Degradation of chlorobenzoates and chlorophenols by methanogenic consortia

Pollution of the environment with chlorinated organic compounds mainly results from (agro)industrial activity. In many studies, biodegradation is examined under anaerobic conditions, because highly chlorinated compounds are more easily degradable under anaerobic than under aerobic conditions. Problems arise because these compounds can inhibit methane formation and the microorganisms which are able to degrade these compounds may be absent in the methanogenic environment, like for instance wastewater treatment plants.This thesis concentrates on chlorinated aromatic compounds and aims to examine their biodegradation kinetics by methanogenic consortia. The biochemical degradation mechanisms of aromatic compounds under anaerobic conditions differ greatly from the aerobic conversions, because under aerobic conditions, oxygen is a reactant, while under anaerobic conditions, other activation steps are involved. The degradation of chlorinated aromatic compounds requires the elimination of chlorine atoms. Under methanogenic conditions, the initial degradation step is a reductive dechlorination. This conversion may yield biological useful energy, if the chlorinated compound is utilized as a terminal electron acceptor.The product of the reductive dechlorination step is an aromatic compound. For some of these compounds, for instance benzoate and phenol, the oxidation to acetate, carbon dioxide and hydrogen is endergonic under standard conditions. Therefore, this conversion can only occur when hydrogen-consuming organisms are present, which in turn depend on hydrogen-producers for substrate supply. This relationship is called syntrophism. Methanogenic archaea can act as syntrophic partners. Hydrogen can also be an electron donor for some dechlorinating microorganisms.Consequently, for the complete degradation of chlorinated aromatic compounds under methanogenic conditions, a consortium of different types of microorganisms is needed. Furthermore, the distance for hydrogen diffusion between syntrophic partners determines the conversion rate of the aromatic compounds. The interbacterial distances are small when the microorganisms exist as aggregated biomass in and/or on carrier materials, or as flocs or granules without the need for carrier materials. In upflow anaerobic sludge blanket (UASB) reactors, the microorganisms are auto-immobilized as granular sludge. These aspects are dealt with in Chapter 1.Chapter 2 describes the toxicity of monochlorophenols (MCPs)to granular sludge from a UASB reactor. Methane production from a mixture of acetate, propionate and butyrate was measured to test the influence of MCPs on the activity of the sludge. Since other substrates in addition to acetate were used, the sensitivity of syntrophs was also determined. The concentration, the isomeric form and the exposure time of the MCP determined the inhibition of methane formation. Different sensitivities were found for sludges, developed in reactors which had received similar influents, which may be due to differences in the population composition or the granule size. After cold storage of the sludge, the methane production rate decreased, and the sensitivity towards 2-chlorophenol (2-CP) increased. However, an activation period after cold storage could reduce the toxic effect. Inhibition of methane production occurred by MCP concentrations that were in the mM-range.The biodegradation of MCPs was studied in samples from different anaerobic environments, most of which were granular sludges from UASB reactors (Chapter 3). 2-CP was transformed by granular sludge, a sediment mixture and a peat slurry. The first intermediary products which accumulated were phenol and most probably 3-CB. This indicated that 2-CP was reductively dechlorinated to phenol as well as carboxylated and dehydroxylated to 3-CB. Two enrichment cultures were obtained from the granular sludge. One converted 2-CP to phenol and later to 3-CB. Unfortunately, this culture lost its 2-CP-degrading activity. The other dechlorinated 2-CP and dichlorophenols specifically at the ortho -position, and did not form 3-CB.Chapters 4, 5 and 6 describe the dechlorination and subsequent mineralization of the model compound 3-CB by a defined consortium, consisting of four microorganisms. Desulfomonile tiedjei reductively dechlorinated 3-CB to benzoate, Syntrophus buswellii oxidized benzoate syntrophically to acetate and hydrogen, Methanospirillum hungatei formed methane from hydrogen and carbon dioxide, and Methanosaeta concilii converted acetate to methane and carbon dioxide. The growth conditions were varied to optimize the dechlorination as well as the mineralization