation of 3-chlorobenzoate coupled to formate oxidation by Desulfomonile tiedjei. 26 cultures which reductively dehalogenate substrates which are not further degraded (e.g., PCBs or HCB). The coupling of dehalogenation to formate or H2 oxidation suggests that energy conservation is via a respiratory process, since neither formate or H2 is known to support substrate-level phosphorylation. Like other sulfate reducers, D. tiedjei appears to have several respiratory electron carriers. Desulfoviridin and cytochrome c3 were isolated from D. tiedjei (DeWeerd et al., 1990a). Naphthoquinone (or menadione) is required by D. tiedjei for dehalogenation but not for fermentative growth (J. Apajalahti, 1989, personal communication). It is not reported whether the vitamin is also required by D. tiedjei for sulfoxy anion metabolism; although the vitamin stimulates growth on pyruvate plus thiosulfate (DeWeerd et al., 1990a). In addition to their probable role in sulfate reduction, some or all of the above electron carriers may participate directly in dehalogenation or in respiratory energy conservation from dehalogenation. Dechlorination directly supported ATP synthesis in stationary phase cultures which were limited by 3GB (Dolfing, 1990) and in cell suspensions (see Chapter 4). In the latter system, the effects respiratory inhibitors and imposed pH gradients suggest that dechlorination and. ATP synthesis are coupled ‘via a chemiosmotic mechanism involving a proton-driven ATPase. Thus, reductive dechlorination may support a novel mode of anaerobic respiration. D. tiedjei appears to have a relatively limited range of electron donors. In addition to pyruvate, formate and H2 mentioned above, D. tiedjei oxidizes C0, lactate, butyrate (Mohn and Tiedje, 27 1990b) 3- and 4-methoxy benzoates and their derivatives, and benzoate (DeWeerd et al., 1990a). Acetate appears to be used, but only slowly (DeWeerd et al.,l990a; Mohn and Tiedje, 1990b). Oxidation of methoxy benzoates is via O-demethylation to corresponding hydroxy benzoates (DeWeerd et al., 1986) which are not further degraded (Mohn and Tiedje, 1990b). The latter activity is characteristic of organisms with the acetyl-COA pathway. The electron donor range of D. tiedjei is typical of sulfate reducing bacteria, and could allow D. tiedjei a terminal position in anaerobic food chains, using products of fermentative organisms. INDUCTION or DEHALOGENATION Initially it was reported that dehalogenation activity in D. tied jei was dependent on growth in the presence of 3C3 (DeWeerd and Suflita, 1989). More recently m-halobenzoates or analogs were also found to specifically induce activity (Cole and Tiedje, 1990). There are a number of inducers which are not substrates (gratuitous) as well as a number of substrates which are not inducers (Figures 2 and 4; J.R. Cole, 1990, personal communication). Inducers must be meta- substituted, but dehalogenation activity can also act at the ortho and para positions. Inducers can have certain meta-substituents which are not transformed (e.g., F, CH3, CF3). If the lack of induction by certain substrates is common, the failure to detect activity is not proof that organisms having activity are not present. Thus, dehalogenating organisms could possibly be undetected in experiments using current methodology. 28 Inducers: COOH, CHon, CONH2 ("OI OH, NHz) (not Cl, 1) Cl, Br, I, F, CH3, CF3 (IlOI CHO, NHz, CN, OH) (not Cl, I) FIGURE 4. Inducers of reductive dehalogenation activity in Desulfomonile tiedjei: one of the substituents shown in both the one and three positions is required. ' 29 INHIBITION OF omocmnou a! SULI-‘OXY muons The relationship between dehalogenation and metabolism of sulfoxy anions byD. tiedjei is not simple. Thiosulfate and sulfite inhibit dechlorination of 308 by growing cells (DeWeerd et a1, 1986; Linkfield and Tiedje, 1990) and by resuspended cells (DeWeerd et al., 1990b)., Sulfate inhibited dechlorination of 3CB by cells growing on one medium (Linkfield and Tiedje, 1990) but not by cells growing on a different medium (J.R. Cole, 1989, personal communication) or by resuspended cells (DeWeerd et al., 1990b). The latter authors found that both 3GB and sulfoxy anions could support H2 consumption by resuspended cells, the latter at a higher rate. With both electron acceptors present an intermediate rate of Hz consumption was observed, suggesting to these authors that dechlorination and sulfoxy anion reduction are enzymatically distinct pathways which compete for limited electron donors. The above results suggest that inhibition of dehalogenation by thiosulfate and sulfite observed in undefined cultures may occur via intraspecific channelling of electrons to electron acceptors. However, if dehalogenating organisms in such undefined cultures resemble D. tiedjei, inhibition by sulfate must involve interspecific competition for electron donors. Tm DeWeerd et al. (1990a) determined the 168 rRNA sequence of Desulfomonile tied jei which clearly indicates that the organism is a member of the delta subdivision of the class Proteobacteria (purple bacteria). The degree of distance between Desulfomonile tied jei and Desulfovibrio desulfuricans was greater than between Desulfomonile 30 tied jei and Desulfuromonas acetoxidans (an elemental sulfur-reducing bacterium) or Desulfobacter spp. It was concluded from the 168 rRNA sequence that Desulfomonile tied jei represents a new genus among the sulfate-reducing bacteria, although comparisons were only made with three other species of that group. The unique physiological characteristics of D. tiedjei (above) and the sequence analysis led DeWeerd et al. (1990b) to assign the name, Desulfomonile tiedjei gen. nov. and sp. nov., to the organism, formerly strain DCB-l. SYNTRDPEY Studies of D. tiedjei suggest possible reasons why reductive dehalogenation is favored in undefined communities. In natural habitats D. tiedjei appears to obtain a .number of nutrients and other factors from other organisms. First, as an obligate anaerobe, D. tied jei requires a reduced, oxygen-free environment created by other organisms. As mentioned above, D. tied jei uses electron donors which probably originate as end products of other anaerobes. Products of D. tied jei which are toxic to the organism, such as sulfide and benzoate, may be removed by other organisms. Five vitamins are stimulatory or, possibly, required by D. tiedjei (DeWeerd et al., 1990a). Initially, D. tiedjei required rumen fluid for growth and dehalogenation activity (Shelton and Tiedje, 1984b). It was latter found that fermentative growth could occur in a defined medium but that dehalogenation activity required a factor which could be provided by rumen fluid, a Propionibacterium sp. in coculture or the culture fluid of the Propionibacterium sp. (Apajalahati et al., 1989). The factor in the culture fluid was extractable and its 31 chemical properties suggested that it was a quinoid compound. The factor could be replaced by l,4-naphthoquinone or menadione (vitamin K3). Thus, D. tiedjei conforms to the general rule of syntrOphy in anaerobic ecosystems. A consequence of such interdependence may be the observed difficulty of isolating other dehalogenating organisms. Successful applications of biological reductive dehalogenation will likely require the use and understanding of these complex anaerobic communities. CONCLUSION A fundamental question eluded to above is whether dehalogenation activity per se was independently selected for (evolved) or whether the activity is coincidentally catalyzed by an enzyme(s) evolved for a different activity (fortuitous). Natural selection for this activity is possible because of the existence of naturally occurring haloaromatic compounds. The ability of D. tiedjei to use dehalogenation for energy metabolism indicates one selective advantage of this activity; detoxication might be another. The specific induction of dehalogenation by D. tied jei suggests that at least the (regulation of this activity is evolved in D. tiedjei. The apparent distinction between the enzymatic pathways of dehalogenation and sulfoxy anion metabolism by D. tied jei indicate that the former activity is not a fortuitous consequence of the latter. The evidence is not conclusive, but it suggests the possibility that, in the case of D. tiedjei, dehalogenation may be an evolved activity . 32 Attempts to find other dehalogenators SULFATE - REDUCING BACTERIA After Desulfomonile tiedjei was identified as a sulfate- reducing bacterium, a variety of other sulfate reducers were tested for the ability to reductively dehalogenate haloaromatic compounds. Linkfield (1985) tested three Desulfovibrio spp. for dechlorination of 3-chlorobenzoate under sulfate-reducing conditions. Later, ten species of the following genera were tested: Desulfovibrio, Desulfobacter, Desulfobacterium and Desulfococcus (see Chapter 5). In the latter test, conditions used were based on optimal conditions for D. tiedjei (i.e., in the presence of required electron donors and vitamins and in the absence of competitive electron acceptors) and substrates tested included halobenzoates and halophenols. No dehalogenation activity was detected. Thus, dehalogenation activity does not appear to be a general property of sulfate reducers; although, it should be noted that a major grOup, gram-positive sulfate reducers, was not tested. ISOLATES ~The difficulty in isolating anaerobes capable of reductive dehalogenation of aromatic compounds has been mentioned above. Only one such strain other than D. tiedjei has been isolated (see Chapter 5). The new strain is from the same sewage sludge sample as D. tiedjei and closely resembles D. tiedjei morphologically and physiologically. Medium selecting for the new isolate was based on the ability of D. tied jei to grow diazotrophically on pyruvate plus thiosulfate (Mohn 6: Tiedje, 1990b). This isolation strategy has 33 failed to obtain dehalogenating organisms from other enrichments, suggesting that in these enrichments the dehalogenating organisms are physiologically distinct from D. tiedjei. Zhang and Wiegel (1990) have recently reported the establishment of stable culture which reductively dechlorinates 2,4- dichlorophenol to 4-chlorophenol without further degrading this product. The dechlorinating organism is believed to be a spore- forming rod, since the ortho-dehalogenation activity was separated from a 2,4-dichlorophenol-mineralizing enrichment by pasteurization. However, the rod has not yet been purified. Cell-free activity (kmunmm In the presence Of the strong reductant, titanium (III) citrate, cofacters common to a variety of bacteria (e.g., cobalamin, factor F,30 and hematin) have recently been found to reductively dehalogenate a variety of compounds. This activity has been observed with alkyl solvents such as polychloromethanes and polychloroethylenes (Holliger, 1990) as well as with hexachlorobenzene (L . Wackett , 1990 , personal communication) . Not all organisms with these cofactors have dehalogenation activity; thus, if these cofactors are involved in dehalogenation by whole cells, they interact with other cell components. The activity of the cofactors suggests the possibility that lysed cells might catalyze some dehalogenation activities Observed in reduced environments; although, activity is typically not found in killed control cultures. 34 CRUDE WI or Desulfomonile tiedjei DeWeerd and Suflita (1989) have obtained cell-free extract from D. tiedjei which dechlorinates 3GB. Activity is heat-labile; thus, it differs from the above activity of heat-stable cofactors. Dehalogenation by the crude extract is dependent on methyl viologen. A number of biological electron carriers tested were unable to replace methyl viologen. Activity appears to be membrane associated (K.A. DeWeerd, 1989, personal communication). As in whole cells (DeWeerd et al., 1990b), thiosulfate and sulfite inhibit cell-free activity while sulfate does not. Kinetic experiments determining the nature of this inhibition might be very useful in resolving the relationship between sulfoxy anion. metabolism and dehalogenation, especially if the dehalogenase can be purified. Reductive dehalogenation by aerobes Reductive dehalogenation also appears to be employed by aerobic bacteria in the degradation of highly chlorinated aromatic rings which are invulnerable to ring-cleaving oxygenases. Even when such organisms are in oxidized environments, their cytoplasm probably has a low redox potential and is favorable for reductive reactions. The pathways of aerobic mineralization of pentachlorophenol by two different organisms, a Flavobacteriwm sp. (Steiert & Crawford, 1986) and Rhodococcus Chlorophenolicus (Apajalahti & Salkinoja-Salonen, 1987) have been shown to involve reductive dehalogenation steps. The degradation pathways are different for the two organisms, but they both commence with hydrolytic dechlorination(s) followed by reductive dechlorinations (Figure 5). For both aerobes, Chlorophenols 35 specifically induced their own dechlorination, but it is not clear whether only the hydrolytic activities are inducible or whether the reductive activities are also inducible. The dechlorination substrate specificity varied for the above aerobes, but again it is not clear whether this is a property of only the hydrolytic activities or also of the reductive activities. Whole cells of the Flavobacterium required 02 for the first reductive dechlorination, while cell extracts of R. Chlorophenolicus did not require 02 for the reductive dechlorinations. A facultative anaerobe, Alcaligenes denitrificans, also employs both hydrolytic and reductive dechlorination reactions for the aerobic mineralization of 2,4-dichlorobenzoate (Van den Tweel et a1. , 1987). It was proposed that reductive dechlorination preceded hydrolytic dechlorination (Figure 5); although, the evidence leaves the possibility that the order is reversed. The consumption of 2,4- dichlorobenzoate, presumably including the reductive dechlorination reaction, was catalyzed by cells grown on 4-iodo-,' 4-bromo- and 4- chlorobenzoates. Thus, the reductive dechlorination activity was not specifically induced by its substrate. Similarly to the Flavobacterium above, whole cells of A. denitrificans required 02 for the dechlorination reactions. In both cases it is possible that energy is required for the activity, but other explanations are also possible. Until the activities of the Flavobacterium and A. denitrificans are tested in- cell extracts, it will not be known whether they differ from that of R. Chlorophenolicus in requirement 36 Rhodococcus chlorophenolicus: OH I OH _ OH CI‘CI HO C10 ”—21 C10 OH 2“ \ OH 02'— C1 C1 C1 C1 I / Cl OH j OH OH OH 2H I \ OH 2H OH //’ OH OH F lavobacterium sp.: OH :Jé: :ég :éf' 0C1 C10 C1 OH Alcaligenes denitrificans: COOH COOH COOH elemo C1 C1 OH Ificmm 5. Initial steps in aerobic degradation pathways which include reductive dehalogenation reactions. 37 of 02. Regardless, these aerobes experience a net gain of energy after oxidation of the resulting aromatic rings. Conclusions 1. In agreement with early studies, recent studies indicate that reductive dehalogenation is the primary step in the biological anaerobic degradation of haloaromatic compounds, including compounds which are extremely toxic and are not known to be biodegraded aerobically. 2. This activity most readily occurs in undefined anaerobic communities suggesting that the responsible organisms may be obligate syntrophs. 3. Such communities may vary fundamentally in composition, as they respond differently to environmental factors, notably including the availability of various electron acceptors. 4. At least one anaerobe, Desulfomonile tiedjei, can gain energy from reductive dechlorination, and the ability of others to do so is consistent with the observed enrichment of this activity. 5. Reductive dehalogenation can be catalyzed by common cofactors in the presence of a strong reductant, but this activity appears distinct from reductive dehalogenation activities in most anaerobic cultures. 6. Reductive dehalogenation also appears to be employed by aerobes in the degradation of haloaromatic compounds which are not initially susceptible to oxidative degradation. 7. Studies of Desulfomonile tiedjei as well as the difficulty of isolating other dehalogenating anaerobes suggest that further study 38 of these environmentally and economically significant organisms may be of fundamental microbiological interest. 39 References Adrian NR, Suflita JM (1990) Reductive dehalogenation of a nitrogen heterocyclic herbicide in anoxic aquifer slurries. Appl. Environ. Microbiol. 56:292-294. Alder AC, Haggblom M, Young LY (1990) Dechlorination of PCBs in sediments under sulfate reducing and methanogenic conditions. Abstr. Ann. Meet. Am. Soc. Microbiol., Q-47, p. 296. Apajalahti JHA, Salkinoja-Salonen MS (1987) Dechlorination and para-hydroxylation of polychlorinated phenols by Rhodococcus Chlorophenolicus. J. Bacteriol. 169:675-681. Apajalahti J, Cole J, Tiedje J (1989) Characterization of a dechlorination cofactor: an essential activator for 3-chlorobenzoate dechlorination by the bacterium DCB-l. Abstr. Ann. Meet. Am. Soc. Microbiol., Q-36, p. 336. Attaway HH, Paynter MJB, Camper ND (1982) Degradation of selected phenylurea herbicides by anaerobic pond sediment. J. Environ. Sci. Health B17:683-699. Bedard DL, Wagner RE, Brennan MJ, Haberl ML, Brown JFJ (1987) Extensive degradation of Aroclors and environmentally transformed polychlorinated biphenyls by Alcaligenes eutrophus H850. Appl. Environ. Microbiol. 