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Growth of Pseudomonas chloritidismutans AW-1T on C7 to C12 n-alkanes with oxygen or chlorate as electron acceptor was studied by genome and proteome analysis. Whole genome shotgun sequencing resulted in a 5 Mbp assembled sequence with a G+C content of 62.5% The automatic annotation identified 4767 protein-encoding genes and a putative function could be assigned to almost 80% of the predicted proteins. The distinct phylogenetic position of P. chloritidismutans AW-1T within the Pseudomonas stutzeri cluster became clear by comparison of average nucleotide identity values of sequenced genomes. Analysis of the proteome of P. chloritidismutans AW-1T showed the versatility of this bacterium to adapt to aerobic and anaerobic growth conditions with acetate or n-decane as substrates. All enzymes involved in the alkane oxidation pathway were identified. An alkane monooxygenase was detected in n-decane-grown cells, but not in acetate-grown cells. The enzyme was found when grown in the presence of oxygen or chlorate, indicating that under both conditions an oxygenase-mediated pathway is employed for alkane degradation. Proteomic and biochemical data also showed that both chlorate reductase and chlorite dismutase are constitutively present, but most abundant under chlorate-reducing conditions

Studies on the occurrence of pharmaceuticals show that the widely used pharmaceuticals ibuprofen and diclofenac are present in relevant concentrations in the environment. A pilot plant treating hospital wastewater with relevant concentrations of these pharmaceuticals was evaluated for its performance to reduce the concentration of the pharmaceuticals. Ibuprofen was completely removed, whereas diclofenac yielded a residual concentration, showing the necessity of posttreatment to remove diclofenac, for example, activated carbon. Successively, detailed laboratory experiments with activated sludge from the same wastewater treatment plant showed bioremediation potential in the treatment plant. The biological degradation pathway was studied and showed a mineralisation of ibuprofen and degradation of diclofenac. The present microbes were further studied in laboratory experiments, and DGGE analyses showed the enrichment and isolation of highly purified cultures that degraded either ibuprofen or diclofenac. This research illuminates the importance of the involved bacteria for the effectiveness of the removal of pharmaceuticals in a wastewater treatment plant. A complete removal of pharmaceuticals from wastewater will stimulate water reuse, addressing the worldwide increasing demand for clean and safe fresh water.

The diffuse pollution by chlorinated organic compounds in river basins is a concern, due to their potential adverse effects on human health and the environment. Organohalides, like hexachlorobenzene (HCB), are recalcitrant to aerobic microbial degradation, and “ Dehalococcoides ” spp. are the only known microorganisms capable of anaerobic transformation of these compounds coupled to their growth. In this study, sediments from four European rivers were studied in order to determine their HCB dechlorination capacities and the role of Dehalococcoides spp. in this process. Only a weak correlation was observed between Dehalococcoides species abundance and HCB transformation rates from different locations. In one of these locations, in the Ebro River sediment, HCB dechlorination could be linked to Dehalococcoides species growth and activity by 16S rRNA-based molecular methods. Furthermore, HCB dechlorination activity in this sediment was found over the full range of ambient temperatures that this sediment can be exposed to during different seasons throughout the year. The sediment contained several reductive dehalogenase ( rdh ) genes, and analysis of their transcription revealed the dominance of cbrA , previously shown to encode a trichlorobenzene reductive dehalogenase. This study investigated the role of Dehalococcoides spp. in HCB dechlorination in river sediments and evaluated if the current knowledge of rdh genes could be used to assess HCB bioremediation potential.

The ability of Dehalococcoides spp. to reduce chlorinated compounds offers a great potential for bioremediation and/or bioaugmentation of contaminated environments. So far, however, our knowledge of the activity of Dehalococcoides spp. in situ is limited to only a few subsurface environments. The aim of this study was to broaden this knowledge to other environments, and we investigated the role of Dehalococcoides spp. in the transformation of chlorinated benzenes and chlorinated ethenes in the Ebro River (Spain) sediments. Lab-scale batch microcosms were used to follow the growth and abundance of Dehalococcoides spp. during the transformation of selected chlorinated compounds. We applied biomolecular tools targeting the 16S rRNA, the 16S rRNA gene and several functional genes involved in dechlorination in combination with chemical measurements. The growth of Dehalococcoides spp. and the differential expression of several reductive dehalogenase genes during the dechlorination process could be demonstrated. Furthermore, 16S rRNA gene-based clone libraries of dechlorinating river sediment showed a complex community structure and indicated the involvement of several additional bacterial genera in the transformation process, underlining the remarkable potential of this rivers' sediment to transform different halo-organic pollutants.

Microbiota on the human skin plays a major role in body odour production. The human microbial and chemical signature displays a qualitative and quantitative correlation. Genes may influence the chemical signature by shaping the composition of the microbiota. Recent studies on human skin microbiota, using 16S rRNA gene sequencing, found a high inter- and intrapersonal variation in bacterial species on the human skin, which is relatively stable over time. Human body odours mediate the attraction of mosquitoes to their blood hosts. Odours produced by skin microbiota are attractive to mosquitoes as shown by in vitro studies, and variation in bacterial species on the human skin may explain the variation in mosquito attraction between humans. Detailed knowledge of the ecology and genetics of human skin microbiota is needed in order to unravel the evolutionary mechanisms that underlie the interactions between mosquitoes and their hosts.