of 3-CB (Chapter 4).In addition, the effects of carrier materials (Chapter 5) and of the immobilization of the consortium in a gel (Chapter 6) are described. In Chapter 4, the composition of the 3-CB-degrading consortium is described. The growth medium was optimized to obtain balanced growth of the consortium. D. tiedjei utilized hydrogen for the dechlorination of 3-CB to benzoate, while hydrogen was only produced when S. buswellii oxidized benzoate. Therefore, to initiate the 3-CB conversion, an additional electron donor, e.g. pyruvate, was required. Furthermore, the substrate spectrum, the toxicity of 3-CB towards individual consortium members and the necessity for buffering capacity were studied. Based on cell counts it was found that a stable consortium consisted of about 18% D. tiedjei , 70% S. buswellii , 10% M. hungatei , and 3% M. concilii .Chapter 5 describes the effects of carrier materials on the 3-CB-degrading consortium. The consortium was incubated with 3-CB and pyruvate in the presence of a range of carrier materials. In the presence of polystyrene, the length of the lag phase for dechlorination was decreased. In the presence of vermiculite and granular sludge the consumption rates of pyruvate by D. tiedjei and acetate by M. concilii were higher than in the their absence. As a result, these three carrier materials decreased the time period for complete mineralization of 3-CB. Attachment of the consortium cells to the carrier materials was not an explanation for the stimulatory effects, because in pure cultures, similar effects were observed. Furthermore, cells adhered only weakly to glass and teflon sheets, and the majority of the cells in the consortium grew in suspension. The conditions for aggregation or adherence may not have been optimal. Nevertheless, an improvement of the pyruvate and acetate consumption rates or the shortening of the lag phase for dechlorination could reduce the time for complete degradation of 3-CB up to 50%.Immobilization of the consortium inκ-carrageenan gel beads is described in Chapter 6. It was demonstrated that 3-CB degradation and growth occurred in the gel beads. Subsequently, cells grown in suspension were concentrated and immobilized in different ratios. An increased dechlorination rate was achieved with extra D. tiedjei cells (for 3-CB and 3,5-dichlorobenzoate) and with extra M. hungatei cells (for 3-CB). Addition of extra S. buswellii cells only resulted in a higher specific activity of D. tiedjei cells. In that case, immobilized cell numbers of D. tiedjei , M. hungatei , and M. concilii were lower than without extra S. buswellii . The interbacterial distances were determined by the cell densities. Therefore, a variation of the cell ratios had to affect the rate of the hydrogen diffusion between S. buswellii and the two hydrogen consumers, D. tiedjei and M. hungatei .The minimal interbacterial distances were calculated and compared with the substrate conversion rates. Indeed, the changes in the calculated distances could, at least partially, explain the changes in the measured substrate conversion rates. Unfortunately, better growth of cells in the proximity of syntrophic partner cells could neither be established with carrier materials (Chapter 5) nor in gel beads (Chapter 6). The performance of the immobilized consortium was not tested in continuous flow reactors (which would be more like wastewater treatment plants), but in batch cultures a high activity was achieved (up to 0.56 mM/h) and only low concentrations of intermediary products accumulated transiently.In conclusion, the toxicity tests depict the sensitivity of granular sludge to MCPs and MCBs. Stored sludge which in practice is used again would perform better when the exposure to toxic compounds is preceded by a reactivation period. Furthermore, as has been frequently observed, 2-CP was most easily biodegraded, while the other two MCPs were not or hardly degraded. Methanogenic degradation of 2-CP has been observed to proceed via phenol. Here we found another pathway via 3-CB. In addition, it was possible to grow a completely mineralizing consortium on 3-CB. However, an external electron donor was important, and, under starting conditions, indispensable.The consortium did not aggregate nor adhere to surfaces, but it could convert 3-CB at a high rate when immobilized in a gel matrix. Dechlorination could be accelerated by an increase of the cell numbers of the dechlorinators or the hydrogenotrophic methanogens and not of the syntrophic benzoate oxidizers. In the presence of carrier materials, stimulation of the growth of the dechlorinating bacteria or the aceticlastic methanogens decreased the time period required for mineralization.