53:1094-1102. Beeman RE, Suflita JM (1987) Microbial ecology of a shallow unconfined ground water aquifer polluted by municipal landfill leachate. Microb. Ecol. 14:39-54; Bosma TNP, van der Meer JR, Schraa G, Tros ME, Zehnder AJB (1988) Reductive dechlorination of all trichloro- and dichlorobenzene isomers. FEMS Microbiol. Ecol. 53:223-229. Boyd SA, Shelton DR, Berry D, Tiedje JM (1983) Anaerobic biodegradation of phenolic compounds in digested Sludge. Appl. Environ. Microbiol. 46:50-54. Boyd SA, Shelton DR (1984) Anaerobic biodegradation of Chlorophenols in fresh and acclimated sludge. Appl. Environ. Microbiol. 47:272-277. Brown JF, Bedard DL, Brennan MJ, Carnahan JC, Feng H, Wagner RE (1987a) Polychlorinated biphenyl dechlorination-in aquatic sediments. Science 236:709-712. 40 Brown JF, Wagner RE, Bedard DL, Brennan MJ, Carnahan JC, May RJ, Tofflemire TJ (1984) PCB transformations in upper Hudson sediments. Northeast. Environ. Sci. 3:167-179. Brown JF, Wagner RE, Feng H, Bedard DL, Brennan MJ, Carnahan JC, May RJ (1987b) Environmental dechlorination of PCBs. Environ. Toxicol. and Chem. 6:579-593. Chiou CT (1989) Theoretical considerations of the partition uptake of nonionic organic compounds by soil organic matter. In: Sawheny BL, Brown K (Eds) Reactions and Movements of Organic Chemicals in Soils. Soil Science Society of America and American Society of Agronomy, Madison,WI, p 1-30. Cole JR, Tiedje JM (1990) Induction of anaerobic dechlorination of chlorobenzoate in strain DCB-l. Abstr. Ann. Meet. Am. Soc. Microbiol., Q-43, p. 295. De Bont JAM, Vorage MJAW, Hartmans S, Van den Tweel WJJ (1986) Microbial degradation of 1,3-dichlorobenzene. Appl. Environ. Microbiol. 52:677-680. DeWeerd KA, Suflita JM, Linkfield TG, Tiedje JM, Pritchard PH (1986) The relationship between reductive dehalogenation and other aryl substituent removal reactions catalyzed by anaerobes. FEMS Microbiol. Ecol. 38:331-339. DeWeerd KA, Suflita JM (1989) Aryl reductive dehalogenation in cell-free extracts of a 3-chlorobenzoate degrading consortium and an isolated bacterium strain DCB-l. Abstr. Ann. Meet. Am. Soc. Microbiol., K-65, p. 255. DeWeerd KA, Concannon F, Suflita JM (1990b) Hydrogen consumption and the reduction of halobenzoates and sulfur oxyanions in resting cells of Desulfamanile tiedjei. Abstr. Ann. Meet. Am. Soc. Microbiol., Q-53, p. 297. DeWeerd KA, Mandelco L, Tanner RS, Woese CR, Suflita JM (1990a) Desulfomanile tiedjei gen. nov. and sp. nov. a novel anaerobic, dehalogenating, sulfate- -reducing bacterium. Arch. Microbiol. (in press). Dolfing J, Tiedje JM (1986) Hydrogen cycling in a three-tiered food web growing on the methanogenic conversion of 3-chlorobenzaate. FEMS Microbiol. Ecol. 38:293-298. Dolfing J, Tiedje JM (1987) Growth yield increase linked to reductive dechlorination in a defined 3-chlorobenzoate degrading methanogenic coculture. Arch. Microbiol. 149:102-105. 41 Dolfing J (1990) Reductive dechlorination of 3-chlorobenzoate is coupled to ATP production and growth in an anaerobic bacterium, strain DCB-l. Arch. Microbiol. 153:264-266. Fathepure BZ, Nengu JP, Boyd SA (1987b) Anaerobic bacteria that dechlorinate perchloroethene. Appl. Environ. Microbiol. 53. Fathepure BZ, Tiedje JM, Boyd SA (1987a) Reductive dechlorination of 4-chloraresorcinol by anaerobic microorganisms. Environ. Toxicol. Chem. 6:929-934. Fathepure B2, Tiedje JM, Boyd SA (1988) Reductive dechlorination of hexachlorobenzene to tri- and dichlorobenzenes in anaerobic sewage sludge. Appl. Environ. Microbiol. 54:327-330. Gibson SA, Suflita JM (1986) Extrapolation of biodegradation results to groundwater aquifers: reductive dehalogenation of aromatic compounds. Appl. Environ. Microbiol. 52:681-688. Haggblom M, Rivera M, Young LY (1990) Biodegradation of chlorinated phenols under sulfate reducing conditions. Abstr. Ann. Meet. Am. Soc. Microbiol., Q-4l, p. 295. Hakulinen R, Salkinoja-Salonen M (1982) Treatment of pulp and paper industry wastewaters in an anaerobic fluidized bed reactor. Process Biochem. 17:18-22. Hassett JJ, Banwart WL (1989) The sorption of nonpolar organics by soils and sediments. In: Sawhney BL, Brown K (Eds) Reactions and Movements of Organic Chemicals in Soils. Soil Science Society of America and American Society of Agronomy, Madison, WI, p 31-44. Halliger C, Schraa G, Stams AJM, Zehnder AJB (1989) Anaerobic reductive dechlorination of 1,2,3-trichlorobenzene in a two phase system. Abstr. 5th Inter. Symp. Microbial Ecol., P-23-3, p. 216. Holliger C, Schraa G, Stams AJM, Stupperich E, Zehnder AJB (1990) Cabamide-dependent reductive dechlorination of 1,2-dichloroethane by Methanosarcina barkeri. Abstr. Ann. Meet. Am. Soc. Microbiol., Q-49, p. 296. Horowitz A, Suflita JM, Tiedje JM (1983) Reductive dehalogenations of halobenzoates by anaerobic lake sediment microorganisms. Appl. Environ. Microbiol. 45:1459-1465. Ide A, Niki Y, Sakamota F, Wantanabe I, Wantanabe H (1972) Decomposition of pentachlorophenol in paddy soil. Agric. Biol. Chem. 36:1937-1944. 42 King GM (1986) Inhibition of microbial activity in marine sediments by a bromophenol from a hemichordate. Nature (London) 323:257-259. King GM (1988) Dehalogenation in marine sediments containing natural sources of halophenols. Appl. Environ. Microbiol. 54:3079-3085. Kirkpatrick D, Biggs SR, Conway B, Finn CM, Hawkins DR, Honda T, Ishida M, Powell GP (1981) Metabolism of N-(2,3-dichloraphenyl)-3,4,5,6-tetrachloraphthalmic acid (Techlofthalam) in paddy soil and rice. J. Agric. Food Chem. 29:1149-1153. Kohring G-W, Zhang X, Wiegel J (1989b) Anaerobic dechlorination of 2,4-dichlorophenol in freshwater sediments in the presence of sulfate. Appl. Environ. Microbiol. 55:2735-2737. Kohring G-W, Rogers JE, Wiegel J (1989a) Anaerobic biodegradation of 2,4-dichlorophenol in freshwater lake sediments at different temperatures. Appl. Environ. Microbial. 55:348-353. Kramer M, Cypionka H (1989) Sulfate formation via ATP sulfurylase in thiosulfate- and sulfite-disproportionating bacteria. Arch. Microbiol. 151:232-237. Kuhn EP, Suflita JM (1989) Dehalogenation of,pesticides by anaerobic microorganisms in soils and groundwater - a review. In: Sawhney BL, Brown K (Eds) Reactions and Movement of Organic Chemicals in Soils. Soil Science Society of America and American Society of Agronomy, Madison,WI p 111-180. Linkfield TC (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. PhD Thesis, Michigan State Univ. Linkfield TG, Suflita JM, Tiedje JM (1989) Characterization of the acclimation period before anaerobic dehalogenation of halobenzoates. Appl. Environ. Microbiol. 55:2773-2778. Linkfield TG, Tiedje JM (1990) Characterization of the requirements and substrates for reductive dehalogenation by strain DCB-l. J. Indust. Microbial. 5:9-16. Mikesell MD, Boyd SA (1985) Reductive dechlorination of the pesticides 2,4-D, 2,4,S-T and pentachlorophenol in anaerobic sludges. J. Environ. Qual. 14:337-340. Mahn WW, Tiedje JM (1990a) Strain DCB-l conserves energy for growth from reductive dechlorination coupled to formate oxidation. Arch. Microbiol. 153:267-271. 43 Mohn WW, Tiedje JM (1990b) Catabolic thiosulfate disproportionation and carbon dioxide reduction in strain DCB-l, a reductively dechlorinating anaerobe. J. Bacteriol. 172:2065-2070. Mohn WW, Linkfield TG, Pankratz HS, Tiedje JM (1990) Involvement of a collar structure in polar growth and cell division of strain DCB-l. Appl. Environ. Microbiol. 56:1206-1211. Moon YH, Kuwatsuka L (1984) Properties and conditions of soils causing the dechlorination of the herbicide benthiocarb (thiobencarb) in flooded soils. J. Pest. Sci. 9:745-754. Mousa MA, Rogers JE (1990) Dechlorination of hexachlorobenzene in two freshwater pond sediments under methanogenic conditions. Abstr. Ann. Meet. Am. Soc. Microbiol., Q-45, p. 296. Quensen III JF, Tiedje JM, Boyd SA (1988) Reductive dechlorination of polychlorinated biphenyls by anaerobic microorganisms from sediments. Science 242:752-754. Quensen III JF, Morris PJ, Boyd SA (1990b) Dehalogenation of PCBs and PBBs by anaerobic microorganisms from sediments. Abstr. Ann. Meet. Am. Soc. Microbiol., Q-44, p. 295. Quensen III JF, Boyd SA, Tiedje JM (1990a) Dechlorination of four commercial Aroclors (PCBs) by anaerobic microorganisms from sediments. Appl. Environ. Microbiol. (in press). Reineke W, Knackmuss H-J (1988) Microbial degradation of haloaromatics. Ann. Rev. Microbiol. 42:263-287. Safe 8, Robertson LW, Safe L (1982) Halogenated biphenyls: molecular toxicology. Can. J. Physiol. Pharmacol. 60:1057-1064. Sahm H, Brunner M, Schoberth SM (1986) Anaerobic degradation of halogenated aromatic compounds. Microb. Ecol. 12:147-153. Sato K, Tanaka H (1987) Degradation and metabolism of a fungicide, 2,4,5,6-tetrachloroisophthalonitrile (TPN) in soil. Biol. Fert. Soils 3:205-209. Schraa G, Boone ML, Jetten MSM, Van Neerven ARW, Colberg PC, Zehnder AJB (1986) Degradation of l,4-dichlorobenzene by Alcaligenes sp. strain A175. Appl. Environ. Microbiol. 52:1374-1381. Sharak Genthner BR, Price WAI, Pritchard PH (1989a) Anaerobic degradation of chloroaromatic compounds in aquatic sediments under a variety of enrichment conditions. Appl. Environ. Microbiol. 55:1466-1471. 44 Sharak Genthner BR, Price WAI, Pritchard PH (1989b) Characterization of anaerobic dechlorinating consortia derived from aquatic sediments. Appl. Environ. Microbiol. 55:1472-1476. Shelton DR, Tiedje JM (1984a) General method for determining anaerobic biodegradation potential. Appl. Environ. Microbiol. 47:850-857. Shelton DR, Tiedje JM (1984b) Isolation and partial characterization of bacteria in an anaerobic consortium that mineralizes 3-chlarobenzoic acid. Appl. Environ. Microbial. 48:840-848. Steiert JG, Crawford RL (1986) Catabolism of pentachlorophenol by a Flavobacterium sp. Biochem. Biophys. Res. Commun. 141:825-830. Stevens TO, Tiedje JM (1988) Carbon dioxide fixation and mixotrophic metabolism by strain DCB-l, a dehalogenating anaerobic bacterium. Appl. Environ. Microbial. 54:2944-2948. Stevens TO, Linkfield TG, Tiedje JM (1988) Physiological characterization of strain DCB-l, a unique dehalogenating sulfidogenic bacterium. Appl. Environ. Microbiol. 54:2938-2943. Struijs J, Rogers JE (1989) Reductive dehalogenation of dichloroanilines by anaerobic microorganisms in fresh and dichlorophenal-acclimated pond sediment. Appl. Environ. Microbiol. 55:2527-2531. . Suflita JM, Horowitz A, Shelton DR, Tiedje JM (1982) Dehalogenation: a novel pathway for the anaerobic biodegradation of haloaromatic compounds. Science 218:1115-1117. Suflita JM, Miller GD (1985) Microbial metabolism of chlorophenolic compounds in ground water aquifers. Environ. Toxicol. Chem. 4:751-758. Tiedje JM, Boyd SA, Fathepure B2 (1987) Anaerobic degradation of chlorinated aromatic hydrocarbons. Developments in Industrial Microbiology 27:117-127. Tsuchiya T, Yamaha T (1984) Reductive dechlorination of 1,2,4-trichlorobenzene by Staphylococcus epidermidis isolated from intestinal contents of rats. Agric. Biol. Chem. 48:1545-1550. Van den Tweel WJJ, Kak JB, De Bont JAM (1987) Reductive dechlorination of 2,4-dichlorobenzoate to 4-chlorobenzoate and hydrolytic dehalogenation of 4-chloro, 4-bromo, and 4-iodobenzoate by Alcaligenes denitrificans NTB-l. Appl. Environ. Microbiol. 53:810-815. 45 Van der Meer JR, Roelofsen W, Schraa G, Zehnder AJB (1987) Degradation of low concentrations of dichlorobenzenes and 1,2,4-trichlorobenzene by Pseudomonas sp. strain P51 in non-sterile soil columns. FEMS Microbiol. Ecol. 45:333-341. Yamada T, Suzuki T (1983) Occurrence of reductive dechlorination products in the paddy field soil treated with CNP (Chloranitrofen). J. Pestic. Sci. 8:437-443. Zhang X, Wiegel J (1990) Sequential anaerobic degradation of 2,4-dichlorophenol in freshwater sediments. Appl. Environ. Microbiol. 56:1119-1127. Chapter 2: Catabolic thiosulfate disproportionation and carbon dioxide reduction in strain DCB-l, a reductively dechlorinating anaerobe 46 JOURNAL OF BACTERIOLOGY. Apr. 1990. p 2065—2070 0021-9193/90/042065 ~06$02 .00/0 Copyright C 1990. American Socrety for Microbiology 47 Vol. 172. N0, 4 Catabolic Thiosulfate Disproportionation and Carbon Dioxide Reduction in Strain DCB-l. a Reductively Dechlorinating Anaerobe WILLIAM W. MOHNl AND JAMES M. TIEDIELI‘ Departments of Microbiology‘ and Crop and Soil .S‘ciences.z Michigan State University. East Lansing. Michigan 48824 Received 27 September 1989/Accepted 3 January 1990 Strain DCB-l is a strict anaerobe capable of reductive dehalogenation. We elucidated metabolic processes in DCB-l which may be related to dehalogenation and which further characterize the organism physiologically. Sulfoxy anions and C02 were used by DCB-l as catabolic electron acceptors. With suitable electron donors, sulfate and thiosulfate were reduced to sulfide. Sulfate and thiosulfate supported growth with formate or hydrogen as the electron donor and thus are probably respiratory electron acceptors. Other electron donors supporting growth with sulfate were C0. lactate, pyruvate. butyrate, and 3-methaxybenzoate. Thiosulfate also supported growth without an additional electron donor. being disproportionated to sulfide and sulfate. In the absence of other electron acceptors. C0, reduction to acetate plus cell material was coupled to pyruvate oxidation to acetate plus C0,. Pyruvate could not be fermented without an electron acceptor. Carbon monoxide dehydrogenase activity was found in whole cells. indicating that CO2 reduction probably occurred via the acetyl coenzyme A pathway. Autotrophic growth occurred on H2 plus thiosulfate or sulfate. Diazotrophic growth occurred. and whole cells had nitrogenase activity. On the basis of these physiological characteristics, DCB-l is a thiosulfate-disproportionating bacterium unlike those previously described. The process of anaerobic reductive dechlorination of aromatic compounds is well documented (23. 28) and is of great interest for degradation of hazardous chemicals. How- ever, very little is known of the specific organisms and the metabolic properties which are responsible for this process. At present only one isolate. strain DCB-l. is known to reductively dehalogenate aromatic compounds under anaer- obic conditions. This organism provudes an opportunity to identify metabolic processes which underlie reductive deha- logenation. Additionally. it would be useful to identify a physiological group to which DCB-l belongs. as other or- ganisms in such a group may be responsible for the reductive dehalogenation activities presently known in uncharacter- ized communities. Strain DCB-l is a gram-negative, strict anaerobe isolated from an enrichment mineralizing 3-chlorobenzaate (3C8) (25). DCB-l is capable of reductive dehalogenation of 3C8 (25). other halogenated benzoates (8). and tetrachloroethyl- ene (12). Growth of DCB-l is stimulated by thiosulfate. sulfate. and sulfite, and with these substrates. sulfide is produced; thus. the organism has been labeled a sulfidogen (26). Strain DCB-l can fix C0,. and this process was reported to,occur simultaneously with fermentative growth on pyruvate (27). Dolfing and Tiedje (9) constructed a defined anaerobic consortium containing DCB-l. a benzoate fermentor. and a methanogen which grows on 3C8. Evi- dence was presented that reductive dechlorination of 3C8 stimulated growth of the consortium (10). Recent evidence indicates that pure cultures of DCB-l can conserve energy for growth from dechlorination of 3C8 coupled to H2 or formate oxidation (8a. 20a). It thus appears that reductive dechlorination of aromatic compounds can serve as a cata- bolic electron-accepting process. This physiological study of DCB-l was undertaken to elucidate metabolic activities which may be related to dehal- ogenation activities and which further classify the organism physiologically. Growth experiments with defined medium ‘ Corresponding author. 2065 identified several previously unknown substrates. A balance of carbon and electrons was determined to investigate the role of CO2 in the previously reported fermentation of pyruvate. Carbon monoxide dehydrogenase (CODH) activ- ity was assayed to indicate whether the acetyl caenzyme A (acetyl-COA) pathway is involved in CO2 reduction. The metabolism of sulfoxy anions was more fully investigated by varying electron donors and quantifying products. MATERIALS AND METHODS Cultures. Strain DCB-l and Methanospirillum sp. strain PM-l were obtained from our laboratory culture collection. All cultures in this study were done in a previously described anaerobic mineral medium (25) with the following modifica- tions. Naphthoquinone (0.6 MA) was added before autoclav- ing (K. A. DeWeerd and J. M. Suflita. Abstr. Annu. Meet. Am. Soc. Microbiol. 