Microbial (per)chlorate reduction is a unique process in which molecular oxygen is formed during the dismutation of chlorite. The oxygen thus formed may be used to degrade hydrocarbons by means of oxygenases under seemingly anoxic conditions. Up to now, no bacterium has been described that grows on aliphatic hydrocarbons with chlorate. Here, we report that Pseudomonas chloritidismutans AW-1(T) grows on n-alkanes (ranging from C7 until C12) with chlorate as electron acceptor. Strain AW-1(T) also grows on the intermediates of the presumed n-alkane degradation pathway. The specific growth rates on n-decane and chlorate and n-decane and oxygen were 0.5 +/- 0.1 and 0.4 +/- 0.02 day(-1), respectively. The key enzymes chlorate reductase and chlorite dismutase were assayed and found to be present. The oxygen-dependent alkane oxidation was demonstrated in whole-cell suspensions. The strain degrades n-alkanes with oxygen and chlorate but not with nitrate, thus suggesting that the strain employs oxygenase-dependent pathways for the breakdown of n-alkanes.

An anaerobic coculture was enriched from a hexachlorocyclohexane (HCH) polluted soil. The coculture reductively dechlorinates the beta-HCH isomer to benzene and chlorobenzene in a ratio of 0.5-2 depending on the amount of beta-HCH degraded. The culture grows with H(2) as electron donor and beta-HCH as electron acceptor, indicating that dechlorination is a respiratory process. Phylogenetic analysis indicated that the coculture consists of two bacteria that are both related to gram-positive bacteria with a low G + C content of the DNA. One bacterium was identified as a Dehalobacter sp. This bacterium is responsible for the dechlorination. The other bacterium was isolated and characterized as being a Sedimentibacter sp. This strain is not able to dechlorinate beta-HCH. The Dehalobacter sp. requires the presence of Sedimentibacter for growth and dechlorination, but the function of the latter bacterium is not clear. This is the first report on the metabolic dechlorination of beta-HCH by a defined anaerobic bacterial culture.

Halorespiring microorganisms have been detected in soils that were not polluted with chlorinated compounds. In this study, we describe alternative electron acceptor utilization by some halorespiring bacteria and phylogenetically related bacteria. It appears that oxidized metals like selenate, arsenate and manganese are rather common electron acceptors for halorespiring species of Desulfitobacterium and Sulfurospirillum and related bacteria. All tested microorganisms are able to reduce anthraquinone-2,6-disulfonate (AQDS) and four tested organisms (Desulfobacterium hafniense DP7, Sulfurospirillum barnesii, Sulfurospirillum deleyianum and Sulfurospirillum arsenophilum) are able to oxidize reduced anthrahydroquinone-2,6,-disulfonate (AH(2)QDS) as well. The characteristic to reduce oxidized metals, and to reduce and oxidize quinone moieties coupled to energy conservation is a likely explanation for the presence of halorespiring microorganisms in unpolluted soils. (C) 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

An anaerobic, halorespiring bacterium (strain PCE-M2(T) = DSM 13726(T) = ATCC BAA-583(T)) able to reduce tetrachloroethene to cis-dichloroethene was isolated from an anaerobic soil polluted with chlorinated aliphatic compounds. The isolate is assigned to the genus Sulfurospirillum as a novel species, Sulfurospirillum halorespirans sp. nov. Furthermore, on the basis of all available data, a related organism, Dehalospirillum multivorans DSM 12446(T), is reclassified to the genus Sulfurospirillum as Sulfurospirillum multivorans comb. nov.

Chlorinated ethenes and chlorinated aromatics are often found as pollutants in sediments, groundwater, and wastewater. These compounds were long considered to be recalcitrant under anaerobic conditions. In the past years however, dechlorination of these compounds has been found to occur under anaerobic conditions at contaminated sites and in wastewater treatment systems. This dechlorination is mainly attributed to halo-respiring bacteria, which are able to couple this dechlorination to energy conservation via electron transport coupled phosphorylation. The dechlorinating activities of the halo-respiring bacteria seem to be confined to the dechlorination of chloroethenes and chlorinated aromatic compounds. In addition, methanogenic and acetogenic bacteria are also able to reduce the chlorinated ethenes via a-specific cometabolic pathways. Although these latter reactions may not be important in the remediation of contaminated sites, they may be of substantial influence in the start-up of remediation processes and in the application of granular sludge from UASB reactors. Specific halo-respiring bacteria may be used to increase the dechlorination activities via bioaugmentation in the case that the appropriate microorganisms are not present at the contaminated site or in the sludge.

Thermophilic anaerobic biodegradation of tetrachloroethene (PCE) was investigated with various inocula from geothermal and nongeothermal areas. Only polluted harbor sediment resulted in a stable enrichment culture that converted PCE via trichloroethene to cis -1,2-dichloroethene at the optimum temperature of 60 to 65°C. After several transfers, methanogens were eliminated from the culture. Dechlorination was supported by lactate, pyruvate, fructose, fumarate, and malate as electron donor but not by H 2 , formate, or acetate. Fumarate and l -malate led to the highest dechlorination rate. In the absence of PCE, fumarate was fermented to acetate, H 2 , CO 2 , and succinate. With PCE, less H 2 was formed, suggesting that PCE competed for the reducing equivalents leading to H 2 . PCE dechlorination, apparently, was not outcompeted by fumarate as electron acceptor. At the optimum dissolved PCE concentration of ∼60 μM, a high dechlorination rate of 1.1 μmol h −1 mg −1 (dry weight) was found, which indicates that the dechlorination is not a cometabolic activity. Microscopic analysis of the fumarate-grown culture showed the dominance of a long thin rod. Molecular analysis, however, indicated the presence of two dominant species, both belonging to the low-G+C gram positives. The highest similarity was found with the genus Dehalobacter (90%), represented by the halorespiring organism Dehalobacter restrictus , and with the genus Desulfotomaculum (86%).

