Reductive dehalogenation by anaerobic bacteria

The understanding of the fate of synthetic halogenated hydrocarbons became a matter of major interest over the last two decades. Halogenated compounds may threaten ecosystems due to their biocide properties. The degradability of halocompounds determines whether they will accumulate in a certain environment or whether they will be transformed to harmless products. A whole range of anthropogenic organohalogen compounds was detected in soils, sediments, surface and subsurface waters, and the atmosphere. Explanations for these accumulations could be that the halocompound is not degradable, that the transformation reactions are too slow, or that adverse environmental conditions prevent degradation. Aerobic bacteria are able to mineralize many halogented compounds under optimal conditions in the laboratory. However, polyhalogenated congeners often persisted aerobic biodegradation. This observation led to an intensification of biodegradation studies under anoxic conditions. Halogenated hydrocarbons were transformed in methanogenic microcosms of soils, sediments, aquifer material, or sewage sludge by reductive dehalogenation. Many of these microcosm studies indicated that the observed reductive dehalogenation reactions were biologically mediated. Nevertheless, only little was known about the micro-organisms which catalyzed these reactions. The aim of this thesis was to investigate the kind of bacteria involved in the reductive dehalogenation and to elucidate the physiological meaning and the biochemistry of the process. The results presented in this thesis showed that the physiological meaning of reductive dechlorination reactions catalyzed by anaerobic bacteria can be two-fold: i) a cometabolic activity and ii) a novel type of anaerobic respiration.Methanogenic bacteria were known to reductively dechlorinated aliphatic C1 and C2 hydrocarbons. The dechlorination of 1,2-dichloroethane (1,2-DCA) by these organisms was characterized in detail in Chapters 2 to 4. Concentrated cell suspensions of methanogenic bacteria reductively dechlorinated 1,2-DCA via two reaction mechanisms: a dihalo-elimination yielding ethene and two hydrogenolysis reactions yielding chloroethane (CA) and ethane, consecutively (Chapter 2). These reactions were catalyzed by hydrogenotrophic as well as acetoclastic methanogens. Stimulation of methanogenesis caused an increase in the amount of dechlorination products formed, whereas the opposite was found when methane formation was inhibited. The observation that the dechlorination occurred independently from the primary substrate metabolized indicated that an enzyme system present in all methanogens was involved in the dechlorination reactions. Possible catalysts of the dechlorination of 1,2-DCA were corrinoids or factor F 430 , two tetra-pyrrole cofactors present in high amounts in methanogens.Cobalamin and the native and diepimeric form of factor F 430 indeed catalyzed the reductive dechlorination of 1,2-DCA to ethene or CA in a buffer with Ti(III) citrate as electron donor (Chapter 3). Ethene was the major product in the cobalamin-catalyzed transformation and the ratio between ethene and CA formed was 25:1. Native F 430 and 12,13-di-epi-F 430 produced ethene and CA in a ratio of about 2:1 and 1:1, respectively. Crude and boiled cell extracts of Methanosarcina barkeri also dechlorinated 1,2-DCA to ethene and CA with Ti(III) citrate as reductant. The catalytic components in boiled extracts were heat- and oxygenstable, and had a low molecular mass. Fractionation of boiled extracts by a hydrophobic interaction column revealed that part of the dechlorinating components had a hydrophilic, and part a hydrophobic character. These chemical properties of the dechlorinating components and spectroscopic analysis of boiled extracts indicated that corrinoids or factor F 430 were responsible for the dechlorinations. The ratio of 3:1 to 7:1 between ethene and CA formed by cell extracts suggested that both cofactors were concomitantly active.Reductive dechlorination of 1,2-DCA could also be performed in cell extracts of methanogens with the physiological electron donor H 2 . Experiments with crude cell extracts of Methanobacterium thermoautotrophicum strain ΔH were carried out to get indications about the involvemnet of protein-bound corrinoids and factor F 430 in the 1,2-DCA dechlorination (Chapter 4). First the effect of MgATP and CoM-S-S-HTP, the heterodisulfide of coenzyme M and 7- mercaptoheptanoylthreonine phosphate, was investigated. Other studies demonstrated that the