1988. [-108. p. 199). Unless otherwise stated. media were buffered with bicarbonate (pi-I 7) by addition of 28 mM NaHCO, and an Nz-CO, (80:20) gas phase before autoclaving. When CO2 was not wanted as a potential carbon source or electron acceptor. media were buli‘ered with 10 mM phosphate (pH 7) and an N 2 gas phase was used. Where indicated. i-l2 was included in the gas phase after autoclaving as iii-CO2 (80:20). introduced with a vacuum! pressure-gassing manifold; CO and CH, were added to the gas phase by syringe. The sulfide reductant in the described medium was replaced with 1 mM cysteine. added before autoclaving, and 0.1 mM titanium citrate (30). added after autoclaving. These reductants were replaced with 0.5 mM dithionite. added after autoclaving. from freshly prepared. filter-sterilized. anaerobic stock solution when cysteine was not wanted as a potential carbon source. All other additions to the media were made after autoclaving from filter-ster- ilized. anaerobic stock solutions. Growth experiments used lO-ml cultures in 26-ml anaerobic tubes with 10% inocula. Products from pyruvate plus CO: were determined in cul- tures buffered by addition of 15 mM NaHCO, and an Nz-COZ (95:5) gas phase and reduced with only cysteine. Products from sulfoxy anions were determined in 25-ml cultures in the 2066 MOHN AND TIEDJE same tubes with 4% inocula. Cultures used for determination of metabolic products were inoculated with cells grown on homologous media. Whole-cell enzyme assays were done with 100-ml cultures in 160-ml serum bottles. All incubations were done at 37°C. Analytical methods. Growth of cultures was measured as optical density at 660 nm (Iightpath ofculture tubes, 1.6 cm). Culture yields were determined as protein concentration by a modification (14) of the assay of Lowry et al. The gasses C02. H2. and CH. were analyzed by gas chromatography with a Carle model AGC-lll gas chromatograph equipped with a 2-m Porapak Q column and a microthermister detec- tor. The carrier for the gas analysis was Ar at 20 ml/min. and the column temperature was 40°C. For COz determinations. cultures were acidified by addition of 3 drops of H280. (pl-i < 3). shaken. and allowed to equilibrate for at least 1 h before analysis. Ethanol and ethylene were analyzed with a Varian model 3700 gas chromatograph equipped with a 2-m Porapak Q column, a flame ionization detector, and, as the carrier. N, at 9 mllmin. For the ethanol analysis. the column was at 170°C and the detector was at 200°C. For the ethylene analysis. the column was at 35°C and the detector was at 170°C. Organic acids were analyzed by the high-pressure liquid chromatography (HPLC) method of Stevens et al. (27), with the column at 60°C. Benzoates were analyzed by the HPLC method for benzoates of Stevens et al. (26). except that the eluent was water-methanol-phosphoric acid (55:45:0.05) and the UV detector was set at 230 nm. Stop- flow UV scanning was performed with a Hewlett Packard model 1050 variable-wavelength detector. Samples for sulf- oxy anion analysis were flushed for 15 min with N2 to remove H28 and stabilized from oxidation by addition of 0.2 mM glycerol (19). Sulfaxy anions were analyzed with a Dionex model 2000i ion chromatograph equipped with a Dionex AS4A column and a conductivity detector. The eluent for sulfoxy anions was 3.0 mM NaHCO,—2.5 mM NaZCO, at 2.4 ml/min. Samples for sulfide assay were diluted 10-fold in 100 mM zinc acetate to precipitate sulfide and prevent loss of hydrogen sulfide (6). Sulfide was assayed by the colorimetric method of Cline (7). Radioisotope methods. The CO2 pool of cultures was labeled with 177 MBq (1.33 uCi) of sodium [“Clbicarbonate (Research Products inc., Mount Prospect. Ill.). incorpora- tion of “CO, into cell material was determined by collecting ' cells on membrane filters (0.45 pm pore size). washing the cells with water, and dissolving the filters in Filter Solv (Beckman Instruments. inc., Fullerton. Calif). Incorpora- tion of “C02 into organic acids was determined by injection of 100 iii of culture fluid on the organic acid HPLC column and collection of eflluent fractions in basic solution. A Packard model 1500 liquid scintillation analyzer was used to measure “C. CODH assay. Cells used for the CODH assay were har- vested by centrifugation (30 min at 1.500 x g) in the culture battles. The cells were washed and suspended. concentrated 20-fold. in anaerobic 30 mM Tris hydrochloride bufl'er (pH 7) under a headspace of N2. Cell suspensions were stored at -20°C until use. CODi-l activity was determined as CO- dependent reduction of methyl viologen by the procedure of Krzycki and Zeikus (18). Nitrogenue assay. For ammonium-free medium, NILCI in the previously described mineral medium was replaced with an equimolar amount of NaCl. leaving N2 as the sole N source. Cells for the nitrogenase assay were harvested as described above and suspended. concentrated 10-fold. in homologous medium. The suspensions of 10 ml were placed 48 J. BACTERIOL. 0.15 1" > 1 .0 d o vvvavv 0.05 i- . Optical density (660 nm) Time (d) FIG. 1. Growth of strain DCB-l on various media. (A) Require- ment for electron acceptor. After two passages on phosphate- butfered medium containing 10 mM pyruvate and no electron acceptor. cells were used to inoculate homologous medium (circles). bicarbonate-buffered medium containing 10 mM pyruvate (squares). and phosphate-buffered medium containing 10 mM pyruvate plus 5 mM thiosulfate (triangles). (B) Effect of electron donors and carbon sources on growth on bicarbonate-buffered medium containing 5 mM thiosulfate. Cultures contained 20 mM formate (circles). no electron donor with 1 mM acetate as a carbon source (squares). or 1.6 atm of hydrogen with CO, as the sole carbon source (triangles). in 23-ml serum bottles. 0.05 atm (ca. 50.5 kPa) acetylene was added to the headspace. and ethylene production in the bottles was measured during incubation at 37°C. RESULTS Pyruvate metabolism. Fermentative growth of DCB-l on pyruvate was tested by growing cultures on phosphate- butfered medium with 10 mM pyruvate. It was found that growth only occurred if a potential electron acceptor was present (Fig. 1A). Growth on pyruvate plus thiosulfate in phosphate-buffered medium indicates that CO2 (other than that produced from pyruvate oxidation) is not required for anabolic purposes. Thus. DCB-l appears to require an electron acceptor for growth on pyruvate. and added CO2 appears to serve this function. In bicarbonate-buffered me- dium reduced with only cysteine. the consumption of 85 t 7 umol of pyruvate was accompanied by the production of 105 z 1 umol of acetate and 43 t 5 umol of total CO2 (values here and below are means of triplicates : standard error). Parallel replicate cultures with [“Clbicarbonate added incor- porated 4.1 t 0.1 umol of C02 into cell material and 23.8 t 0.3 umoi of CO2 into acetate. No other “C-labeled products were detected. The balances of carbon and electrons be- tween substrates and products were near 100% (Table 1) and confirm the function of CO, as an electron acceptor. Lactate was not produced in the above cultures. but on similar media with stronger reductants (e.g., sodium sulfide or titanium citrate), lactate was found as a product of pyruvate in lesser amounts than acetate. Other organic acids. ethanol. and H2 were never detected as products of pyruvate. Thus. on VOL. 172. 1990 TABLE 1. Carbon and electron balance for strain DCB-l grown on pyruvate plus CO3“ 49 CATABOLIC PROCESSES 1N STRAIN DCB-l 2067 TABLE 3. Metabolism of sulfoxy anions by strain DCB-i during 16 days of incubation on bicarbonate-buffered medium Amt used Carbon ()rR Balance (umol x Compound (umol) lumol) value” OIR value) Substrates Pyruvate 85 255 +1 +85 CO, 28 28 +2 + 56 Total 283 +141 Products Acetate _ 105 210 0 0 CO,r 71 71 +2 +142 Cell C 4 4 0 0 Total ' 285 +142 ‘ Recovery was 101%. determined as amount of carbon or as carbon and electron balance. ’ OIR value is the oxidation state of carbon. calculated by the method of Gottschalk (13). ‘ Product CO, is net CO, produced plus CO3 consumed. media with no electron acceptor other than C02. DCB-l primarily oxidizes pyruvate while it reduces C0,. Growth did not occur if pyruvate was omitted from the medium. indicating that cysteine could not support growth. Growth did not occur on bicarbonate-buffered medium with 10 mM lactate, either with or without 10 mM acetate. Use of electron acceptors. Because DCB-l could not grow on phosphate-buffered medium with pyruvate. apparently due to the lack of an electron acceptor, this medium was used to test potential electron acceptors. Furthermore. since pyruvate oxidation can support substrate-level phosphory- lation (SLP). this medium indicated general (i.e., both res- piratory and nonrespiratary) electron acceptors. Putatively respiratory electron acceptors were distinguished by substi- tuting formate for pyruvate as an electron donor. since DCB-l can oxidize formate (data shown below). but formate cannot support SLP. With formate. CO2 was provided as an additional carbon source. Sulfaxy anions served both as general and as putatively respiratory electron acceptors (Table 2). Dithionite appeared to serve as an electron acceptor. since growth was propor- tional to dithionite concentration. but because of the chem- ical instability of dithionite. other sulfur compounds may be partially or completely responsible for the growth observed. Fumarate supported relatively slow growth and low yields with pynivate. the doubling time being 17 days. Fumarate did not support respiratory growth (Table 2). and succinate TABLE 2. Use of general and putatively respiratory electron acceptors by strain DCB-l Use as electron acceptor“ Concn Test com M (mhl) General‘ Respiratory‘ Sulfate 5 + + Thiosulfate 5 + + Sulfite 3 + + Dithionite 0.5. 1. 2 +" ND Fumarate 5 + — Nitrate 5 - ND CO, (0.2 atm) + - " Symbols: +. duplicate cultures were successfully grown through three serial transfers on the medium indicated; -. criterion for + not met; ND. not done. ' Phosphate-buffered medium containing 10 mM pyruvate. ‘ Bicarbonate-buffered medium containing 10 mM formate. "We did not conduct a serial transfer experiment. but cell yield was correlated with dithionite concentration. Mean (mM) 2 SE“ Electron Electron “0"” “WW E'mm“ Sulfide Sulfate (20 mM) (.1li acceptor produced produced consumed Formate Sulfate 2.5 : 0.2 2.3 .t 0.1 Formate Thiosulfate 0.8 r 0.1 1.5 t 0.2 0.0 t 0.0 None” Thiosulfate 2.4 r 0.0 2.5 2 0.0 1.7 t 0 1 " Data are means of triplicates : standard error. " None provided other than thiosulfate and acetate (1 mM). which was required. presumably as a carbon source. did not accumulate during growth on pyruvate plus fuma- rate. Metabolism of sulfoxy anions. Both sulfate and thiosulfate were reduced stoichiometrically to sulfide when formate was provided as an electron donor (Table 3). Sulfide production from cysteine was negligible. as controls without sulfate or thiosulfate had less than 0.1 mM sulfide after autoclaving and less than 0.2 mM sulfide after inoculation and incubation. Sulfate and thiosulfate were stable in uninoculated controls. while 1.0 mM sulfide was partially oxidized during the 16-day incubation and formed 0.2 mM thiosulfate. There was a linear relationship between limiting amounts of sulfate or thiosulfate (i.e., 1 mM or less) and total growth. Thus. sulfate and thiosulfate clearly served as respiratory electron acceptors. Thiosulfate was disproportionated ta sulfide and to a lesser amount of sulfate when formate was omitted (Table 3). There was again a linear relationship between limiting amounts of thiosulfate and total growth. Growth occurred through at least five passages on medium containing 5 mM thiosulfate and 1 mM acetate. the latter substrate being required. presumably as a carbon source. Thiosulfate dis- proportionation therefore supports grOWth of DCB-l. al- though some acetate oxidation may have occurred. account- ing for the production of more sulfide than sulfate. Apparent growth rates on thiosulfate were similar with and without formate (Fig. 18). In the presence of 20 mM pyruvate. both sulfide and a smaller amount of sulfate were found as products of thiosulfate (data not shown). Together. these results indicate that the fate of thiosulfate may depend on thermodynamic conditions for growth of DCB-l (i.e., the availability of electron donors and their strength as reduc- tants). The toxicity of the sulfide product apparently limited growth and consumption of sulfate or thiosulfate. Starting concentrations of 3 mM sulfate and thiosulfate were never completely consumed. even though electron donors were supplied in excess. Cultures never produced 3 mM or more sulfide. When cultures growing on 20 mM formate and 3.5 mM thiosulfate were flushed with Nz-CO2 in late log phase. removing H28. growth continued longer than in unflushed control cultures. Flushing increased thiosulfate consumption from 30 to 88 umol and final cell protein from 0.78 to 0.97 mg. Cultures incubated on medium containing 10 mM pyru- vate. 0.05% yeast extract. and various initial sulfide concen- trations grew only when the sulfide concentration was 2 mM or less. Test of syntrophic growth. To determine whether an H,- consuming organism could substitute for an electron accep- tor. DCB-l and Methanospiri‘lliim sp. strain PM-l were cocultured on bicarbonate-bufi'ered medium with pyruvate. Growth occurred. but there was no significant production of 2068 MOHN AND TIEDJE l I I 1 J 0.04" 0.8 i- E A up ~De C I E + « 0.033 : 0.6~ ‘ 3 3 l a g 0“ .- 1 0.02 '2 g L fl 0“, 8 C 0.2 p (O '4 0.0‘ B 1 s 1 s O 0"0 s to is 20 0'00 fime (d) FIG. 2. Consumption of a limiting amount of pyruvate with transient accumulation of acetate during growth of strain DC B-l on 5 mM sulfate as an electron acceptor. Data are means of triplicates with standard errors (bars). 00.. Optical density. methane. Microscopic observation of the coculture revealed only DCB-l cells. Thus. even under favorable conditions (i.e., low H, partial pressure). DCB-l did not produce H, from pyruvate. Use of electron donors and carbon sources. The use of electron donors was tested with 5 mM sulfate as an electron acceptor. The criteria for use were significant (by the Stu- dent t test. a = 0.05) stimulation of both growth (measured as optical density) and sulfide production relative to those in control cultures without electron donors. The following electron donors were used (initial concentration): H, (1.6 atm). formate (10 mM). CO (0.1 atm). lactate (10 mM). pyruvate (10 mM). butyrate (10 mM). and 3-methoxyben- zoate (2.5 mM). The following were not used: methane (0.1 atm). methanol (2.5 mM). ethanol (2.5 mM). acetate (10 mM). propionate (10 mM). glycerol (10 mM). malate (10 mM). fructose (5 mM). or glucose (5 mM). Conditions used here to test electron donors differed from conditions used previously to test substrates (26) in several ways: (i) sulfate was provided here as an electron acceptor. (ii) naphthoqui- none was provided here. and (iii) ruminal fluid was not provided here. Growth with CO was very slow relative to growth with other electron donors. An initial concentration of 10 mM pyruvate was incompletely oxidized to acetate during growth on sulfate thiosulfate. but at a lower initial concentration with excess sulfate. the acetate product sub- sequently disappeared (Fig. 2). Sulfide accumulation may have prevented complete oxidation of the higher concentra- tion of pyruvate. Thus. with sulfate as an electron acceptor. DCB-l may completely oxidize pyruvate despite its inability to grow when transferred to medium with sulfate and acetate as a sole electron donor (see above). Consumption of a low initial concentration of 3-methoxybenzoate with excess sul- fate was accompanied by stoichiometric production of 3- hydroxybenzoate. which was not degraded further (Fig. 3). indicating that only the methoxyl group of that substrate was used. The identity of the 3-hydroxybenzoate end product was confirmed by comparison of its UV spectrum with that of an authentic standard. Both lactate and pyruvate served as sole carbon sources. supporting serial transfers on phos- phate-bufi'ered. dithionite-reduced medium with 5 mM thio- sulfate. Autotrophic growth was possible. as growth occurred through at least five serial transfers each on dithionite- reduced. bicarbonate-buffered medium containing 1.6 atm of H, plus either 5 mM thiosulfate or 5 mM sulfate. The apparent doubling time of autotrophic cultures with thiosul- fate was longer than that of other cultures with thiosulfate 50 J. BACTERlOL. .0 o a A A . E 1 E -I 0.03 8 v c m-hydroxybenzoote . 3 .9 " on - 0.02 g o s t v 3 .. 0.013 8 .9. m—methoxybenzoote . g QM Time (d) FIG. 3. Consumption ofalimiting amountof 3-methoxybenzoate with accumulation of 3-hydroxybenzoate as an end product during growth of strain DCB-l on 5 mM sulfate as an electron acceptor. Data are means of triplicates with standard errors (bars). 0.0.. Optical density. (Fig. 18). AutotrOphic growth on thiosulfate did not occur without an additional electron donor. CODH activity. CODH activity was assayed to determine whether the acetyl-CoA pathway could be responsible for CO, fixation by DCB-l. Activity was detected in cells growth on all media tested (Table 4). These activities are lower than those reported for some other sulfate reducers believed to be using the acetyl-CoA pathway (24). but the differences in most cases are less than an order of magni- tude. Factors contributing to the differences may be the slower growth rate of DCB-l and the testing of whole rather than permeabilized DCB-l cells. The addition of 1 mM potassium cyanide to a suspension of cells grown on 11,. C0,. and 5 mM thiosulfate caused an 86% inhibition of ‘CODH activity. Growth of DCB-l did not occur with 10 mM thiosulfate plus CO as a sole carbon source. Test of homoacetogenic growth. On bicarbonate-buffered medium with no additional electron acceptor. growth did not occur with 1.6 atm of H, or 10 mM formate. Growth did occur on this medium with 5 mM 3-methoxybenzoate through three serial transfers but then stopped. Acetate was not produced from 3-methoxybenzoate. Since the carbon sources provided in these media were sufficient for growth with thiosulfate. it appears that DCB-l did not gain sufficient energy for growth from these substrates by homoacetogen- e5is. Dlaaotrophlc growth. Growth of DCB-l occurred on 10 mM pymvate plus 5 mM thiosulfate through at least five serial transfers with N, as the sole N source. Serial transfers could not be maintained if N, was replaced by Ar. The growth rate was lower on N, than on ammonium. Cells grown on N, had nitrogenase activity. measured as produc- tion of 0.24 umol of ethylene min" g of protein“ from acetylene. This activity is probably inducible. as cells grown on ammonium produced no ethylene. TABLE 4. CODH activity in whole cells of DCB-l grown on various bicarbonate-buffered media Substrate (concn. mM) Reductant Activity“ Pyruvate (10) Cysteine 81 H, (1.6 atm) + thiosulfate (5) Dithionite 363 Formate (10) + thiosulfate (5) Dithionite 479 Pyruvate (10) + thiosulfate (5) Dithionite 721 " Activity in micromoles of CO per minute per gram of protein. values are means of triplicates. VOL. 172. 