The potential of granular sludge from upflow anaerobic sludge blanket (UASB) reactors for bioremediation of chlorinated pollutants was evaluated by using carbon tetrachloride (CT) as a model compound. Granular sludges cultivated in UASB reactors on methanol, a volatile fatty acid mixture, or sucrose readily degraded CT supplied at a concentration of 1,500 nmol/batch (approximately 10 μM) without any prior exposure to organohalogens. The maximum degradation rate was 1.9 μmol of CT g of volatile suspended solids −1 day −1 . The main end products of CT degradation were CO 2 and Cl − , and the yields of these end products were 44 and 68%, respectively, of the initial amounts of [ 14 C]CT and CT-Cl. Lower chlorinated methanes accumulated in minor amounts temporarily. Autoclaved (dead) sludges were capable of degrading CT at rates two- to threefold lower than those for living sludges, indicating that abiotic processes (mediated by cofactors or other sludge components) played an important role in the degradation observed. Reduced components in the autoclaved sludge were vital for CT degradation. A major part (51%) of the CT was converted abiotically to CS 2 . The amount of CO 2 produced (23%) was lower and the amount of Cl − produced (86%) was slightly higher with autoclaved sludge than with living sludge. Both living and autoclaved sludges could degrade chloroform. However, only living sludge degraded dichloromethane and methylchloride. These results indicate that reductive dehalogenation, which was mediated better by living sludge than by autoclaved sludge, is only a minor pathway for CT degradation. The main pathway involves substitutive and oxidative dechlorination reactions that lead to the formation of CO 2 . Granular sludge, therefore, has outstanding potential for gratuitous dechlorination of CT to safe end products.

The biotransformation of 3-chlorobenzoate (3CB) by attached cells of Pseudomonas sp. strain B13 was studied in percolation columns operated at various flow rates and biomass contents. Steady-state residual effluent concentrations were compared with predictions from spreadsheet models describing the combined action of microbial and mass transfer kinetics. The effluent concentrations were successfully predicted above a critical ratio of flow rate and biomass. Below this critical point the steady-state residual effluent concentrations were higher than predicted and this deviation increased with decreasing flow rate:biomass ratios. The results are discussed in the light of convection-diffusion processes limiting the transport of both substrate and oxygen to the attached cells.

A hydrogen-consuming methanogenic enrichment showed complete reduction of ethene to ethane. The ethene reduction was coupled to the formation of methane and addition of bromoethane sulfonate completely inhibited the reduction of ethene. At an ethene concentration of 0.8% in the gas phase (approximately 90 μmol l−1 in the liquid phase), the formation of ethane from ethene was completely inhibited and methane formation was inhibited by 85%. Three dominant microorganisms were present in the enrichment. A highly motile, oval-shaped homoacetogenic bacterium, and two long, rod-shaped methanogens. One methanogen was irregularly crooked and the other one was Methanospirillum-like. The acetogen and the irregularly crooked methanogen were isolated, but in pure culture they did not reduce ethene. So far, we have been unable to isolate the other methanogen.

The genomes of the Betaproteobacteria Alicycliphilus denitrificans strains BC and K601T have been sequenced to get insight into the physiology of the two strains. Strain BC degrades benzene with chlorate as electron acceptor. The cyclohexanol-degrading denitrifying strain K601T is not able to use chlorate as electron acceptor, while strain BC cannot degrade cyclohexanol. The 16S rRNA sequences of strains BC and K601T are identical and the fatty acid methyl ester patterns of the strains are similar. Basic Local Alignment Search Tool (BLAST) analysis of predicted open reading frames of both strains showed most hits with Acidovorax sp. JS42, a bacterium that degrades nitro-aromatics. The genomes include strain-specific plasmids (pAlide201 in strain K601T and pAlide01 and pAlide02 in strain BC). Key genes of chlorate reduction in strain BC were located on a 120 kb megaplasmid (pAlide01), which was absent in strain K601T. Genes involved in cyclohexanol degradation were only found in strain K601T. Benzene and toluene are degraded via oxygenase-mediated pathways in both strains. Genes involved in the meta-cleavage pathway of catechol are present in the genomes of both strains. Strain BC also contains all genes of the ortho-cleavage pathway. The large number of mono- and dioxygenase genes in the genomes suggests that the two strains have a broader substrate range than known thus far., This research was supported by the Technology Foundation, the Applied Science Division (STW) of the Netherlands Organization for Scientific Research (NWO), project number 08053, the graduate school WIMEK (Wageningen Institute for Environment and Climate Research, which is part of SENSE Research School for Socio-Economic and Natural Sciences of the Environment, www.wimek-new.wur.nl and www.sense.nl), SKB (Dutch Centre for Soil Quality Management and Knowledge Transfer, www.skbodem.nl) and the Consolider project CSD-2007-00055. The research was incorporated in the TRIAS (TRIpartite Approaches 469 toward Soil systems processes) program (http://www.nwo.nl/en/research-and-results/programmes/alw/trias-tripartite-approach-to-soil-system-processes/index. html). Flávia Talarico Saia was supported by a FAPESP (the State of São Paulo Research Foundation) scholarship (2006-01997/5). The work conducted by the DOE JGI is supported by the Office of Science of the United States Department of Energy under contract number DE-AC02-05CH11231. Alfons Stams acknowledges support by an ERC (European Research Counsil) advanced grant (project 323009). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

The minimum substrate concentration required for growth, Smin, was measured for Pseudomonas sp. strain B13 with 3-chlorobenzoate (3CB) and acetate in a recycling fermentor. The substrates were provided alone or in a mixture. Smin values predicted with kinetic parameters from resting-cell batches and chemostat cultures differed clearly from the values measured in the recycling fermentor. When 3CB and acetate were fed as single substrates, the measured Smin values were higher than the individual Smin values in the mixture. The Smin in the mixture reflected the relative energy contributions of the two substrates in the fermentor feed. The energy-based maintenance coefficients during zero growth in the recycling fermentor were comparable for all influent compositions (mean +/- standard deviation, 0.34 +/- 0.07 J mg [dry weight]-1 h-1). Maintenance coefficient values for acetate were significantly higher in chemostat experiments than in recycling-fermentor experiments. 3CB maintenance coefficients were comparable in both experimental systems. The parameters for 3CB consumption kinetics varied remarkably with the experimental growth conditions in batch, chemostat, and recycling-fermentor environments. The results demonstrate that the determination of kinetic parameters in the laboratory for prediction of microbial activity in complex natural systems should be done under conditions which best mimic the system under consideration.