corrinoid-containing methyl-tetrahydromethanopterin: coenzyme M methyltransferase and the factor F 430 -containing methyl-coenzyme M reductase required an ATP-dependent reductive activation in invitro systems of M. thermoautotrophicum strain ΔH. The methyltransferase could in addition be activated by CoM-S-S-HTP. The dechlorination of 1,2-DCA to ethene and CA by crude cell extracts of M. thermoautotrophicum strain ΔH with H 2 as electron donor was stimulated by MgATP. CoM-S-S-HTP together with MgATP partially inhibited ethene production but stimulated CA production as compared to MgATP alone. Michaelis-Menten kinetics for initial product formation rates with different 1,2-DCA concentrations indicated the enzymatic character of the dechlorination. Apparent K m 's for 1,2-DCA of 89 and 119 μM, and V max 's of 34 and 20 pmol/min per ing protein were estimated for ethene and CA production, respectively. 3-Bromopropanesulfonate, a specific inhibitor for methyl-CoM reductase, completely inhibited dechlorination of 1,2-DCA. Purified methyl-CoM reductase, together with FAD and a crude fraction of component A, an enzyme system which reduces the nickel of factor F 430 in methyl-CoM reductase, converted 1,2- DCA to ethene and CA with H 2 as electron donor. These results showed that, at least partially, the in vivo dechlorination was based on the activity of the methyl-CoM reductase.The enrichment and characterization of bacteria which possibly use the reductive dechlorination as a novel type of anaerobic respiration is described in Chapter 5 and 6. Trichloro- and dichlorobenzenes were reductively dechlorinated in columns packed with river Rhine sediment. Enrichments inoculated with material from these percolation columns reductively dechlorinated hexachlorobenzene, pentachlorobenzene, all three isomers of tetrachlorobenzene, 1,2,3- trichlorobenzene (1,2,3-TCB), and 1,2,4-trichlorobenzene in the presence of lactate, glucose, ethanol, or isopropanol as electron donors (Chapter 5). A stable consortium grown on lactate as energy and carbon source in the presence of 1,2,3-TCB dechlorinated this isomer stoichiometrically to 1,3-dichlorobenzene. Dechlorinating activity could only be maintained when an electron donor was added. Lactate, ethanol, and hydrogen appeared to be the best suited substrates. For further enrichment of the 1,2,3-TCB dechlorinating bacteria, a two-liquid-phase (hexadecane/water) system was used with hydrogen as electron donor and 1,2,3-TCB or CO 2 as electron acceptor. Methanogens and acetogens were the major substrate-competing (H 2 /CO 2 ) microorganisms in the two-liquid-phase system. Inhibition of methanogenesis by 2-bromoethanesulfonic acid did not influence dechlorination, and acetogens which were isolated from the enrichment did not have dechlorinating activity. These results indicated that bacteria were present using 1,2,3-TCB as terminal electron acceptor. Although dechlorination was found in dilutions down to 10 -8from the twoliquid-phase system, attempts to isolate a bacterium in pure culture able to use 1,2,3-TCB as terminal electron acceptor failed.A microscopically pure culture, "PER-K23", was enriched from material of an anaerobic packed- bed column which reductively transformed tetrachloroethene (PCE) to ethane via trichloroethene (TCE), cis -1,2-dichloroethene ( cis -1,2-DCE), chloroethene, and ethene (Chapter 6). PER-K23 catalyzed the dechlorination of PCE via TCE to cis -1,2-DCE and coupled this reductive dechlorination. to growth. H 2 or formate were the only energy sources which supported growth with PCE or TCE as electron acceptors. In the absence of PCE or TCE, no growth occurred. Nor O 2 , NO 3-, NO 2-, SO42-, SO32-, S 2 O32-, S, fumarate, or CO 2 could replaced PCE or TCE as electron acceptors with H 2 as electron donor. PER-K23 was also not able to grow fermentatively on any of the organic compounds tested. Electron balances showed that all electrons derived from H 2 or formate consumption could be recovered in dechlorination products and biomass formed. PER-K23 is a Gram-negative rod with one lateral flagellum. Analysis of the 16S ribosomal RNA of PER-K23 demonstrated that this bacterium belongs to the subdivision of species with Gram-negative cell walls within the phylum of Gram-positive bacteria. Based on physiological and molecular properties of the isolate, we propose Dehalobacter as the name of the genus of this newly described reductive dehalogenating bacterium. The type species, Dehalobacter restrictus sp. nov., is named after the restricted spectra of electron donors and acceptors utilized.