1990 DISC USS ION Stevens et al. (26) reported that various sulfoxy anions stimulated the growth of DCB-l on pyruvate with the formation of undetermined amounts of sulfide and so classi- fied the organism as a sulfidogen. Here. we show that sulfate was reduced stoichiometrically to sulfide (Table 3); thus. DCB-l is a sulfate-reducing bacterium. Since H, and formate are not known to support SLP. the ability of DCB-l to grow with either of these electron donors and sulfate (Table 2) indicates a respiratory process. like that found in other sulfate reducers (2). Like most other sulfate reducers. DC B- 1 will also grow on thiosulfate. sulfite. and. possibly. dithio- nite (Table 2). Use of dithionite as a reductant probably explains the previous finding that DCBol grew on lactate plus acetate (26). which we did not find on cysteine-titanium citrate-reduced medium. Growth of DCB-l was also sup- ported by disproportionation of thiosulfate to sulfate and sulfide (Table 3). Growth by this type of lithotrophic fermen- tation has been reported for Desulfovibrr’o sulfodismurans. D. desulfuricans. and two other strains of sulfate reducers (3. 4. 17). Sulfide appears to be relatively toxic to DCB-l compared with other sulfate reducers. However. toxicity of 2 mM sulfide has also been reported for Desulforomaculum Spp. (16). In open natural systems. such sensitivity may not be a great disadvantage, as sulfide might be prevented from accumulating by precipitation. diffusion. and volatilization of H,S. However. such sensitivity to sulfide could greatly bias traditional enrichment and isolation procedures against such organisms. The apparent use of fumarate as an electron acceptor by DCB-l (Table 2) seems not to be respiratory. as it is in many other organisms. including some sulfate reducers (5). De- spite the fact that DCB-l can reduce nitrate to nitrite (25). nitrate did not serve as a catabolic electron acceptor for DCB-l (Table 2) as it can for some other sulfate reducers (20). Desulfovi‘bri'o desulfuri'cans. which can grow by sulfite disproportionation. can use both fumarate and nitrate as electron acceptors. DCB-l was previously reported to ferment pyruvate (26); however. the fermentation balance was not complete. The carbon recovery was near 100%. but the recovery of reduc- ing equivalents was low. We found that DCB-l is unable to grow on pyruvate without some electron acceptor (e.g.. sulfoxy anions or C0,). Specifically. DCB-l did not ferment pyruvate to acetate. C0,. and H, as can many sulfate reducers. and although lactate was formed by DCB-l or by nonbiological reactions under certain conditions (i.e.. in the presence of strong reductants). DCB-l did not ferment pyruvate to equimolar amounts of lactate. acetate. and C0,. Our results indicate that pyruvate oxidation (equation 1. below). in the absence of any other electron acceptor. is balanced by C0, reduction to acetate and cell material (equation 2). For simplification. we assume that cell carbon has the same oxidation state as that of acetate (0/R value = 0). Our data fit the net equation (equation 3) well and indicate a balance of carbon and electrons between substrates and products (Table 1). In agreement with these results. Stevens and Tiedje (27) reported that during metabolism of pyruvate by DCB-l. 20% of the acetate formed was from C0,. The proposed catabolic mode for DCB-l (equation 3) is not a fermentation in the strict sense of the term. as a substrate is not being simultaneously oxidized and reduced to yield a net balance of reducing equivalents in the substrate and the products. Like a fermentation. however. this mode may provide energy for growth entirely from SLP. Lactate could 51 CATABOLIC PROCESSES IN STRAIN DCB-l 2069 not be used in the preceding manner or be fermented by DCB-l under the conditions provided. 4CH3COCOOH + 4 H,O—-4CH.COOH + 4C0, + 8 H (I) 8 H + 2 CO,- CHiCOOH + 2 H30 (2) 4 CH3COCOOH + 2 H3O -0 5 CHiCOOI-I + 2 CO, (3) The finding of CODH (acetyl-CoA synthase) activity sug- gests that CO, fixation by DCB-l occurs via the acetyl-CoA pathway. as in Desulfovi‘bri‘o baarsi'i‘ (15). Operation of the reductive pentose phosphate pathway is unlikely. as DCB-l was found to lack ribulose bisphosphate activity (T. 0. Stevens. M.S. thesis. Michigan State University. East Lan- sing. 1987). The acetyl-CoA pathway probably accounts for several other activities which we identified in DCB-l. The pathway may allow DCB-l to reduce CO, to acetate. as do homoacetogens and some Desulforomaculum spp. (16); however. unlike homoacetogens and Desulforomaculum ori- enri‘s. DCB-l apparently can only gain energy by coupling CO, reduction to oxidations which support SLP (i.e.. pyru- vate oxidation). The slow oxidation of C0 and. possibly. acetate by DCB-l may also be catalyzed by the acetyl-CoA pathway. as in other sulfate reducers (24). and is consistent with the reversible nature of the pathway. DeWeerd et al. (8) have shown that DCB-l metabolizes 3-methoxybenzoate by O-demethylation. an activity found in acetogens (l) and Desulforomaculum spp. (16) and also thought to be cata- lyzed by enzymes of the acetyl-CoA pathway. Finally. the ability of DCB-l to dechlorinate tetrachloroethylene may be due to the acetyl-CoA pathway. as Egli et al. (11) have proposed a correlation of this pathway and the ability of anaerobes to dechlorinate volatile alkanes. although it should be noted that this correlation was based on dechlo- rination of tetrachloromethane. Thus. several of our findings can be explained by the acetyl-CoA pathway. Diazotrophic growth is another property of DCB-l com- mon to many other sulfate reducers. This capacity is known among several members each of the genera Desulfovibri'o (22). Desulforomaculum (21). and Desulfobacrer (29). The rate of acetylene reduction found for DCB-l is lower than. but within an order of magnitude of. rates reported for members of the above genera. Strain DCB-l is a sulfate reducer which has physiological characteristics found in a broad range of members of this group but does not fit well into any known physiological subset of the group. The morphology of DCB-l is unique by virtue of a collar structure (25). It remains to be determined whether DCB-l is a representative of a physiological group of sulfate reducers yet to be characterized and. if so. whether other reductive dehalogenators are also in such a group. This study will hopefully facilitate attempts to find other organisms related to DCB-l. Recent evidence indi- cates that reductive dechlorination of benzoates by DCB-l is a respiratory process yielding energy which supports growth (8a. 20a). Presumably DCB-l did not evolve under natural selection for a respiratory system dedicated to chlorinated substrates. Since sulfoxy anions are the only other puta- tively respiratory substrates for DCB-l presently known. it appears most likely that the same respiratory system may be coupled to sulfoxy anion reduction and reductive dehaloge- nation of benzoates. It is possible that other sulfate reducers are capable of reductive dehalogenation under certain con- ditions. especially sulfur compound disproportionaters. which appear to have the most catabolic similarity to DCB-l. Although some Desulfovibn‘o spp. have been tested for 2070 MOHN AND TIEDJE dechlorination activity without success (T. G. Linkfield. Ph.D. thesis. Michigan State University. East Lansing. 1985). this possibility needs to be more thoroughly investi- gated. ACKNOWLEDGMENTS We thank John Kemner for assistance with the CODH assay and J. G. Zeikus for the use of the facilities for that assay. We thank Joyce Wildenthal for assistance with the protein assay and Edwin Stegmann for help in developing analytical methods for sulfur compounds. We thank James R. Cole and Friedhelm Bak for helpful discussions. This research was supported by Environmental Protection Agency grant R-813892. ADDENDUM IN PROOF The name Desulfomom'le ri'edjei' has recently been pro- posed for strain DCB-l (K. A. DeWeerd. L. Mandelco. R. S. Tanner. C. R. Woese. and J. M. Suflita. Arch. Microbiol.. in press). This new genus is indicated by physiological charac- terization as well as 16S rRNA sequence analysis of strain DCB-l. LITERATURE CITED 1. Bacbe. R.. and N. Pfennig. 1981. Selective isolation of Aceto- bacterium woodi'i' on methoxylated aromatic acids and determi- nation of growth yields. Arch. Microbiol. 130:255-261. 2. Badzlong. W.. and R. K. “latter. 1978. Growth yields and growth rates of Desulfovi'bri‘o vulgaris (Marburg) growing on hydrogen plus sulfate and hydrogen plus thiosulfate as the sole energy sources. Arch. Microbiol. 117:209-214. 3. Bak. F.. and H. Cyplonka. 1987. A novel type of energy metabolism involving fermentation of inorganic sulfur com- pounds. Nature (London) 326:891-892. 4. Bali, 17.. and N. Pfesinlg. 1987. Chemolithotrophic growth of Desulfovibri'o sulfodi'smurans sp. nov. by disproportionation of inorganic sulfur compounds. Arch. Microbiol. "72184-189. 5. Barton. 1... J. LeGall. and H. D. J. Peck. 1970. Phosphorylation coupled to oxidation of hydrogen with fumarate in extracts of the sulfate reducing bacterium. Desulfovibri'o gigas. Biochem. Biophys. Res. Commun. 41:1036—1042. 6. Brock. T. D.. M. 1.. Brock. T. L. Bott. and M. R. Edwards. 1971. Microbial life at 90’C; the sulfur bacteria of Boulder Spring. J. Bacteriol. 107:303-314. 7. Cllne. J. D. 1969. Spectrophotometric determination of hydro- gen sulfide in natural waters. Limnol. Oceanogr. 14:454-458. 8. DeWeerd. K. A.. J. M. Snfllta. T. Linkfield. J. M. Tiedje. and P. H. Pritchard. 1986. The relationship between reductive de- halogenation and other aryl substituent removal reactions cata- lyzed by anaerobes. FEMS Microbiol. Ecol. 38:331-339. 8a.Dollng. J. 1990. Reductive dechlorination of 3—chlorobenzoate is coupled to ATP production and growth in an anaerobic bacterium. strain DCB-l. Arch. Microbiol. [53:264—266. 9. 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. 10. Dolfing. J.. and J. M. Tiedje. 1987. Growth yield increase linked to reductive dechlorination in a defined 3-chlorobenzoate de- grading methanogenic coculture. Arch. Microbiol. [49:102-105. 11. Egll. C.. T. Tech-n. R. Sebalta. A. M. Cook. and T. . 1988. Transformation of tetrachloromethane to dichlorometh- 52 J. BACTERIOL. ane and carbon dioxide by Arrmhricren‘um woodi'r‘. Appl. En- viron. Microbiol. 54:2819-2824. l2. Fathepure. B. 2.. J. P. Nengu. and S. A. Boyd. 1987. Anaerobic bacteria that dechlorinate perchloroeihene. Appl. Environ. Mi- crobiol. 53:2671-2674. l3. Gottschalk. G. 1986. Bacterial metabolism. 2nd ed.. p. 232. Springer-Verlag. New York. 14. Hanson. R.. and J. Phillips. 1981. Chemical composition. p. 358. In P. Gerhardt (ed.). Manual of methods for general microbiol- ogy. American Society for Microbiology. Washington. DC. 15. Jansen. K.. G. Fuchs. and R. K. Thauer. 1985. Autotrophic C0, fixation by Desulfovibri'o baarsii': demonstration of enzyme activities characteristic for the acetyl-CoA pathway. FEMS Microbiol. Lett. 28:311-315. 16. Klemps. R.. H. Cyplonlta. F. Wlddel. and N. Pfennig. 1985. Growth with hydrogen. and further physiological characteristics of Desulforomaculum species. Arch. Microbiol. [43:203-208. 17. Kramer. M.. and H. Cyplonlta. 1989. Sulfate formation via ATP sulfurylase in thiosulfate- and sulfite-disproportionating bacte- ria. Arch. Microbiol. [51:232-237. 18. Krzyekl. J. A.. and J. G. Zeikus. 1984. Characterization and purification of carbon monoxide dehydrogenase from Metha- nosarcina barkeri. J. Bacteriol. [58:231-237. 19. Lindgren. M.. and A. Cedergren. 1982. Conditions for sulfite stabilization and determination by ion chromatography. Anal. Chim. Acta 141:279-286. 20. McCready. R. G. L.. W. D. Gould. and F. D. Cook. 1983. Respiratory nitrate reduction by Drsulfow‘brr‘o sp. Arch. Micro- biol. [35:182-185. 20a.Mohn. w. w.. and J. M. Tiedje. 1990. Strain DCB-l conserves energy for growth from reductive dechlorination coupled to formate oxidation. Arch. Microbiol. 153:267-271. 21. Postgate. J. R. 1970. Nitrogen fixation by sporulating sulphate- reducing bacteria including rumen strains. J. Gen. Microbiol. 63:137-139. 22. Postgate. J. R.. and H. M. Kent. 1985. Diazotrophy within Desulfovi'brio. J. Gen. Microbiol. [31:2119-2122. 23. Reineke. W.. and H.-J. Knackmuss. 1988. Microbial degradation of haloaromatics. Annu. Rev. Microbiol. 42:263-287. 24. Sehauder. R.. B. Elkmanns. R. K. Thauer. I". Wlddel. and G. F ueln. 1986. Acetate oxidation to C0, in anaerobic bacteria via a novel pathway not involving reactions of the citric acrd cycle. Arch. Microbiol. 145:162-172. 25. 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. 26. Stevens. T. 0.. T. G. Llnkfield. and J. M. Tiedje. 1988. Physio- logical characterization of strain DC B-1. a unique dehalogenat- ing sulfidogenic bacterium. Appl. Environ. Microbiol. 54:2938- 2943. 27. Stevens. T. 0.. and J. M. Tiedje. 1988. Carbon dioxide fixation and mixotrophic metabolism by strain DCB-l. a dehalogenating anaerobic bacterium. Appl. Environ. Microbiol. 54:2944—2948. 28. Tiedje. J. M.. S. A. Boyd. and B. Z. Fathepure. 1987. Anaerobic degradation of chlorinated aromatic hydrocarbons. Dev. lnd. Microbiol. 27:117-127. 29. Wlddel. F. 1987. New types of acetate-oxidizing. sulfate-re- ducing Desulfobacrer speCies. D. Iii-drogenophiliis sp. nov.. D. lulu: sp. nov.. and D. cun'ams sp. nov. Arch. Microbiol. I48:286—291. 30. Zehnder. A. J. 8., and K. thrmann. 1976. Titaniumllll) citrate as a nontoxic oxidation-reduction buffering system for the culture of obligate anaerobes. Seience 194:1165-1166. Chapter 3: Strain DCB-l conserves energy for growth from reductive dechlorination coupled to formate oxidation 53 Arch Microbiol (l990) l53.267 -27l 54 Archives of 1‘ Springer-Verlag 1990 Strain DCB—l conserves energy for growth from reductive dechlorination coupled to formate oxidation William W. Mohn‘ and James M. Tiedje U Departments of Microbiology' and Crop and Sml Scienccs" Michigan State Univ. East Lansing. Ml 48824. USA Abstract. Strain DCB-l is a strict anaerobe capable of the reductive dechlorination of chlorobenzoates. The effect of dechlorination on the yield of pure cultures of DCB-l was tested. Cultures were incubated with formate or H, as electron donors and CO, as a putative carbon source. Re~ lative to control cultures with benzoate. cultures which dechlorinated 3-chlorobenzoate and 3.5-dichlorobenzoate had higher yields measured both as protein and cell density. 0n the media tested the apparent growth yield was 1.7 to 3.4 g cell protein per mole Cl‘ removed. Dechlorination also stimulated formate oxidation by growing cultures. Resus- pended cells required an electron donor for dechlorination activity. with either formate or elemental iron serving this function. Resuspended cells did not require an electron ac- ceptor for formate consumption. but reductive dechlorina- tion of 3CB to benzoate stoichiometrically stimulated oxi- dation of formate to C0,. These results indicate that DC B—l conserves energy for growth by coupling formate. and prob- ably, H, oxidation to reductive dechlorination. Key words: Anaerobic degradation — Reductive dehalo- genation — Chlorobenzoate — Growth yield — Anaerobic respiration Dolfing and Tiedje (1987) demonstrated that the cell yield of a defined anaerobic consortium was greater with 3-chlorobenzoate (3CB) as a substrate than with benzoate. This finding indicates that the initial reaction in the degra- dation of 3C B. reductive dechlorination to benzoate. stimu- lates growth of the consortium. Thermodynamic data were also presented in that study showing that the reductive dechlorination of 3CB coupled to H, oxidation is exergonic under the physicochemical conditions of the consortium (210' =- -112 kJ per mole 3CB). Together this evidence suggests a novel mode of catabolism whereby energy is con- served during anaerobic reductive dechlorination. Because the consortium contained three organisms which were nu- tritionally interdependent. it could not be definitely con- cluded that the dechlorinating organism. strain DC B- 1. con- served energy from reductive dechlorination. Recently. we discovered that the addition of Pro- pi'oni'bacteri'um sp. culture fluid ( PC F) stimulates growth and dechlorination activity in pure cultures of DCB-l with Offprint requests to .' J. M. Tiedje Nonstandard abbreviations: 3CB. 3-chlorobenzoate; 350C B. 3.5- dichlorobenzoate; PC F. Propr'om’bacteri’um sp. culture fluid pyruvate and C0, as carbon and energy sources (Apajalahti J. Cole J and Tiedje JM. Abstr. Ann. meeting Amer. Soc. Microbiol.. 