A bacterial strain that was able to mineralize 2,4,6-trichlorophenol was isolated from a chlorophenol-fed percolator and was identified as a member of the genus Rhodococcus on the basis of chemotaxonomic characteristics and 16S RNA phylogenetic inference data. This organism (strain MBS1T [T = type strain]) exhibited a typical irregular rod-coccus cycle, and the cells had fimbria-like structures on their surfaces. The diagnostic cell wall amino acid was meso-diaminopimelic acid, and the sugars were arabinose and galactose; the mycolic acids contained 46 to 54 carbon atoms. The main menaquinone was MK-8(H2), and MK-9(H2) was a minor component. The cellular phospholipids were phosphatidylethanolamine, phosphatidylinositol, phosphatidylinositolmannoside, phosphatidylglycerol, and diphosphatidylglycerol. Tuberculostearic acid was present. The whole-cell fatty acids were straight-chain acids with 14 to 18 C atoms. The G+C content of the DNA was 67.4 mol%. This organism grew on sucrose, pyruvate, and 2,4,6-trichlorophenol, and it oxidized a large number of carbon compounds, including catechol, 3-hydroxyphenylacetic acid, and phenol. It also exhibited beta-galactosidase, urease, and 2-acetyl-lactate decarboxylase activities. On a phylogenetic tree that was based on 16S ribosomal DNA gene sequences strain MBS1T was found among the rhodococci on an independent branch. On the basis of the chemotaxonomic and phenotypic characteristics of strain MBS1T and its phylogenetic position we suggest that this bacterium should be placed in a new species, Rhodococcus percolatus; the specific epithet was chosen because the organism was isolated by using an enriched percolator. The type strain is strain MBS1.

The physiological meaning of reductive dechlorination reactions catalyzed by anaerobic bacteria can be explained as a co-metabolic activity or as a novel type of respiration. Co-metabolic activities have been found mainly with alkyl halides. They are non-specific reactions catalyzed by various enzyme systems of facultative as well as obligate anaerobic bacteria. In contrast, the reductive dechlorinations involved in metabolic respiration processes are very specific reactions. Only a limited number of alkyl and aryl chlorinated compounds is presently known to function as a terminal electron acceptor in a few, recently isolated bacteria. Metabolic dechlorination rates are in general several orders of magnitude higher than co-metabolic ones. Both reaction types are suitable for the anaerobic treatment of waste streams.

A microscopically pure enrichment culture of a gram-negative anaerobic bacterium, in the present article referred to as PER-K23, was isolated from an anaerobic packed-bed column in which tetrachloroethene (PCE) was reductively transformed to ethane via trichloroethene (TCE), cis-1,2-dichloroethene (cis-1,2-DCE), chloroethene, and ethene. PER-K23 catalyzes the dechlorination of PCE via TCE to cis-1,2-DCE and couples this reductive dechlorination to growth. H2 and formate were the only electron donors that supported growth with PCE or TCE as an electron acceptor. The culture did not grow in the absence of PCE or TCE. Neither O2, NO3-, NO2-, SO4(2-), SO3(2-), S2O3(2-), S, nor CO2 could replace PCE or TCE as an electron acceptor with H2 as an electron donor. Also, organic electron acceptors such as acetoin, acetol, dimethyl sulfoxide, fumarate, and trimethylamine N-oxide and chlorinated ethanes, DCEs, and chloroethene were not utilized. PER-K23 was not able to grow fermentatively on any of the organic compounds tested. Transferring the culture to a rich medium revealed that a contaminant was still present. Dechlorination was optimal between pH 6.8 and 7.6 and a temperature of 25 to 35 degrees C. H2 consumption was paralleled by chloride production, PCE degradation, cis-1,2-DCE formation, and growth of PER-K23. Electron balances showed that all electrons derived from H2 or formate consumption were recovered in dechlorination products and biomass. Exponential growth could be achieved only in gently shaken cultures.(ABSTRACT TRUNCATED AT 250 WORDS)

The ability of Dehalococcoides spp. to reduce chlorinated compounds offers a great potential for bioremediation and/or bioaugmentation of contaminated environments. So far, however, our knowledge of the activity of Dehalococcoides spp. in situ is limited to only a few subsurface environments. The aim of this study was to broaden this knowledge to other environments, and we investigated the role of Dehalococcoides spp. in the transformation of chlorinated benzenes and chlorinated ethenes in the Ebro River (Spain) sediments. Lab-scale batch microcosms were used to follow the growth and abundance of Dehalococcoides spp. during the transformation of selected chlorinated compounds. We applied biomolecular tools targeting the 16S rRNA, the 16S rRNA gene and several functional genes involved in dechlorination in combination with chemical measurements. The growth of Dehalococcoides spp. and the differential expression of several reductive dehalogenase genes during the dechlorination process could be demonstrated. Furthermore, 16S rRNA gene-based clone libraries of dechlorinating river sediment showed a complex community structure and indicated the involvement of several additional bacterial genera in the transformation process, underlining the remarkable potential of this rivers' sediment to transform different halo-organic pollutants.

Microbiota on the human skin plays a major role in body odour production. The human microbial and chemical signature displays a qualitative and quantitative correlation. Genes may influence the chemical signature by shaping the composition of the microbiota. Recent studies on human skin microbiota, using 16S rRNA gene sequencing, found a high inter- and intrapersonal variation in bacterial species on the human skin, which is relatively stable over time. Human body odours mediate the attraction of mosquitoes to their blood hosts. Odours produced by skin microbiota are attractive to mosquitoes as shown by in vitro studies, and variation in bacterial species on the human skin may explain the variation in mosquito attraction between humans. Detailed knowledge of the ecology and genetics of human skin microbiota is needed in order to unravel the evolutionary mechanisms that underlie the interactions between mosquitoes and their hosts.

Reductive dechlorination of 1,2-dichloroethane (1,2-DCA) to ethylene and chloroethane (CA) by crude cell extracts of Methanobacterium thermoautotrophicum delta H with H2 as the electron donor was stimulated by Mg-ATP. The heterodisulfide of coenzyme M (CoM) and 7-mercaptoheptanoylthreonine phosphate together with Mg-ATP partially inhibited ethylene production but stimulated CA production compared Mg-ATP alone. The pH optimum for the dechlorination was 6.8 (at 60 degrees C). Michaelis-Menten kinetics for initial product formation rates with different 1,2-DCA concentrations indicated the enzymatic character of the dechlorination. Apparent Kms for 1,2-DCA of 89 and 119 microM and Vmaxs of 34 and 20 pmol/min/mg of protein were estimated for ethylene 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 flavin adenine dinucleotide and a crude component A fraction which reduced the nickel of factor F430 in methyl-CoM reductase, converted 1,2-DCA to ethylene and CA with H2 as the electron donor. In this system, methyl-CoM reductase was also able to transform its own inhibitor 2-bromoethanesulfonate to ethylene.

Soil column experiments were performed to obtain insight in the different biological and physico-chemical processes affecting biodegradation of halogenated compounds under natural conditions in a water infiltration site. Lower chlorinated aromatic compounds could be degraded under aerobic conditions, whereas highly chlorinated compounds and chlorinated aliphatic compounds were mainly transformed under anaerobic conditions. Microorganisms which derive energy from reductive dechlorination were enriched and characterized. It was found that microbes could adapt to using chlorinated benzenes by evolution of new enzyme specificities and by exchange of genetic material. For halogenated pollutants, which are generally hydrophobic, sorption processes control the concentration available for biodegradation. The effects of very low concentrations of halogenated compounds on their biodegradability are described. The use of isolated bacterial strains to enhance biodegradation was evaluated with respect to their temperature-related activity and to their adhesion properties.

Hexachlorobenzene (HCB), pentachlorobenzene (QCB), all three isomers of tetrachlorobenzene (TeCB), 1,2,3-trichlorobenzene (1,2,3-TCB), and 1,2,4-TCB were reductively dechlorinated by enrichment cultures in the presence of lactate, glucose, ethanol, or isopropanol as the electron donor. The enrichment cultures originated from percolation columns filled with Rhine River sediment in which dechlorination of TCBs and dichlorobenzenes (DCBs) occurred. A stable consortium obtained by transfer on lactate as the energy and carbon source in the presence of 1,2,3-TCB dechlorinated this isomer stoichiometrically to 1,3-DCB. Dechlorinating activity could only be maintained when an electron donor was added. Lactate, ethanol, and hydrogen appeared to be the best substrates. Optimal temperature and pH for dechlorination were 30 degrees C and 7.2, respectively. The specificity of the enrichment on lactate and 1,2,3-TCB was tested after approximately 60 transfers (after 2.5 years). HCB and QCB were stoichiometrically dechlorinated to 1,3,5-TCB and minor amounts of 1,2,4-TCB. 1,3,5-TCB was the sole product formed from 1,2,3,5-TeCB, while 1,2,3,4-TeCB and 1,2,4,5-TeCB were converted to 1,2,4-TCB. 1,2,4-TCB, 1,3,5-TCB, and the three isomers of DCB were not dechlorinated during 4 weeks of incubation. For further enrichment of the 1,2,3-TCB-dechlorinating bacteria, a two-liquid-phase (hexadecane-water) system was used with hydrogen as the electron donor and 1,2,3-TCB or CO2 as the electron acceptor. Methanogens and acetogens were the major substrate-competing (H2-CO2) 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 culture did not have dechlorinating activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Background Host-seeking of the African malaria mosquito, Anopheles gambiae sensu stricto, is guided by human odours. The precise nature of the odours, and the composition of attractive blends of volatiles, remains largely unknown. Skin microbiota plays an important role in the production of human body odours. It is hypothesized that host attractiveness and selection of An. gambiae is affected by the species composition, density, and metabolic activity of the skin microbiota. A study is presented in which the production and constituency of volatile organic compounds (VOCs) by human skin microbiota is examined and the behavioural responses of An. gambiae to VOCs from skin microbiota are investigated. Methods Blood agar plates incubated with skin microbiota from human feet or with a reference strain of Staphylococcus epidermidis were tested for their attractiveness to An. gambiae in olfactometer bioassays and indoor trapping experiments. Entrained air collected from blood agar plates incubated with natural skin microbiota or with S. epidermidis were analysed using GC-MS. A synthetic blend of the compounds identified was tested for its attractiveness to An. gambiae. Behavioural data were analysed by a χ2-test and GLM. GC-MS results were analysed by fitting an exponential regression line to test the effect of the concentration of bacteria. Results More An. gambiae were caught with blood agar plates incubated with skin bacteria than with sterile blood agar plates, with a significant effect of incubation time and dilution of the skin microbiota. When bacteria from the feet of four other volunteers were tested, similar effects were found. Fourteen putative attractants were found in the headspace of the skin bacteria. A synthetic blend of 10 of these was attractive to An. gambiae. Conclusions The discovery that volatiles produced by human skin microorganisms in vitro mediate An. gambiae host-seeking behaviour creates new opportunities for the development of odour-baited trapping systems. Additionally, identification of bacterial volatiles provides a new method to develop synthetic blends, attractive to An. gambiae and possibly other anthropophilic disease vectors.