1989). Strain DCB-l was found to consume H, (Linkfield and Tiedje 1989). use thiosulfate for growth (Stevens et al. 1988) and fix C0, (Stevens and Tiedje 1988). We subsequently found that putatively autotrophic growth could occur on hydrogen or formate with a suitable electron acceptor such as thiosulfate. and that whole cells of DCB-l have carbon monOXIde dehydrogenase activity (Mohn and Tiedje. submitted). DeWeerd et al. (1986) found that 3.5- dichlorobenzoate (3SDCB) could also be dechlorinated by DCB-l. These advances have allowed the following study in which DCB-l was grown in pure culture with either H, or formate as an electron donor and 3CB or 35DCB as an electron acceptor. Resuspended cells were used to test vari- ous electron donors for dechlorination. and to determine the stoichiometry of formate oxidation coupled to 3CB dechlo- financn. Materials and methods Culture methods Strain DCB-l was isolated from a sewage sludge enrichment grown on 3-chlorobenzoate (Shelton and Tiedje 1984). All cultures were grown on reduced anaerobic mineral medium prepared as described by Shelton and Tiedje (1984). with the following modifications. The mineral salts included 0.5 mg/l CoCl, - 6 H,0 and 0.05 mg/l Na,SeO3. Before autoclaving. the vitamin mixture of Wolin et al. (1963) plus 50 pg/I naphthoquinone (DeWeerd K. personal communication) were added. To reduce the medium. 1 mM cysteine was added before autoclaving. and 0.1 mM titanium citrate (Zehnder and Wuhrmann 1976) was added after autoclaving. All amendments after autoclaving were from anaerobic. fil- ter—sterilized stock solutions. All cultures were 50 ml. incu- bated in l60-ml serum bottles at 37 C. The medium for stock cultures had 0.01% yeast extract and l0°/o Propiom'hacteri'um sp. culture fluid (PFC). 10 mM pyruvate acid and 0.5 mM sodium 3-chlorobenzoate added before autoclaving. Dechlorination was monitored in stock cultures. and 0.5 mM 3C8 was again added after dechlorina- tion of the initial amount. After the total 1 mM 3CB was dechlorinated. cultures were in stationary phase and were used as inocula for experimental cultures or were resus- pended for cell suspension experiments. Propi‘om'bacteri‘um sp. was grown on the same mineral medium with 0.05% yeast extract and 20 mM lactic acid added before autoclaving. The culture fluid was filtered 268 55 Table l. The effect of reductive dechlorination on yield of DC B-l cultures grown on several media” PC F Electron Electron Time DeCl‘ Protein Cells: ml Growth donor acceptor (days) (mM) (pg/ml) ( x 10 ' ‘1 yield“ —- Form BA 72 - 6.9 i- 0.7 68 1» l6 — — Form 3C8 72 1.9 12211.0" 160:10“ 2.8 — Form 3SDCB 72 1.4 11.6i04” 130:10' 3.4 + Form BA 44 - 11.2 :05 86: 5 — + Form 3CB 44 3.8 17510.8“ 300:90‘ 1.7 + H, BA 36 — 101:0.1 not 0 — + H, 3CB 36 1.9 13.6:06" 160110' 1.8 ' Abbreviations: PCF. Propiom'bactertum sp. culture fluid; OD. optical dcnsnty at 660 nm; Form. formate; BA. benzoate; 3CB. 3-chloro- benzoate; 35DCB. 3.5-dichlorobenzoate " All values are means of triplicates i standard error; except for duplicate 3SDCB cultures ‘ Dechlorination as mM Cl ' removed ‘ Growth yield as the protein yield in g from dechlorination (dechlorinating cultures less corresponding controls) per mole Cl ' removed ‘ P< 0.2. “ P< 0.02, probabilities that dechlorinating cultures are not significantly different than corresponding controls by Student‘s f-lCSl (0.22 pm) under anaerobic conditions and stored at 4°C under N ,. Experimental cultures also had the same mineral medium with the following amendments for the indicated treatments. PC F. 5%, was added before autoclaving. H, was added after autoclaving by changing the headSpace to 2 atm H, -CO, (80:20) using a gassing manifold. Sodium formate. 10 mM. and sodium benzoate or sodium chlorobenzoates. 1 mM. were added after autoclaving. All experimental cultures were in triplicate. Because of the reported toxicity of 3CB to DCB-l (Shelton and Tiedje 1984), chlorobenzoates were added incrementally to cultures. Concentrations of benzo- ates were monitored. and chlorobenzoate concentrations were maintained between 0.1 and 1 mM by addition from stock solutions. Cultures without PCP received 5% inocula. and those with PCP received 10% inocula. Cell suspensions Stock cultures were harvested by centrifugation (1500 x g) in culture bottles. The cells were washed and resuspended. concentrated approximately fivefold. in 20 mM TRIS - HCl buffer pH 7.0 under N ,. This buffer was deoxygenated by boiling under N,. but no reductant was added. All treat- ments with cell suspensions were done in triplicate and were incubated at 37°C. Analytical methods Organic acids were analyzed by HPLC using the method of Stevens et al. (1988) with the column temperature at 60°C. Benzoic acids were analyzed by HPbC using the method of Stevens et al. (1988) except that the eluent was methanol - 0.10% phosphoric acid (55 :45), the column was at 40° C. the UV detector was set at 230 nm and 500 MM 2.4-dichlorobenzoate was added to samples before filtration as an internal standard. Total CO, was determined by acidi- fying cell suspensions with H ,SO‘ (pH < 2). allowing 30 min for equilibration, measuring CO, in the headspace and cal- culating dissolved C0,. A 0.20-ml headspace sample was injected on a Carle model AGC-111 gas chromatograph equiped with 6 m Porapak Q column and microthermister detector; the carrier was Ar at 20 ml/min. Protein concen- tration was determined by the method of Lowry as described by Hanson and Phillips (1981). Cell density was determined with a Burker—Turk counting chamber and a Leitz Orthoplan 2 microscope using 640 x magnification and phase contrast illumination; samples for counting were fixed with 5% for- malin and concentrated 10-fold by centrifugation. Examin- ation of cultures for purity was with the same microscope using 1570 x magnification. Dry weight was determined by collecting 35 ml samples on tared membrane filters (0.45 um) and drying to constant weight at 80"C. Raults Experimental cultures Reductive dechlorination of 3CB or 35DCB occurred on all media used.'and there was complete recovery of the aromatic ring as benzoate and residual chlorobenzoates (93- 105% recovery). Cultures were incubated until the turbidity of the dechlorinating cultures was clearly above that of the controls. The yields, as protein and cell density. of re- ductively dechlorinating cultures were significantly higher than those of corresponding control cultures with benzoate added. indicating both an increase in biomass and cell num- ber due to dechlorination (Table 1). Comparison of dry weights. cell- densities and protein concentrations indicate that the mean dry weight per cell was 1.8 pg. and protein was a mean of 49% of dry weight; these expected proportions support the accuracy of the protein and cell density detenni- nations and are similar to values found for DCB-l grown on other media. The stoichiometric recovery of benzoate from chlorobenzoates in the dechlorinating cultures. as well as the presence of benzoate in control cultures. indicate that reductive dechlorination. and not utilization of the benzoate product. is responsible for the differences in yield observed. The growth yield per mole Cl ’ removed from 35DC B was similar to, and not lower than. that from 3C B in correspond- ing cultures (Table 1); thus. the molar growth yield from 35DCB is approximately double that from 3C B. This diiTer- ence is consistent with conservation of energy for growth from the removal of each chlorine atom from 350C B. Syntrophic interactions. such as those with the other members of the defined consortium or those with a Pro- piom’bacterium sp.. affect growth and dechlorination activity 0 ri't‘1'1't't'1 ‘ -l A1 a -I . -t S “ 1 ‘ -I .5. . 52 _/’_g_______ 4 p - ,. 10 - '- 03P ‘ L . §lr ‘ L . 4!- - 2i- " LA . o iozosotosoao‘ro' 11ME(doya) Fig. I. Formate consumption and dechlorination by DCB-l cul- tures. Panel A. without Propioniboeterium sp. culture fluid. panel B. with culture fluid. Open symbols. formate consumption; closed symbols. dechlorination as Cl‘ removed from 3C8 or 3SDCB. Circles. control cultures with benzoate. squares. cultures with 3C B; triangles. cultures with 3SDCB of DC B-l. Because such effects would greatly complicate interpretation of these experiments. we carefully checked the purity of our cultures. The stock cultures used to inoculate experimental cultures were also used to inoculate the me- dium for Propionibacterium sp. After 2 weeks incubation growth was not apparent. and microscopic examination revealed only cells having the morphology characteristic of DCB-l. indicating that the stock cultures were not contami- nated with Propionibacterium sp. or any other organism capable of lactate fermentation. At the end of incubation. experimental cultures were also examined. and only cells with the morphology of DCB-l were found. The initial protein concentrations of cultures sup- plemented with PCF and formate was 8.9 ug/ml. and that of cultures supplemented with PCP and H, was 7.9 ug/ml. Growth therefore occurred in both dechlorinating cultures and controls; although. growth was higher in dechlorinating cultures than in corresponding controls. the difference being approximately fourfold between those with formate. and threefold between those with H, (Table 1). Assuming growth was logarithmic. dechlorinating cultures with formate and H, had doubling times of 45 and 46 days. respectively. To test the coupling of formate oxidation to reductive dechlorination. formate consumption was measured in ex- perimental cultures. Formate consumption was markedly increased by the presence of 3CB or 3SDCB (Fig. 1); although. control cultures also consumed formate. The molar difference in formate consumption between the dechlorinating cultures and controls approximated Cl‘ re- moval. but was slightly higher (Fig. 1). Organic acids. includ- ing acetate. were not produced in amounts sufficient to bal- ance formate consumption observed (i.e. less than 0.5 mM). 269 cutoaoasnzom: (mu) L_1 . j a J k 1 0"’o 10 20 so 40 TIME (hours) Fig. 2. Dechlorination of 3C8 by resuspended DCB-l cells (148 ug protein per ml) in the presence of various electron donors. Circles. control; squares. 10 mM formate; triangles. 1 atm H,; diamonds. 5 mg/ml iron powder Concentration (mM) 110 TIME (hours) Fig. 3. Formate consumption and dechlorination of 3C 8 by resus- pended DCB-l cells. Open symbols. formate consumption; closed symbols. dechlorination measured as benzoate produced. Circles. control suspensions without 3C B; squares. suspensions with 3C B Cell suspensions Cell suspensions were used to more directly test the coupling of formate oxidation to dechlorination. DC 84 cells retained reductive dechlorination activity when washed and resus- pended in deoxygenated. but unreduced buffer under N, (Fig. 2). An electron donor was required for activity. and either formate or elemental iron could serve this function. the latter indicating that the electron donor need not also be a proton donor. An electron acceptor was not required for formate consumption by resuspended cells; however. the addition of 3CB to such suspensions stimulated formate consumption (Fig. 3). These results are consistent with those from growing cultures. The stimulation of oxidation of for- mate to CO, was stoichiometrically equal to the reductive dechlorination of 3C8 to benzoate (Table 2). Acetate was not produced by cell suspensions. Disctnsion The ability of formate to support reductive dechlorination in cell suspensions (Fig. 2) and the stimulation of formate consumption by dechlorination in growing cultures (Fig. 1) 57 270 Table 2. Substrate consumption and product formation after 34 h incubation of resuspended DCB-l cells provided with 26.8 timol formate with or without 5.2 umol 3CB‘ Treatment Formate CO, 3C8 Benzoate consumed produced consumed produced Without 3C8 9.0 t 0 2 8 1 t 0.1 — — with3CB 15.3:03 13.4:05 52:00 58:01 Difference due to dechlorination 5.7 i 0.5 5.3 i 0.6 5.2 t 0 .0 5.8 :01 ‘ Values are means oftriplicates in timol + standard error and cell suspensions (Fig. 3) strongly support the coupling of formate oxidation and dechlorination by DCB-l. The differences in formate consumption between dechlorinating cells and non-dechlorinating controls. relative to dechlorina- tion. in both growing cultures (Fig. 1) and cells suspensions (Table 2) suggest that the two processes are stoichiometri- cally related according to the following equations: HCOO' + 3CB‘ + H,O -. HCO,’ + benzoate' + H+ + Cl‘ 2HCOO‘ + 3SDCB' + 2 H,O 4 2HCO§ + benzoate‘ + 2H+ + 20'. Another oxidant than 3CB or 35DCB is required to explain the formate consumption in non-dechlorinating cultures and cell suspenSions and the stimulation of formate consumption in dechlorinating cultures and cell suspensions somewhat in excess of that predicted by the above equations. Fixation of CO, probably accounts for at least some formate oxidation in the growing cultures. We have found that CO, fixation consumes a significant portion of the reducing equivalents produced by DC B-l cultures oxidizing pyruvate (Mohn and Tiedje. submitted). Since acetate was not formed in cultures or cell suspensions. homoacetogenesis cannot account for formate consumption. Formate consumption by resus- pended DCB—l cells without 3CB resembles the ability of washed and starved Desulfovibrt'o desulfuricans cells to con- sume H, without an electron acceptor (Postgate 1949) and may indicate oxidation potential endogenous to the cells. The failure of H, to serve as an electron donor in cell suspensions does not rule out the coupling of H, oxidation to reductive dechlorination in growing cultures. since a lack of induction. or conditions in the cell suspensions. may have prevented uptake hydrogenase activity. In growing cultures containing H, plus 3CB. no electron donor other than H, was present in quantities sufficient to support the dechlorina- tion observed. Results of this study indicate that. in pure culture. strain DC B-1 can couple oxidation of formate and. probably. of hydrogen to reductive dechlorination of 3CB or 3SDCB. and can conserve energy for growth from these redox couples. Since oxidation of neither electron donor is thought to support substrate-level phosphorylation. it is likely that energy conservation is via electron transport phosphoryla- tion. Because chlorobenzoates are not substrates normally available in natural habitats. the ability of DCB-l to cata- bolically utilize chlorobenzoates is probably not due to natu- ral selection for such utilization. Catabolism ofchlorobenzo- ates probably is due to a respiratory system. either known or novel. having another terminal electron acceptor. Stevens et al. (1988) have shown that DCB-l can utilize sulfoxy anions by what may be a respiratory process resembling that found in sulfate reducing bacteria. It remains to elaborate the respiratory system of DC B-1 and to demonstrate coupling of that system to reductive dechlorination. In the accompanying paper. Dolfing also provides evi- dence that DCB-l can conserve energy for growth from reductive dechlorination. The two studies are conSistent. but as one would expect. the different culture conditions of the studies resulted in different growth rates and growth yields. Unique to Dolfing's medium were 5 mM acetate and the filter-sterilized vitamins. naphthoquinone. nicotinamide and thiamine. Unique to our medium was PCF. Also. Dolfing‘s medium was reduced with cysteine and sulfide. while ours was reduced Wllh cysteine and titanium citrate. Dolfing re- ports a doubling time of8 to 10 days for DCB~1 growing on 3C8; while in our study it was approximately 45 days. The faster dechlorination observed in our cultures with PCF relative to those without PCF (Fig. 1) indicates that some factor provided by PCF may be limiting dechlorination. and thereby. growth on our media. A 45-day doubling time is long relative to most laboratory batch cultures. but is not long relative to those experienced by bacteria in many natu- ral systems. Components provided by Dolfing's medium may have decreased the energy requirement for growth (Yup) relative to our cultures. Also. the slower growth of our cultures would have increased the ratio of energy re- quirement for maintenance functions to energy requirement for growth relative to Dolfing's cultures. These two factors may contribute to the difference in growth yield values re- ported by Dolfing (6.0 g protein per mole 3CB) and by us (1.7 — 3.4 g protein per mole 3C8). Dolfing and Tiedje (1986) reported a doubling time of 3 days for the defined consouium growing on 3CB. The shorter doubling time of the consortium may indicate that syntrophic conditions are more favorable; one possibly sig— nificant factor is the removal of the benzoate product in syntrophic cultures. The growth yields reported here are close to that of 1.9 g protein per mole 3CB reported for the defined consortium (Dolfing and Tiedje 1987). Recent work in this laboratory (Quensen et a1. 