Hexachlorobenzene (HCB) has been widely used in chemical manufacturing processes and as a pesticide. Due to its resistance to biological degradation, HCB has mainly accumulated in freshwater bodies and agricultural soils. “ Dehalococcoides ” spp., anaerobic dechlorinating bacteria that are capable of degrading HCB, were previously isolated from river sediments. Yet there is limited knowledge about the abundance, diversity, and activity of this genus in the environment. This study focused on the molecular analysis of the composition and abundance of active Dehalococcoides spp. in HCB-contaminated European river basins. 16S rRNA-based real-time quantitative PCR and denaturing gradient gel electrophoresis in combination with multivariate statistics were applied. Moreover, a functional gene array was used to determine reductive dehalogenase ( rdh ) gene diversity. Spatial and temporal fluctuations were observed not only in the abundance of Dehalococcoides spp. but also in the composition of the populations and rdh gene diversity. Multivariate statistics revealed that Dehalococcoides sp. abundance is primarily affected by spatial differences, whereas species composition is under the influence of several environmental parameters, such as seasonal changes, total organic carbon and/or nitrogen content, and HCB contamination. This study provides new insight into the natural occurrence and dynamics of active Dehalococcoides spp. in HCB-contaminated river basins.

The amount of energy that can be conserved via halorespiration by Desulfitobacterium dehalogenans JW/IU-DC1 was determined by comparison of the growth yields of cells grown with 3-chloro-4-hydroxyphenyl acetate (Cl-OHPA) and different electron donors. Cultures that were grown with lactate, pyruvate, formate, or hydrogen as an electron donor and Cl-OHPA as an electron acceptor yielded 3.1, 6.6, 1.6, and 1.6 g (dry weight) per mol of reduction equivalents, respectively. Fermentative growth on pyruvate yielded 14 g (dry weight) per mol of pyruvate oxidized. Pyruvate was not fermented stoichiometrically to acetate and lactate, but an excess of acetate was produced. Experiments with 13 C-labeled bicarbonate showed that during pyruvate fermentation, approximately 9% of the acetate was formed from the reduction of CO 2 . Comparison of the growth yields suggests that 1 mol of ATP is produced per mol of acetate produced by substrate-level phosphorylation and that there is no contribution of electron transport phosphorylation when D. dehalogenans grows on lactate plus Cl-OHPA or pyruvate plus Cl-OHPA. Furthermore, the growth yields indicate that approximately 1/3 mol of ATP is conserved per mol of Cl-OHPA reduced in cultures grown in formate plus Cl-OHPA and hydrogen plus Cl-OHPA. Because neither formate nor hydrogen nor Cl-OHPA supports substrate-level phosphorylation, energy must be conserved through the establishment of a proton motive force. Pyruvate ferredoxin oxidoreductase, lactate dehydrogenase, formate dehydrogenase, and hydrogenase were localized by in vitro assays with membrane-impermeable electron acceptors and donors. The orientation of chlorophenol-reductive dehalogenase in the cytoplasmic membrane, however, could not be determined. A model is proposed, which may explain the topology analyses as well as the results obtained in the yield study.

ortho-Chlorophenol reductive dehalogenase of the halorespiring Gram-positive Desulfitobacterium dehalogenans was purified 90-fold to apparent homogeneity. The purified dehalogenase catalyzed the reductive removal of a halogen atom from the ortho position of 3-chloro-4-hydroxyphenylacetate, 2-chlorophenol, 2,3-dichlorophenol, 2,4-dichlorophenol, 2,6-dichlorophenol, pentachlorophenol, and 2-bromo-4-chlorophenol with reduced methyl viologen as electron donor. The dechlorination of 3-chloro-4-hydroxyphenylacetate was catalyzed by the enzyme at a Vmax of 28 units/mg protein and a Km of 20 microM. The pH and temperature optimum were 8.2 and 52 degrees C, respectively. EPR analysis indicated one [4Fe-4S] cluster (midpoint redox potential (Em) = -440 mV), one [3Fe-4S] cluster (Em = +70 mV), and one cobalamin per 48-kDa monomer. The Co(I)/Co(II) transition had an Em of -370 mV. Via a reversed genetic approach based on the N-terminal sequence, the corresponding gene was isolated from a D. dehalogenans genomic library, cloned, and sequenced. This revealed the presence of two closely linked genes: (i) cprA, encoding the o-chlorophenol reductive dehalogenase, which contains a twin-arginine type signal sequence that is processed in the purified enzyme; (ii) cprB, coding for an integral membrane protein that could act as a membrane anchor of the dehalogenase. This first biochemical and molecular characterization of a chlorophenol reductive dehalogenase has revealed structural resemblance with haloalkene reductive dehalogenases.

A soil from a former chemical redistribution company, contaminated with mainly chlorinated aliphatics, was studied for bioremediation purposes. Groundwater analyses revealed that the original pollutants, i.e. tetrachloroethene (PCE) and trichloroethene (TCE), were present at levels ranging from 2.3 to 122 mg/L. Dichloroethene (DCE), vinylchloride (VC), ethene and ethane were also detected at significant concentrations although they had never been introduced to the soil. Relatively high concentrations of cis-DCE as compared to trans-DCE and 1,1-DCE indicated that a slow in situ biodegradation had taken place by reductive dechlorination. Laboratory experiments with flow-through soil columns were performed to determine the optimal conditions for the enhancement of reductive dechlorination by the indigenous dechlorinating population. The addition of single electron donors to artificial groundwater resulted in the dechlorination of PCE to TCE and cis-DCE, whereas complete dechlorination to ethene was solely achieved with compost extract added to native groundwater.

The biotransformation of 3-chlorobenzoate (3CB) by attached cells of Pseudomonas sp. strain B13 was studied in percolation columns operated at various flow rates and biomass contents. Steady-state residual effluent concentrations were compared with predictions from spreadsheet models describing the combined action of microbial and mass transfer kinetics. The effluent concentrations were successfully predicted above a critical ratio of flow rate and biomass. Below this critical point the steady-state residual effluent concentrations were higher than predicted and this deviation increased with decreasing flow rate:biomass ratios. The results are discussed in the light of convection-diffusion processes limiting the transport of both substrate and oxygen to the attached cells.

A bacterial culture (LET-13) was enriched, which uses toluene as sole carbon and energy source, and manganese oxide as terminal electron acceptor. The culture is able to degrade a variety of substituted monoaromatic compounds like p-hydroxy-benzylalcohol, p-hydroxy-benzaldehyde, p-hydroxy-benzoate, phenol and the three isomers of cresol. Benzene, ethylbenzene, all xylenes and naphthalene were not degraded under the experimental conditions used. Based on the results of growth experiments and the detection of intermediates, it is concluded that toluene is degraded via a methyl hydroxylation. A possible side reaction can lead to the formation of cresol. The organisms in the culture look similar; motile rods, which are Gram-negative, oxidase-negative and catalase-negative. The culture was partly identified by phylogenetic analysis of cloned rDNA sequences. The phylogenetic analysis showed that at least two major groups of bacteria are present. One group of bacteria shows 70–80% similarity (based on 16S rRNA gene sequence data) with the Bacteroides-Cytophaga group, and one group consists of members of the β-subclass of the Proteobacteria.

Toluene degradation occurred in anaerobic flow-through sediment columns filled with contaminated sediment and sludge to which either amorphous or highly crystalline manganese oxide was added. An enrichment culture from these sediment columns was able to grow on toluene under strictly anaerobic conditions in the presence of manganese oxide. The oxidation of toluene was coupled to the production of CO2 and to the reduction of Mn(IV). Of the different manganese oxides tested, the rate was slowest with crystalline manganese oxide. After several transfers of the enrichment culture, its ability to degrade toluene became less and it was ultimately lost, unless sterilized Rhine river sediment was present in the medium. Direct contact between the bacteria and the manganese oxide was found to be advantageous for a rapid toluene degradation. The degradation rate could be further increased by adding organic ligands such as oxalic acid or nitrilotriacetic acid.

The transformation of 3-chlorobenzoate (3CB) and acetate at initial concentrations in the wide range of 10 nM to 16 mM was studied in batch experiments with Pseudomonas sp. strain B13. Transformation rates of 3CB at millimolar concentrations could be described by Michaelis-Menten kinetics (K(infm), 0.13 mM; V(infmax), 24 nmol (middot) mg of protein(sup-1) (middot) min(sup-1)). Experiments with nanomolar and low micromolar concentrations of 3CB indicated the possible existence of two different transformation systems for 3CB. The first transformation system operated above 1 (mu)M 3CB, with an apparent threshold concentration of 0.50 (plusmn) 0.11 (mu)M. A second transformation system operated below 1 (mu)M 3CB and showed first-order kinetics (rate constant, 0.076 liter (middot) g of protein(sup-1) (middot) min(sup-1)), with no threshold concentration in the nanomolar range. A residual substrate concentration, as has been reported for some other Pseudomonas strains, could not be detected for 3CB (detection limit, 1.0 nM) in batch incubations with Pseudomonas sp. strain B13. The addition of various concentrations of acetate as a second, easily degradable substrate neither affected the transformation kinetics of 3CB nor induced a detectable residual substrate concentration. Acetate alone also showed no residual concentration (detection limit, 0.5 nM). The results presented indicate that the concentration limits for substrate conversion obtained by extrapolation from kinetic data at higher substrate concentrations may underestimate the true conversion capacity of a microbial culture.

Reductive dechlorination of tetrachloroethene (perchloroethylene; PCE) was observed at 20 degrees C in a fixed-bed column, filled with a mixture (3:1) of anaerobic sediment from the Rhine river and anaerobic granular sludge. In the presence of lactate (1 mM) as an electron donor, 9 microM PCE was dechlorinated to ethene. Ethene was further reduced to ethane. Mass balances demonstrated an almost complete conversion (95 to 98%), with no chlorinated compounds remaining (less than 0.5 micrograms/liter). When the temperature was lowered to 10 degrees C, an adaptation of 2 weeks was necessary to obtain the same performance as at 20 degrees C. Dechlorination by column material to ethene, followed by a slow ethane production, could also be achieved in batch cultures. Ethane was not formed in the presence of bromoethanesulfonic acid, an inhibitor of methanogenesis. The high dechlorination rate (3.7 mumol.l-1.h-1), even at low temperatures and considerable PCE concentrations, together with the absence of chlorinated end products, makes reductive dechlorination an attractive method for removal of PCE in bioremediation processes.

In this paper a model is presented which can be used to simulate the behaviour of xenobiotic chemicals in soil columns with respect to their physical and chemical properties. Terms describing biological transformation of xenobiotics are also included in the model. It incorporates microbial growth following Monod kinetics and a chemotactic response of the transforming bacteria towards the xenobiotic substrate. The model appeared to yield good simulations of an experiment by Van der Meer et al. (1987) who investigated the degradation of 1,2-dichlorobenzene in soil columns inoculated with Pseudomonas sp. strain P51. The behaviour and the fate of 1,4-dichlorobenzene as found by Kuhn et al. (1985) can also be simulated using this model but their results were also adequately simulated using a simple second order model. The results generated by the model correspond to kinetic parameters obtained in other studies. It is concluded that the model is a useful tool for the investigation of the activity of bacteria degrading xenobiotics in soil columns, provided that the microbial parameters can be determined in independent experiments, and that the active microbial mass in the soil can be measured.