1988) found biological dechlorination of polychlorinated biphe- nyls (PCBs) added to river sediments with a history of PCB contamination. but found no activity in sediments upstream of the contamination. Thus. natural selection for dechlorinating populations seems possible. The finding that dechlorination can support growth identifies a possible mechanism for such selection. An improved understanding of this phenomenon may prove very useful both in under- standing the response of natural systems to chlorinated pol- lutants and in developing methods for biological remeo diation of chlorinated wastes. A(‘k”(HF/('(lgt'nu'nls’ We thank Joyce Wildcnthal for technical assis- tance. and thank Juha Apajalahti. James Cole and Stephen Boyd for helpful discussion This research was supported by Environmental Protection Agency grant R.813892. References DeWeerd KA. Suflita JM. Linkfield T. Tiedje JM. Pritchard PH (1986) The relationship between reductive dehalogenation and other aryl substituent removal reactions catalyzed by anaerobes. FEMS Microbiol Ecol 38:331—339 Dolfing J. TICde JM (1986) Hydrogen cycling in a three-tiered food web growmg on the methanogenic conversion of 3-chloro- benzoate FEMS Microbiol Ecol 38:293-298 Dolfing J. Tiedje JM (1987) Growth yield increase linked to re- ductive dechlorination in a defined 3-chlorobenzoate degrading methanogenic coculture. Arch Microbiol I49: 102 — I05 Hanson R. Phillips J (1981)Chcmicalcomposuion. In: Gerhardt P (ed) Manual of Methods for General Microbiology. Society for Microbiology. Washington. DC. p 358 Linkfield TG. TlCde JM (1989) Characterization of the require- ments and substrates for reductive dehalogenation by strain DCB-l. J lndustr Microbiol (in press) 58 271 Postgate J (1949) Competitive inhibition of sulphate reduction by selenate. Nature (Lond) 164:670-671 Quensen 111 IF. Tiedje JM. Boyd SA (1988) Reductive dechlorina- tion of polychlorinated biphenyls by anaerobic microorganisms from sediments. SCiencc 242: 752 — 7S4 Shelton DR. Tiedje JM (1984) Isolation and partial characteriza- tion of bacteria in an anaerobic consortium that mineralizes 3-chlorobenzoic aetd. Appl Ethron Microbiol 48840—848 Stevens TO. Tiedje JM (1988) Carbon dioxide fixation and mixotrophic metabolism by strain DCB—I. a dehalogenating anaerobic bacterium. Appl EnViron Microbiol 54 2944-2948 Stevens TO. Linkfield TG. Tiedje JM ( 1988) Physiological character. ization of strain DCB-l. a unique dehalogenating sulfidogenic bacterium. Appl Environ Microbiol 542938 —2943 Wolin EA. Wolin MJ. Wolfe RS (1963) Formation of methane by bacterial extracts. J Biol Chem 238.2882 - 2886 Zehnder AJB. Wuhrmann K (1976) Titanium (Ill) Citrate as a nontoxic oxidation-reduction buffering system for the culture of obligate anaerobes. Science 194: 1165- 1166 Received April 19. 1989/Accepted August 1. 1989 Chapter 4: Evidence for chemiosmotic coupling of reductive dechlorination and ATP synthesis in Desulfomonile tiedjei S9 60 Introduction Desulfomonile tiedjei (formerly strain DCB-l) is a sulfate reducing bacterium (DeWeerd et al., 1990; Mohn and Tiedje, 1990a) which is capable of the reductive dehalogenation of chlorobenzoates (Shelton and Tiedje, 1981i; DeWeerd et al., 1986; Cole, 1990) and chloroethylenes (Fathepure et al., 1987). This organism was shown to conserve energy for growth from reductive dechlorination of 3- chlorobenzoate (3GB) and. 3,5-dichlorobenzoate (Dolfing, 1990; .Mohn and Tiedje, 1990b). The addition of 3C8 also supported ATP production in stationary phase cultures which had been limited by 3GB (Dolfing, 1990). Dechlorination of 308 was shown to be coupled to formate and, probably, to H2 oxidation (Mohn and Tiedje, 1990b). Since those electron donors are not known to support substrate-level phosphorylation, it seems likely that a chemiosmotic (respiratory) mechanism might couple dechlorination and ATP synthesis. Such a process would represent a novel mode of anaerobic respiration. Of possible significance to such a mechanism, the electron carrier cytochrome c3 has been identified in D. tiedjei (DeWeerd et al., 1990) and dechlorination activity appears to be membrane associated in this organism (K.A. DeWeerd, 1989, personal communication). Respiratory inhibitors originally used in mitochondrial systems have been successfully used to study anaerobic chemiosmotic mechanisms in a phylogenetically diverse range of bacteria. Extensive 'work of this nature on. methanogenic bacteria. has been reviewed by Daniels et a1. (1984). Respiratory inhibitors have also been used with sulfate-reducing bacteria (Barton et al., 1970; Kramer 61 and Cypionka, 1989; Steenkamp and Peck, 1981), acetogenic bacteria (Ivey and Ljungdahl, 1986), dissimilatory iron reducing bacteria (Arnold et al., 1986) and fermentative bacteria (Cox and Henick- Kling, 1989; Russell and Strobel, 1989). The following inhibitors have been found broadly effective in various of the above studies and were employed in this study: the uncouplers, pentachlorophenol (PCP), 2,4-dinitrophenol (DNP), and carbonyl cyanide m- chlorophenylhydrazone (CCCP); the ionophores, monensin and gramicidin: and the proton-driven. .ATPase inhibitor, N,N'- dicyclohexylcarbodiimide (DCCD). This study examined the effects of respiratory inhibitors and imposed pH gradients on resuspended D. tiedjei cells in order to test the hypothesis that a chemiosmotic mechanism couples reductive dechlorination and ATP synthesis. D. tiedjei was also maintained through serial transfers on defined medium with formate plus 308 as growth substrates, providing further evidence reductive dechlorination supports energy metabolism. Materials and Methods MEDIA. Cultures were grown on formate plus 3C8 in a mineral medium plus the trace element solution for Desulfobulbus (Widdel and Pfennig, 1984) with the following modification and additions: CaCl2 was reduced to 0.1 mM. 10 mM HEPS buffer (hemisodium salt), 1 mg/l resazurin, 12 mM sodium formate, 6 mM sodium acetate, 1 mM BCB, 3 _pg/l NaZSeoa, 8 ug/l NaZWO‘, 30 mM NaHCOa, 1 mM cysteine, vitamins and 0.1 mM titanium (III) citrate. The gas phase was Nz-CO2 (95:5). The pH was adjusted to 7.5 before autoclaving. The vitamins included 500 62 pg/l nicotinamide, 200 pg/l naphthoquinone, 50 pg/l thiamine and 50 pg/l lipoic acid (DeWeerd et al., 1990) added from 1000-fold concentrated, filter-sterilized stock solution dissolved in NaOH. Titanium citrate was prepared according to Zehnder and Wuhrmann (1976) and added from filter-sterilized stock solution. When depleted, 3GB was replenished from filter-sterilized stock solution. Stock cultures used to provide resuspended cells were grown on the mineral medium of Shelton and. Tiedje (1984) with the following modifications and additions: the reductant was changed to 1 mM cysteine plus 0.1 mM titanium citrate, the vitamins were changed to the above mixture, 10 mM HEPS buffer (hemisodium salt), 20 mM sodium pyruvate and 1 mM 3GB. Pyruvate was added from filter-sterilized stock solution. The pH, gas phase and 3C8 additions were as above. All inocula were 10%, except for cultures used for the growth curve (Fig. l) which had 5% inocula. All incubations of cultures and cell suspensions were at 37°C. CELL susrmcszous. Cells in log phase and actively dechlorinating 3GB were harvested by centrifugation in the culture vessels (l60-ml serum bottles) for 60 min at 2500 rpm at h'C. The pellet was washed in A‘C buffer containing, 20 mM HEPS or 20 mM Tris-H01 buffer, 1 mg/l resazurin, 1 mM Nazs and 0.5 mM titanium citrate, pH 7.5 under a gas phase of N2. Cells were centrifuged again for 30 min and resuspended in buffer, concentrated 3- to 5-fold (final protein concentration 90 to 186 pg/l). 3GB was added from a neutralized stock solution (pH 7.5). When used, inhibitors were added 20 min prior to 3GB addition from ethanol stock solutions and all treatments, including controls 63 received the same amount (0.5%) of ethanol. HCl was added from a 5 M stock solution. ANALYSES. Benzoates were analyzed by high pressure liquid chromatography as described previously (Stevens et al., 1988) except the eluent consisted of water-acetone-phosphoric acid (66:33:0.l) and the UV detector was set at 230 nm. Organic acids were analyzed by high pressure liquid chromatography as described previously (Stevens et al., 1988) with the column at 60°C. ATP was extracted as follows: a 0.20-ml sample was added to 0.80 ml 95°C 20 mM Tris-H01 pH 7.8, incubated 5 min at that temperature and filtered (0.45 pm). ATP was assayed with luciferin-luciferase reagent in Tris-aspartate buffer (Sigma Chemical Co., St. Louis, MO) using a Chem-Clo photometer. A linear standard curve was obtained over the range of ATP concentrations assayed. Protein concentration was determined by a modification of the Lowry assay (Hanson and Phillips, 1981). Results D. tiedjei grew through at least five serial transfers on defined medium with formate plus 3C8. Growth was dependent on 3C8 and benzoate accumulated as a product (Fig. 1). The final protein concentrations in control cultures and in cultures with 3C8 were 9 and 68 pg/ml, respectively. Cultures with 3C8 consumed 12 mM formate and produced 1.6 mM acetate. Upon addition of 3C8 to resuspended cells, dechlorination immediately began and the cellular ATP pool rapidly increased approximately 3-fold (Fig. 2). Benzoate accumulated stoichiometrically as a product of 308. The dechlorination rate and 64 0.04 0.03 0.02 0.01 Concentration (mM) Optical density (660 nm) 5.0 - .. - enzootea ' 4.0 L 4 L d 3.0 - _ L «I 2.0 - .. L J 1 at 1 3C8 n ().C) ‘* l ‘7 I I I , 0 10 20 30 40 Time (days) kamm 1. Desulfomonile tiedjei dependence on 3C8 for growth (A) and metabolism of 3C8 (B) on defined medium with formate plus 3GB as substrates. Data are means of triplicates with standard error bars. 65 TE ? I l I I t I t 200 3 e 8 * ' 150 °‘ fL L '~ I. 'c» '0: 6 L g i E E L - 100 3 '6 4 t S E : '4 V 5 2 i- + ~ 50 3 o. L 8 2 0 L '__ L l L I ‘ , O E -20 0 20 40 60 at; Time (minutes) . Ffimmr 2. Reductive dechlorination and consequent ATP pool increase in Desulfomonile tiedjei. Cells were resuspended in Tris buffer. At 0 min 3C8 was added to one suspension (open symbols) but not to the control (closed circles). Circles, ATP; squares, dechlorination measured as benzoate accumulation. 66 the ATP pool remained constant for at least one hour; thus, dechlorination and ATP pools were measured 1 h after addition of 3C8 to similar suspensions in order to determine the effects of respiratory inhibitors. Uncouplers including PCP, DNP and CCCP inhibited both dechlorination and the ATP increase, the latter being extremely potent and causing complete inhibition of both at a concentration of 5 imL. At a relatively low concentration, which caused only partial inhibition, PCP had a greater effect on ATP synthesis than on dechlorination (Table l). The ionophore, monensin, had an effect similar to PCP; another ionophore, gramicidin, increased the dechlorination rate and had little effect on the ATP pool increase. The proton-driven. .ATPase inhibitor’ DCCD inhibited. ‘both dechlorination and the ATP increase at high concentrations, but only inhibited the ATP increase at low concentrations (Fig. 3). [At the lowest concentration tested (50 pM), DCCD reduced the increase in ATP due to 3C8 but actually increased the dechlorination rate (Table 1). An imposed pH gradient caused an immediate increase in the ATP pool by approximately half in resuspended cells (Fig. 4). Preincubation of the cells for 20 min with 1 mM KSCN, a permeant ion, had no apparent effect. Discussion The serial transfer of D. tiedjei on defined, medium with formate plus 3C8 indicates that all growth requirements were provided, and the dependence of growth on 308 which was converted to benzoate (Fig. 1) leaves little doubt that 3C8 functions as a 67 .uouuo ounocnum H mounoaamfiuu no names one monum>~ .uouusn mam: cw coocommamou mums maaooa Rm ~.n mm. H mé mo. H 34 as 2: 536336 m.N o.~ «m. H v.n mo. H om.o Ea ooa .CHmcmGOS N.~ o.N on. H v.n OH. H mm.o :5 OH .mom w.H 0." mm. H v.m no. H h~.H :5 om .QUUQ m.n m.n mm. H m.¢ «do. H oo.H Houucoo I o.o «we. H v.H I 00006 mom 02 cofluocwuoanoou Acfiououm Acaououm “cwououn al\ "ommouoca mad oa\Hoacv afl\H0lcv S\Holsv aqua“ onoouo:« mad mad coauocduoHnooa Hucoaucoua .Hflfififldfl «Aflmmflmudmwma no naaoo ooocommsmou ca soaumuucoocoo med use :ofiuocfiuoanooo co muouanancw abououanuou no mucouum .H gamma 68 S" o .0 .p .0 lo 8 3 P (nmol mg“ protein) a—a ATP Dechlorination (nmol h“ mg" protein) T 0—9 Dgchlorinotjon P I L J , f 0 < 0 200 400 600 800 1 008° DCCD (uM) Flaunt 3. Effect of DCCD on dechlorination rate and ATP concentration. Cells were resuspended in Tris buffer. 0.0 ' I ' I ' I ' I ' I ' L . 7;“ 0.8 r- - 0‘ L . E _ 0.6 - 3 a .i O . E C 0.4 - - V L E .- < 0.2 - - L n +KSCN . 0.0 1 I n_ I 1 J L o J —IKSCN n -2 -‘l 0 1 2 3 Time (min) F160!!! 4. ATP pool increase in Desulfomonile tiedjei resulting from an imposed pH gradient. Cells were resuspended and starved in REPS buffer without formate. At 0 min the pH was reduced from 7.5 to 2.9 by RC1 addition. 70 catabolic electron acceptor. Despite some acetate production in the cultures of this study, D. tiedjei could not grow acetogenically on formate plus C02 alone. This inability is in agreement with previous findings (DeWeerd et al., 1990; Mohn and Tiedje, 1990b). The acetate production might account for the formate consumption observed which was in excess of that required for dechlorination; however, the energetic consequences of such a process are not clear. The coupling of formate oxidation to dechlorination was previously shown in cultures and cell suspensions (Mohn and Tiedje, 1990b), and, as in this study, formate consumption was in excess of that required for dechlorination. The ATP increase in resuspended cells upon addition of 3GB (Fig. 2) agrees with previous findings of Dolfing (1990) using stationary phase cultures and indicates that dechlorination and ATP synthesis are somehow coupled. Uncouplers and ionophores are both able to dissipate a proton-motive force (AP). In cell suspensions of D. tiedjei, these agents had the common effect of reducing the ATP increase relative to the dechlorination rate (Table 1), thus, apparently reducing the efficiency of dechlorination-dependent ATP synthesis. Such an effect suggests chemiosmotic coupling of dechlorination and ATP ' synthesis in which dechlorination supports formation of AP which then supports ATP synthesis. An alternative hypothesis is that dechlorination supports ATP synthesis which then supports formation of AP; however, in the latter case, uncouplers and ionophores would not be expected to affect the efficiency of dechlorination-dependent ATP synthesis. The similar effect of such a 71 variety of agents lessens the possibility that the observations are artifacts of unexpected activities of these agents. However, the possibility exists that the dechlorinating enzyme, which is believed to be membrane associated (K.A. DeWeerd, 1989, personal communication), could be directly inhibited by these agents. While these results suggest the involvement of H+ as a coupling ion, they do not rule out the additional significance of other ions. The finding that low concentrations of DCCD do not inhibit dechlorination while they do inhibit the ATP increase (Fig. 3; Table 1) suggests that the action of DCCD is specific to a proton-driven ATPase which is involved in converting AP to ATP. The existence of such an ATPase in D. tiedjei is further supported by the finding that an imposed pH gradient (inside alkaline) also causes an increase in ATP (Fig. 14). It would be very desirable to test whether this pH driven ATP increase is also sensitive to DCCD. The inability of uncouplers, ionophores and DCCD to completely inhibit ATP synthesis while allowing dechlorination may indicate that either ATP or AP are required for dechlorination. ‘ Possible explanations would be that dechlorination requires active transport or an activation step. These two situations have been proposed for dissimilatory sulfate reduction (Cypionka, 1987) and methanogenesis (Mountfort, 1978), respectively. The above results provide a consistent body of evidence supporting chemiosmotic coupling of reductive dechlorination and ATP synthesis in. D. tiedjei. The general agreement of the above results is critical, as the use of respiratory inhibitors provides only indirect 72 evidence which is subject to unexpected activities of the inhibitors. Lancaster (1986) has even argued that results with methanogens similar to those reported here with D. tiedjei are not inconsistent with ATP synthesis via substrate-level phosphorylation. It would be informative to obtain more direct evidence such as measurement of dechlorination-dependent proton extrusion, isolation of ATPase activity or isolation of dechlorinating membrane vesicles. The latter might be especially useful in better understanding the dechlorination reaction. However, such evidence may be difficult to obtain owing to the slow growth rate and low yields of D. tiedjei in laboratory culture. 73 Rererences Arnold RG, DiChristina TJ, Hoffmann R (1986) Inhibitor studies of dissimilative Fe(III) reduction by Pseudomonas sp. Strain 200 ("Pseudomonas ferrireductans"). Appl. Environ. Microbiol. 52:281-289. Barton L, LeGall J, Peck HDJ (1970) Phosphorylation coupled to oxidation of hydrogen with fumarate in extracts of the sulfate reducing bacterium, Desulfovibrio gigas. Biochem. Biophys. Res. Commun. 41:1036-1042. Cole JR, Tiedje JM (1990) Induction of anaerobic dechlorination of chlorobenzoate in strain DCB-l. Abstr. Ann. Meet. Am. Soc. Microbiol., Q-43, p. 295. Cox DJ, Henick-Kling T (1989) Chemiosmotic energy from malolactic fermentation. J. Bacteriol. 171:5750-5752. Cypionka H (1987) Uptake of sulfate, sulfite and thiosulfate by proton-anion symport in Desulfovibrio desulfuricans. Arch. Microbiol. 148:144-149. Daniels L, Sparling R, Sprott GD (1984) The bioenergetics of methanogenesis. Biochim. Biophys. Acta 768:113-163. DeWeerd KA. Concannon F, Suflita JM (1990) Hydrogen consumption and the reduction of halobenzoates and sulfur oxyanions in resting cells of Desulfomonile tiedjei. Abstr. Ann. Meet. Am. Soc. Microbiol., Q-53, p. 297. DeWeerd KA, Suflita JM, Linkfield TG, Tiedje JM, Pritchard PH (1986) The relationship between reductive dehalogenation and other aryl substituent removal reactions catalyzed by anaerobes. FEMS Microbiol. Ecol. 38:331-339. Dolfing J (1990) Reductive dechlorination of 3-chlorobenzoate is coupled to ATP production and growth in an anaerobic bacterium, strain DCB-l. Arch. Microbiol. 153:264-266. Fathepure BZ, Nengu JP, Boyd SA (1987) Anaerobic bacteria that dechlorinate perchloroethene. Appl. Environ. Microbiol. 53. Hanson R, Phillips J (1981) Chemical composition. In: Gerhardt P (Ed) Manual of Methods for General Microbiology. Society for Microbiology, Washington, DC, p 358. Kramer M, Cypionka H (1989) Sulfate formation via ATP sulfurylase in thiosulfate- and sulfite-disproportionating bacteria. Arch. Microbiol. 151:232-237. 74 Lancaster JRJ (1986) A unified scheme for carbon and electron flow coupled to ATP synthesis by substrate-level phosphorylation in the methanogenic bacteria. FEBS Lett. 199:12-18. Ivey DM, Ljungdahl LG (1986) Purification and characterization of the Fl-ATPase from Clostridium thermoaceticum. J. Bacteriol. 165:252-257. Mohn WW, Tiedje JM (1990a) Catabolic thiosulfate disproportionation and carbon dioxide reduction in strain DCB-l, a reductively dechlorinating anaerobe. J. Bacteriol. 172:2065-2070. Mohn WW, Tiedje JM (1990b) Strain DCB-l conserves energy for growth from reductive dechlorination coupled to formate oxidation. Arch. Microbiol. 153:267-271. Mountfort D0 (1978) Evidence for ATP synthesis driven by a proton gradient in Methanosarcina barkeri. Biochem. Biophys. Res. Commun. 85:1346-1351. Russell JB, Strobel HJ (1989) Effect of ionophores on ruminal fermentation. Appl. Environ. Microbiol. 55:1-6. Shelton DR, Tiedje JM (1984) Isolation and partial characterization of bacteria in an anaerobic consortium that mineralizes 3-chlorobenzoic acid. Appl. Environ. Microbiol. 48:840-848. Steenkamp DJ, Peck HDJ (1981) Proton translocation associated with nitrite respiration in Desulfovibrio desulfuricans. J. Biol. Chem. 256:5450-5458. Stevens T0, Linkfield TG, Tiedje JM (1988) Physiological characterization of strain DCB-l, a unique dehalogenating sulfidogenic bacterium. Appl. Environ. Microbiol. 54:2938-2943. Widdel F, Pfennig N (1984) Genus Desulfobulbus. In: Krieg NR, Holt JG (Eds) Bergey's Manual of Systematic Bacteriology, Vol 1. Williams and Wilkins 00., Baltimore, p 676. Zehnder AJB, Wuhrmann K (1976) Titanium(III) citrate as a nontoxic oxidation-reduction buffering system for the culture of obligate anaerobes. Science 194:1165-1166. Chapter 5: Attempts to identify or isolate dehalogenating anaerobes 75 76 Introduction Recent advances in our understanding of the physiology of the dehalogenating anaerobe, Desulfomonile tiedjei (formerly strain DCB-l) (DeWeerd et al., 1990; Dolfing, 1990; Mohn and Tiedje, 1990a; Mohn and Tiedje, 1990b; Stevens and Tiedje, 1988; Stevens et al., 1988) suggested new strategies for finding other dehalogenating anaerobes. First, the identification of DCB-l as a sulfate-reducing bacterium suggested screening existing isolates of this physiological group for dehalogenation activity. A very limited attempt had previously been made by Linkfield (1985) using three Desulfovibrio spp. grown under sulfate-reducing conditions with 3-chlorobenzoate. Emphasis in the present study was given to genera with physiological similarities to DCB-l and to substrates known to. be readily dehalogenated in enrichment cultures. The conditions selected for testing dehalogenation activity were those found to be favorable for this activity in DCB-l. The second strategy was to attempt to isolate from existing enrichment cultures dehalogenating anaerobes which have physiological similarities to DCB-l. These efforts yielded a new strain from the same source as DCB-l which is morphologically and physiologically similar to DCB-l. Dehalogenating anaerobes were not obtained from other enrichment cultures. Materials and Methods Emcm CULTURES. Strains were isolated from four enrichment cultures (Table l). Sewage sludge was collected prior to 1983 from 77 TABLE 1. Enrichment cultures used as sources for isolates. Name Source Substrate Medium Sed 388 Lake sediment 3-bromobenzoate RAMM Slg 388 Sewage sludge 3-bromobenzoate RAMM Mang 3GB Estuary sediment 3-chlorobenzoate SS-RAMM Ditch 2CP Flooded ditch 2-chlorophenol W+P an anaerobic digester (Shelton and Tiedje, 1984). Lake sediment was collected from the highly eutrophic Wintergreen Lake, Kalamazoo Co., Mich. prior to 1985 by T.G. Linkfield. Estuary sediment was collected from a mangrove swamp in the Florida Keys in 1987 by T.0. Stevens. Sediment was collected from a flooded ditch near Lake Lansing, Ingham Co., Mich. in 1989. Enrichments from these sources have been maintained in our laboratory since those times. The Reduced Anaerobic Mineral Medium (RAMM) medium is from Shelton and Tiedje (1984). The SS-RAMM medium is the above mineral medium with added sea salts (220 mM NaCl, 26 mM MgC12, 10 mM KCl and 5 mM CaClz). The W+P mineral medium is one formulated for Desulfobulbus spp. (Widdel and Pfennig, 1984). The W+P medium was buffered (pH 7.0) with 30'mM NaHCO3, and had a headspace of Nz-COZ (95:5). Media were reduced with 1.0 mM cysteine added before autoclaving and 0.1 mM titanium (III) citrate added after autoclaving, unless otherwise stated (Table 2). A vitamin mixture was added to all media after autoclaving; this included vitamins generally required by anaerobic bacteria (Wolin et al., 1963). Halobenzoates were added at 1 mM and 78 .adasuoca on» ma om>umm scans musuaso unmanoauco on» no ocsomaoo DaumaoueoHaz an» vacanucoo omaa awoaza eduusao Iamz .ucduodedu no Am.ov duacoasuae I I m m .Ama dumuadmonau .AoH. oud>su>a madam an «a I on Add denuded .Aoav essence «a unmannedu an .m.ov muscoazuae I I I e .Amv ounuasmoacu .Am.mv mucoucdnaxosudaIn sen: unneededu mm Am.ov ouncoanuae .xmv I I I m duouanmoanu .AHV denuded .Aoav ouoauou seam unneededu mo Am.ov evanescent m c I m .AHV denuded .Aoav ououasmoanu ens uceuozoau ma Adv oumuuao I I I I .HHHV gaseous» .Aaoaa efisau amass am A I I I snowmanuau .Aoaa oum>=uma wand e e m s .Aaoav essay cuss» ..oav oun>su>a mm Ha Ha I Ha Aoav oue>su>m m can use man an: man dam man com HAIEV muaocomaoo :owuew>ounn4 uaaanowuca some Bonn mmuaaomw no .02 .mmPMHONH NO mhflgg 0C0 QCOHHMHOQH MON ”0%” Ufinvw: IN Sgfi. 79 replenished after disappearance. 2-Chlorophenol was added at 0.25 mM and replenished after disappearance. Haloaromatic compounds were analyzed by high-pressure liquid chromatography as described by Stevens et a1. (1988) except that the eluent was water-acetonitrile- phosphoric acid (66:33:0.1) and the UV detector was set at 230 nm for halobenzoates and 218 nm for 2-chlorophenol. Enrichment cultures were subcultured by 10% transfers into homologous media. ISOLATIONS. Strains were isolated from the above enrichments by serial dilution into agar shakes. Mineral media homologous to the corresponding enrichments were used with 1.5% agar and various substrates (Table 2). In addition to the above vitamins, others required by Desulfomonile tiedjei for dehalogenation (Apajalahati et al., 1989; DeWeerd et al., 1990) were also added. The latter vitaminswere nicotinamide (500 pg/l), 2,4-naphthoquinone (200 pg/l) and thiamine (50 pg/l) dissolved in NaOH solution. Colonies were picked with a syringe and transferred to homologous liquid media containing the haloaromatic compound of the original enrichment. Dehalogenation activity of the isolates was assayed by incubating the liquid cultures for at least two weeks after stationary phase was reached, assaying the haloaromatic substrates and comparing the values to those of uninoculated control cultures. When CO2 was omitted from the mineral medium, the phosphate concentration was increased to 10 mM. CULTURES or SULFATE-REDUCING BACTERIA. The sulfate reducers used in this study (Table 3) were cultured on W+P medium described above with various substrates (Table 4). Stock cultures had the higher sulfate .aswooa was» so auscuconouoazOIm mo sawumcamoHonao you wanna» >Hcon 80 H m4 4mm xsmoua>ma .m omnn 2mm maawnnocomouoan Hmuomnomasmon mm .mm xsmmum>mo .m ommnn 0094 mcouo>wuaafi msooooouaamoo Hmd .mm .Hmm X5mmum>0o .m omen 2mm «sauna: adwuouomnomaamoa mm .Hm: xsmauo>ma .m «mun Smn anoanmouuouso asfinmuoaQOHHamma mg .m woman was» nldna oco: mg .45. sound 35 «I45. .28: <29 .m xom .m Hoomna acousamwoouasm oHunw>0uH5mmo m cowuoaaaoo and mm .mu ofiunw>ouasmoo awn cowuoaaaoo and Hmm .mm owuna>ouaamoa mm cowuomaaou nod Haw .mm oHunfl>ouasmoo .nmq Lama .m :oHuoaHHoo and man acaodusuasmoo ofiunw>ouaamoo .nmq.idmm .m cowuooaaoo and «we mcooauamaammo ofiunw>ouasmoa save: mousom .o: :«uuum mama .au«>wuoo cowuacomoHenou you moved» mauouomn acwoscoulououasm .n mqmda 81 Thmm 4. Media used for growth of sulfate-reducing bacteria. Abbreviation Components (mM) P pyruvate (20) HS Hz-CO2 (80:20, 2 atm), sulfate (1 or 5) FS formate (20), sulfate (1 or 5) AS acetate (20), sulfate (1 or 5) LS lactate (20), sulfate (1 or 5) BS benzoate (3), sulfate (1 or 5) ThA thiosulfate (5), acetate (1) concentrations, and dehalogenation test cultures had the lower. All sulfate-containing media also had 0.1 mM NaZSZO‘ as an extra reductant and potential nutrient. Media for strains of Desulfobacterium, Desulfobacter and. Desulfococcus had additional NaCl (6 g/l) and MgClz.6Hg) (1.3 g/l). Dehalogenation activity was assayed as described above. POTENTIAL ELECTRON ACCEPTORS. An important consideration in attempts to identify or isolate dehalogenating anaerobes is the possible effect on dehalogenation of any available electron acceptors (see Chapter 1). Specific concerns in this study were sulfoxy anions commonly used as substrates (i.e., SO", 8203', 503') and media reductants (i.e., 820,-, NaZS). The latter reductant was considered to be of concern because upon chemical oxidation it can yield 8203'. The preceding compounds were avoided or minimized unless specifically desired as electron acceptors. Cysteine plus titanium (III) citrate were therefore usually used as medium reductants. 82 Results and Discussion SCREENING or SULFATE-REDUCING BACTERIA. Sulfate reducers were screened under conditions thought most likely to allow dehalogenation activity. For the reasons stated above, sulfate reducers were tested with excess electron donor and limiting sulfate, and those capable of fermentative growth were additionally tested in the absence of sulfoxy anions. The former condition presumably allowed any inhibitory effect of sulfate to terminate after its exhaustion. Three haloaromatic compounds known to be readily dehalogenated in enrichment cultures were separately tested with each sulfate reducer, 3-chlorobenzoate (0.5 mM), 2-chlorophenol (0.25 mM) and 4- chlororesorcinol (0.25 mM). A total of 52 tests were performed (Table 3), but no dehalogenation activity was detected. Of course it is possible that the organisms tested might have activity under different conditions, or that other sulfate reducers available in pure culture, but not tested here, might have activity. It is perhaps more likely that other sulfate reducers not available in pure culture, and more closely related to D. tiedjei than those available, account for dehalogenation activity in certain enrichment cultures. This possibility was addressed by the following attempts to isolate from various enrichment cultures organisms with physiological similarities to D. tiedjei. ENRICMNTS. The Sed 3BB and Slg 3BB enrichment cultures were modified for this study by the addition of 20 mM formate, 1 mM acetate and 1 mM bromoethane sulfonate. The rationale was to select for organisms like D. tiedjei which can grow with formate as an 83 electron donor and halobenzoates as an electron acceptor. Acetate was an additional carbon source and bromoethane sulfonate was used to prevent methanogens from growing on the formate. The enrichments were serially transferred on this medium twice, retaining dehalogenation activity, and the resulting enrichments were used as inocula for isolations. ISOLATIONS. Over 114 isolates were obtained and tested for dehalogenation activity. Media used (Table 2) selected for various physiological groups and were all previously found to support D. tiedjei. Several general observations were made on the various media. P and PR media selected pyruvate fermenters. These isolates were usually large (5 mm diameter) cocci in clumps which were somewhat translucent under phase-contrast optics. The Man 3GB enrichment yielded very long (>30 mm) filaments on this medium. These morphotypes appeared to be only minor components of the enrichment cultures. These isolates did not show dehalogenation activity. Prif medium was employed in an attempt to isolate spirochetes observed in the Ditch 2GP enrichment. However, this medium yielded only motile rods which showed no dehalogenation activity. RT medium was used to select for organisms adapted to mixotrophic growth on substrates commonly at low concentrations in anaerobic habitats. Small colonies were obtained on this medium, but these did not grow when transferred to homologous liquid medium. 84 ThA medium selected thiosulfate disproportionaters. Most of these isolates were motile vibrios resembling Desulfovibrio sulfodismutans which could ferment pyruvate. Such isolates were tested for dehalogenation activity on P medium, but none showed activity. Two of these isolates from the Sed 3BB enrichment (ThA-2 and ThA-3) were further tested with the sulfate reducer cultures (Table 3). A large rod resembling D. tiedjei was obtained from the Sed BBB enrichment on ThA medium, but this culture was contaminated with cocci. The rod could not be purified in further agar shakes or by passage on various selective media, suggesting that perhaps it was dependent on the cocci. On P medium the cocci outgrew the rod and no dehalogenation activity was observed. MbTh medium was intended to select thiosulfate reducers which could O-demethylate and possibly further degrade methoxybenzoate. Again, motile vibrios which could ferment pyruvate were obtained, and again, no dehalogenation activity was detected on P medium. FA medium was used in an attempt to select organisms capable of using formate as an electron donor and haloaromatic compounds as an electron acceptor (acetate was an additional carbon source). None of the isolates from this medium showed dehalogenation activity. Presumably the isolates (mostly rods) were homoacetogens growing on formate plus C02. PTth medium selected diazotrophic organisms. The Sed 3BB enrichment yielded motile vibrios which could ferment pyruvate but which had no dehalogenation activity on P medium. The Slg 3BB enrichment yielded large rods. These rods had visible collars and 85 were indistinguishable from Desulfomonile tiedjei by phase-contrast microscopy. These isolates fermented pyruvate and dehalogenated 3BB on P medium. One isolate (DCB-2) was selected for further study. Strain DOB-2 also dehalogenated 3GB on P medium (Fig. 1). In contrast to D. tiedjei, the DCB-2 culture also consumed the benzoate product after transient accumulation. On this medium D. tiedjei produced 18 mM acetate plus 1.2 mM lactate; while, DCB-2 produced 22 mM acetate plus 1.9 mM lactate. These cultures did not produce methane. Also in contrast to D. tiedjei (see Chapter 4), DCB-2 did not grow on FA medium with 3GB. DCB-2 does appear able to use the same electron acceptors as D. tiedjei (see Chapter 2), since sulfate, thiosulfate and 002, but not nitrate, stimulate growth on pyruvate (CO2 was omitted when testing other electron acceptors). The DCB-2 culture appeared pure, but these results remain to be confirmed after repurification of DCB-2. The possibility exists that a benzoate- degrading contaminant caused the observed benzoate consumption. The new isolate, DCB-2, thus is very similar to D. tiedjei morphologically and physiologically, but may have significant differences. DCB-2 probably warrantS' further physiological characterization. Molecular methods (e.g., comparing restriction fragment length polymorphisms or testing 16S rRNA homology) would also be very helpful in comparing DCB-2 to D. tiedjei. - Cucumnxm. With the caveat that not all culture conditions or substrates could be tested, it appears that reductive dehalogenation is not a general property of sulfate-reducing bacteria and is not a specific property of members of that group tested here. It should be 86 I l l l I DOB-2 on - 1.0 0.12 h- J L "' 0.8 J 0.08 i— - 0.6 A L '- E ,1 - 0.4 ’2‘ O 0.04L- / E V :3 L Benzoate'] 0'2 c V 0 00 / 308 .3 >‘ e I I I 0.0 O y C ‘6 a. tiedjei 3°"5° t°- 1.0 b 5 012 . 5 .0 L OD - 0.8 g ‘5 o 9- 0 *5. 0.08 - 1 0 6 0 L / - 0.4 0.044 I . L - 0.2 3GB ‘ 0.00 ' ' ' r . 0.0 I, O 5 10 15 20 25 30 Time (days) Ffimmrln Growth on pyruvate and metabolism of 3-chlorobenzoate by strain DCB-2 and Desulfbmonile tiedjei. Data are means of triplicates with standard error bars. 87 noted that a major subgroup, the gram-positive sulfate reducers, were not tested. Enrichment cultures with halobenzoates as sole substrates maintain diversity even after long periods and multiple transfers, as demonstrated by the variety of physiological groups isolated. Isolates with a variety of physiological characteristics common to D. tiedjei were selected from such enrichment cultures, but in only one case was this selection specific for a dehalogenating organism. In that case selection was for diazotrophy, and the enrichment culture was the source of D. tiedjei. This isolate appears to be very closely related to D. tiedjei. It seems likely that dehalogenating organisms in the other enrichment cultures are physiologically different than D. tiedjei and cannot grow on the media used. The possibility exists, however, that the dehalogenating organisms are similar to D. tied jei but are outnumbered by other organisms which grew on the media used. Future attempts to isolate reductively dehalogenating anaerobes will probably require new means of selection, optimally selection for dehalogenation activity itself. 88 References Apajalahti J, Cole J, Tiedje J (1989) Characterization of a dechlorination cofactor: an essential activator for 3-chlorobenzoate dechlorination by the bacterium DCB-l. Abstr. Ann. Meet. Am. Soc. Microbiol., Q-36, p. 336. DeWeerd KA. Mandelco L, Tanner RS, Woese CR, Suflita JM (1990) Desulfomonile tiedjei gen. nov. and sp. nov., a novel anaerobic, dehalogenating, sulfate-reducing bacterium. Arch. Microbiol. (in press). Dolfing J (1990) Reductive dechlorination of 3-chlorobenzoate is coupled to ATP production and growth in an anaerobic bacterium, strain DCB-l. Arch. Microbiol. 153:264-266. Linkfield TG (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. PhD Thesis, Michigan State Univ. Mohn WW, Tiedje JM (1990a) Strain DCB-l conserves energy for growth from reductive dechlorination coupled to formate oxidation. Arch. Microbiol. 153:267-271. Mohn WW, Tiedje JM (1990b) Catabolic thiosulfate disproportionation and carbon dioxide reduction in strain DCB-l, a reductively dechlorinating anaerobe. J. Bacteriol. 172:2065-2070. Shelton DR, Tiedje JM (1984) Isolation and partial characterization of bacteria in an anaerobic consortium that mineralizes 3-chlorobenzoic acid. Appl. Environ. Microbiol. 48:840-848. Stevens T0, Linkfield TG, Tiedje JM (1988) Physiological characterization of strain DCB-l, a unique dehalogenating sulfidogenic bacterium. Appl. Environ. Microbiol. 54:2938-2943. Stevens T0, Tiedje JM (1988) Carbon dioxide fixation and mixotrophic metabolism by strain DCB-l, a dehalogenating anaerobic bacterium. Appl. Environ. Microbiol. 54:2944-2948. Widdel F, Pfennig N (1984) Genus Desulfobulbus. In: Krieg NR, Holt JG (Eds) Bergey's Manual of Systematic Bacteriology, Vol 1. Williams and Wilkins Co., Baltimore, p 676. Wolin EA, Wolin MJ, Wolfe RS (1963) Formation of methane by bacterial extracts. J. Biol. Chem. 238:2882-2886. APPENDIX Involvement of a collar structure in polar growth and cell division of strain DOB-1 The following was collaborative work. My contribution was to provide the scanning electron microscopy samples, to perform and analyze the cell measurements, to make the figures (excluding the prints), and to write the manuscript. 89 APPLIED AND ENVIRONMENTAL MICROBIOLOGY. May 1990. p. 1206-1211 0099-2240/90/0512060630100/0 Copyright © 1990. American Society for Microbiology 90 Vol. 56, No. 5 Involvement of a Collar Structure in Polar Growth and Cell Division of Strain DCB-l WILLIAM W. MOHN.| TIMOTHY G. LINKFIELD,2 H. STUART PANKRATZ.l AND JAMES M. TIEDJE‘J‘ Departments of Microbiology1 and of Crop and Soil Sciences.2 Michigan Stale University. East Lansing. Michigan 488244325 Received 20 October 1989/Accepted 11 December 1989 Microscopic methods were used to investigate the unique collar structure of the gram-negative sulfate- reducing bacterium, strain DCB-l. Polar cell growth apparently occurred from the collar. When the daughter cell was approximately equal in length to the mother cell and the collar was thus centrally located, cell division occurred within the collar region. Division was by a novel mechanism which conserved the collar of the mother cell and gave rise to a new collar of the daughter cell. Cells of DCB-l were also found to contain stacked internal membranes and glycogen bodies. Strain DCB- 1 was isolated from an anaerobic enrichment culture which mineralized 3- chlorobenzoate (8). Apparently. this isolate is a unique type of sulfate reducing bacterium (6b, 9, 10). The isolate is capable of reductive dehalogena- tion of 3-chlorobenzoate (8), other halogenated benzoates (3), and tetrachloroethylene (4) and is of special interest since it is the only pure culture capable of anaerobic reduc- tive dehalogenation of aromatic compounds. Dechlorination has recently been shown to provide energy to DCB-l as a terminal electron-accepting process (3a, 6a Strain DCB-l is a large, gram-negative rod with a unique morphological feature: a collar which girdles each cell (8). To our knowledge, no similar structure has been observed in other bacteria, with the possible exception of an unidentified organism from sediment of a hypereutrophic lake (2). The purpose of this study was to better characterize this collar and its origin. The collar was found to be involved in a unique mechanism of cell division. MATERIALS AND METHODS Cultures. Strain DCB-l was obtained from our laboratory culture collection. Cultures were grown to late log phase on a previously described reduced anaerobic mineral medium (8) with 20 mM pyruvate and 10 mM thiosulfate as sub- strates. Transmission electron micrographs. Agar-embedded cells were fixed overnight in cold 2.5% glutaraldehyde in 0.1 M NaZHPO,-KH2PO, (pl-l 7.2), washed in the phosphate buffer, and postfixed for 1 h at room temperature in 1% 050‘ in the phosphate buffer. The cells were dehydrated through a graded ethanol series and propylene oxide and were embedded in Poly/Bed 812 (Polysciences Inc., Warrington, Penn.). Thin sections were cut with a diamond knife mounted on an Ultotome III (LKB Instruments, Inc., Rock- ville, Md.). Sections were stained with uranyl acetate and lead citrate and were examined with a Philips EM 300 microscope. Scanning electron micrographs. Cells were fixed in gluta- I'aldehyde. mounted on a glass cover slip by using poly- L- -lysine. and dehydrated by the procedure of Klomparens et ‘ Cori’esponding author. 1206 FIG. 1. Scanning electron micrographs of strain DCB-l showing the collar in various locations. OE Open end of collar; TM, terminus of mother cell: TD, terminus of daughter cell. Bar = 1 pm. 91 VOL, 56. I990 GROWTH AND DIVISION OF DCB-l 1207 3 I a". _J h . -b FIG. 2. Transmission electron micrographs of strain DCB~1 showing the collar in various locations and internal structures. OE, Open end of collar; lM. internal membrane; GL, glycogen bodies. Bars = 0.1 (A and B) and 0.5 (C and D) pm. 1208 MOHN ET AL. FIG. 3. Transmission electron micrographs showing membranes of strain DCB-l. lM. Stacked internal membranes; OM. membrane; CM. cytoplasmic membrane. Bars = 0.1 (A) and 0.5 (B) al. (6). Samples were mounted on aluminum stubs, coated with gold (20—nm thickness) in an Emscope Sputter Coater model SC 500 purged with argon, and examined with a Japan Electron Optics Limited model JSM-JSCF scanning electron microscope. All cell measurements were performed on scan- ning electron micrographs with a randomly selected popula- tion of 28 cc ls. Assay of glycogen. Glycogen was isolated, hydrolyzed, and assayed as glucose as described by Hanson and Phillips (5). RESULTS AND DISCUSSION Cells of strain DCB-l were straight or slightly curved rods 3.2 to 8.7 pm long and 0.5 to 0.7 pm wide, in agreement with the initial report of Shelton and Tiedje (8). Each cell had a collar which varied in its location (Fig. I). The width of the collar (measured parallel to the length of the cells) was 0. 3 to 0. 5 pm. The collar had a definite orientation, with an open end apparent on both scanning and transmission electron micrographs (Fig.1 and 2). In all cells observed, the open end of the collar was the end closest to a cell terminus The collar was faintly visible by phase-contrast microscopy. The 92 APPL. ENVIRON. MICROBIOL. cell wall of DCB-l appeared to have the bimembrane struc- ture common to gram-negative procaryotes (Fig. SB). It was unclear whether the cytoplasmic membrane extended into the collar of all cells as was suggested In an earlier study (8). Occasionally, in cells with terminal collars the interior of the collar appeared to be more electron dense than the cytoplasm (Fig. 2A). In the majority of cells. including those with terminal collars, the interior of the collar and the cytoplasm were of equal electron density, and the cytoplas- mic membrane was not clearly discernible throughout the region of the collar. The collar is shown by transmission electron micrographs to be the site of cell division (Fig. 4). Division was not observed at other locations. Division apparently involved (i) invagination of the membrane, which formed a small second collar beneath the first oriented in the opposite direction (Fig. 4A and B); (ii) further invagination. separating the mother and daughter cells (Fig. 4C and D); and (iii) comple- tion of the new cell termini, leaving an open space between the cells (Fig. 4E and F). The cells presumably separated as the new collar slid out of the old. Each cell produced from this mode of division had a terminal collar. This sequence of events was not based on synchronous cultures; thus. the order of events is not certain, and the duration of each step is unknown. Although the presence of the collar makes this division process remarkable, the separation of the cells and completion of terminal cell walls may occur by the same mechanism as that found in other gram-negative bacteria. Membrane-bound bodies, which may be homologous to blebs formed by Escherichia coli (1), appeared in the space between the newly divided cells (Fig. 4E and F) The observations that division occurred at the collar region and that all cells presumably originated with terminal collars suggest that growth of strain DCB-l is polar, with daughter cells elongating from within the collars of mother cells. This mode of growth was further substantiated by analysis of cell measurements. The relative collar location was defined as the distance from the open end of the collar to the terminus of the presumed daughter cell divided by the total length of the cell (Fig. ID). Thus, a terminal collar has a relative location of 0, and a central collar has a relative location of approximately 0.5. Polar growth should result in movement of this relative position from 0 to approximately 0.5. Consistent with this prediction, cell collars appeared at different relative locations with about equal frequency, and collars were not found beyond the center of the cell (Fig. 5). Assuming that mother cells do not vary too greatly in length, polar growth should also result in terminal collars on the shortest (most recently divided) cells and central collars on the longest. The relative collar location (Y) would be related to the total cell length (X) by the following equation: Y = 1 —- (b/X), where b is the initial total cell length (i.e.. the mother cell length). This equation was fit to the observed data using the Marquadt method of nonlinear regression (Fig. 6). All assumptions for regression analysis were met. and the fit was significant (alpha = 0.01) with an r2 of 0.81. Thus, the observed cell dimensions were consistent with polar growth. Variability not accounted for by the equation was most likely due to variation in the initial total cell length (b). The only alternative explanation for our observations Is migration of the collar from the termini to the centers of cells during intercalary growth but that seems unlikely if the collar includes the rigid peptidoglycan layer. The direct observation of growing cells and the labeling of cell wall components to determine where growth of the wall occurs were not possible because of the strict requirement for 1209 GROWTH AND DIVISION OF DCB-l 93 FIG. 4. Transmission electron micrographs showing the collar region of dividing cells of strain DCB-l. OE, Open end of collar; IV. imagination; DC, daughter cell collar; BL. blebs. Bar = 0.1 pm. VOL. 56. 1990 1210 MOHN ET AL. 2 ‘ j r: . / - / / o ‘/ B / / "E: 2_W V / 3 / /// Z 4/ /// / 0 I l 0.0 0.1 0.2 0.3 0.4 0.5 Relative collar location FIG. 5. Histogram showing distribution of collar locations in a randomly selected population of 28 cells. anaerobiosis and the low growth rate (1,, = 4d) of DCB-l. Our interpretation of growth and division of strain DCB-l is summarized in Fig. 7. Another feature observed in DCB-l cells was internal membranes having a stacked configuration (Fig. 2A and 3). Other bacteria known to have internal membranes include sulfate reducers (7), anoxic phototrophs, methylotrophs. and nitrifiers. Many members of these groups are known to exhibit polar growth (e.g., Rhodopseudomonas spp.. Meth- ylosinus spp., and Nirrobacrer spp.). Polar growth also occurs in other groups that have complex membrane struc- tures such as prosthecae (e.g., Caulobacrer spp.). Polar growth can be understood as adaptive for such organisms, since intercalary growth would disrupt membrane struc- tures. Electron-transparent spheres such as‘that in Fig. 3 were observed only rarely and did not normally accompany internal membranes. The sphere was not distinctly mem- brane bound, and its composition is unknown. An additional feature visible in the transmission electron micrographs is electron-dense, spherical bodies which oc- curred preferentially near the poles of DCB-l cells (Fig. 2). C LY: O s b" 0 O.4-r"= 3 i 8 =0 0.2- o L O > 2.15 0.0F .2 It!» i _.2 t l . l 2 4 6 8 Cell length (pm) FIG. 6. Relative collar location as a function of cell length for the same population of cells as in Fig. 5 with nonlinear regression fit of polar-growth model. 94 APPL. ENVIRON. MICROBIOL. l\ Dillohtor @ /" I I / Mother Coll dlvlolon Polar growth main FIG. 7. Proposed mode of growth and division of strain DCB-l. These bodies have the appearance of glycogen. The extract- able polysaccharide of DCB-l was determined to be 80% glycogen. The collar of strain DCB-l appeared to be intimately involved in cell division. No other function for the collar was apparent. This unique morphological feature distinguishes DCB-l from other procaryotes, as does its unique combina- tion of metabolic characteristics (6b, 9, 10). It remains to be determined whether the morphology and physiology of DCB-l define a novel group of sulfate-reducing bacteria. ACKNOWLEDGMENTS We thank Marie-Claude Fortin for help with the polar—growth model. We thank Stanley Flegler and The Center for Electron Microscopy for the scanning electron micrographs. This work was supported by U.S. Environmental Protection Agency grant R-813892. LITERATURE CITED 1. Bartlett. l. D. J.. and R. G. [-3. Murray. 1974. Electron micro- scope study of septum formation in Escherichia coli strains 8 and Rh during synchronous growth. J. Bacteriol. "9:10:49. 1056. 2. Caldwell, D. E., and J. M. Tiedje. 1975. A morphological study of anaerobic bacteria from the hypolimnia of two Michigan lakes. Can. J. Microbiol. 2|:362—376. 3. DeWeerd, K. A., J. M. Suflita. 'l‘. Linkfield. J. M. Tiedje. and P. H. Pritchard. 1986. The relationship between reductive de— halogenation and other aryl substituent removal reactions cata- lyzed by anaerobes. FEMS Microbiol. Ecol. 38:331-339. 3a.Dolfing, J. 1990. Reductive dechlorination of 3-chlorobenzoate is coupled to ATP production and growth in an anaerobic bacterium, strain DCB-l. Arch. Microbiol. [53:264—266. 4. Fathepure. B. 2., J. P. Nengu, and S. A. Boyd. 1987. Anaerobic bacteria that dechlorinate perchloroethene. Appl. Environ. Mi- crobiol. 53:2671—2674. 5. Hanson, R. S. and J. A. Phillips. 1981. Chemical composition. p. 328-364. In P. Gerhardt (ed.). Manual of methods for general bacteriology. American Society for Microbiology. Washington. DC 6. Klomparens, K. I... S. L. Flegler, and G. R. Hooper. 1986. Procedures for transmission and scanning electron microscopy for biological and medical science. 2nd ed. Ladd Research Industries. Burlington, Vt. 6a.Molru, W. W., and J. M. Tiedje. 1990. Strain DCB-l conserves VOL. 56. 1990 energy for growth from reductive dechlorination coupled to formate oxidation. Arch. Microbial. 153:267—271. 6b.Mohn, W. W., and J. M. Tiedje. I990. Catabolic thiosulfate disproportionation and carbon dioxide reduction in strain DCB- 1. a reductively dechlorinating anaerobe. J. Bacteriol. 172: 2065-2070. . Razanava, E. P.. T. N. Nazina, and A. S. Galushka. 1988. Isolation of a new genus of sulfate-reducing bacteria and de- scription of a new species of this genus. Desulfomit‘robium apsheronum gen. nov., sp. nov. Microbiology (Engl. Trans]. Mikrobiologiya) 57:514-520. 95 8. 10. GROWTH AND DIVISION OF DCB-l 1211 Shelton. D. R.. and J. M. Tiedje. 1984. Isolation and partial characterization of bacteria in an anaerobic consortium that mineralizes 3-chlorobenzoic acrd. Appl. Environ. Microbiol. 48:840—848. . Stevens. T. 0.. T. G. Linkfield. and J. M. Tiedje. 1988. Physio- logical characterization of strain DCB-I. a unique dehalogenat- ing sulfidogenic bacterium. Appl. Environ. Microbiol. 54:2938— 2943. Stevens. T. 0., and J. M. Tiedje. 1988. Carbon dioxide fixation and mixotrophic metabolism by strain DCB-l. a dehalogenating anaerobic bacterium. Appl. Environ. Microbiol. 54:2944—2948. MICHIGAN STATE UNIV. LIBRARIES IIHIHWIMllll"I"lllllwllllill""lllllmllllWWll 31293008914297