A newly isolated Pseudomonas species, strain P51, growing aerobically on all dichlorobenzene isomers and on 1,2,4-trichlorobenzene as sole carbon and energy sources was tested for its ability to mineralize these components also in a non-sterile soil environment. Untreated sand from the river Rhine in which none of the dichloro- and trichlorobenzenes were degraded was placed in a percolation column and inoculated with Pseudomonas sp. strain P51. The column was fed continuously with synthetic river water containing the chlorinated compounds at concentrations between 10 μg/1 and 1 mg/1. The inoculated microorganisms were able to degrade the chlorinated benzenes and survived for at least 60 days in the column. For each compound a specific threshold concentration was observed below which no further degradation took place, and which was independent of the initial concentration. These threshold were 6 ± 4 μg/1 for 1,2-dichlorobenzene, 20 ± 5 μg/1 for 1,2,4-trichlorobenzene and more than 20 μg/1 for the 1,3- and 1,4-isomers. Repeated inoculation of the column with strain P51 did not affect this minimal concentration. In noninoculated soil columns the native microbial population adapted to degrade 1,2-dichlorobenzene after a long lag phase, and reduced it from 25 μg/1 to a threshold concentration of 0.1 μg/1.

In this study, the adhesion of bacteria differing in surface hydrophobicity was investigated. Cell wall hydrophobicity was measured as the contact angle of water on a bacterial layer collected on a microfilter. The contact angles ranged from 15 to 70 degrees. This method was compared with procedures based upon adhesion to hexadecane and with the partition of cells in a polyethylene glycol-dextran two-phase system. The results obtained with these three methods agreed reasonably well. The adhesion of 16 bacterial strains was measured on sulfated polystyrene as the solid phase. These experiments showed that hydrophobic cells adhered to a greater extent than hydrophilic cells. The extent of adhesion correlated well with the measured contact angles (linear regression coefficient, 0.8).

An organism, identified as an Alcaligenes sp., was isolated from an enrichment culture in which 1,4-dichlorobenzene served as the sole carbon and energy source. During growth with 1,4-dichlorobenzene in pure culture, stoichiometric amounts of chloride were released. Growth experiments and oxygen uptake rates with other chlorinated aromatic compounds revealed a high degree of specificity of the initial dioxygenase. cis-1,2-Dihydroxycyclohexa-3,5-diene oxidoreductase and 1,2-pyrocatechase, but not 2,3-pyrocatechase, were found in cell extracts, while 3,6-dichlorocatechol and (2,5-dichloro)muconic acid could be detected as intermediates during degradation of 1,4-dichlorobenzene. It is proposed that dioxygenases are involved in the initial steps of 1,4-dichlorobenzene degradation, while ring opening proceeds via ortho cleavage.

All three isomers of trichlorobenzene were reductively dechlorinated to monochlorobenzene via dichlorobenzenes in anaerobic sediment columns. The dechlorination was specific: 1,2,3- and 1,3,5-trichlorobenzene were solely transformed to 1,3-dichlorobenzene, while 1,4-dichlorobenzene was the only product of 1,2,4-trichlorobenzene transformation. Microorganisms were responsible for the observed transformations. Since monochlorobenzene and dichlorobenzene are mineralized by bacteria in the presence of oxygen, the process of reductive dechlorination may be an important initial step to obtain complete mineralization of otherwise recalcitrant trichlorobenzenes. This is especially true for the 1,3,5-isomer, which seems to resist biodegradation in oxic environments.

The relationship between physiochemical surface parameters and adhesion of bacterial cells to negatively charged polystyrene was studied. Cell surface hydrophobicity and electrokinetic potential were determined by contact angle measurement and electrophoresis, respectively. Both parameters influence cell adhesion. The effect of the electrokinetic potential increases with decreasing hydrophobicity. Cell surface characteristics determining adhesion are influenced by growth conditions. At high growth rates, bacterial cells tend to become more hydrophobic. This fact can be of ecological significance for controlling the spread of bacteria throughout the environment.

Aliphatic and aromatic hydrocarbons are two groups of compounds that are widespread pollutants. The aerobic microbial degradation of aliphatic and aromatic hydrocarbons proceeds in general fast and has been widely studied, while the biodegradation in anoxic environments is often incomplete, proceeds at lower rates and is less characterized. Chlorate reduction is a unique process, which yields molecular oxygen upon microbial reduction in anoxic environments. This can be of practical importance, since the oxygen released can be incorporated into the anaerobically recalcitrant compounds by oxygenases to form hydroxylated derivatives, which can be further degraded easily either aerobically or anaerobically. We have found that Pseudomonas chloritidismutans AW-1T, which is a known chlorate-reducing bacterium, can combine the oxidation of n-alkanes and the reduction of chlorate. Similarly this bacterium can combine the degradation of benzoate and catechol with chlorate reduction. We studied the physiological and biochemical properties of this bacterium. With the help of proteogenomics we annotated the key proteins involved in alkane and benzoate oxidation with chlorate. Our findings suggest that oxygen released during chlorate reduction can be used to degrade the anaerobically recalcitrant compounds and chlorate reduction has a very high potential for bioremediation of anoxic soils.

Chlorinated hydrocarbons belong to the most frequently encountered contaminants in soil and groundwater. Many of them were found to be toxic and recalcitrant, which causes a potential threat to the environment. Therefore, it is of great importance that sites contaminated with chlorinated hydrocarbons are remediated.In this thesis we focus on anaerobic microbial oxidation of low-chlorinated hydrocarbons. These low-chlorinated hydrocarbons can enter soil and groundwater by direct release or by partial dechlorination of higher chlorinated hydrocarbons. Their fate strongly depends on microbial processes in soil and groundwater systems. Since many contaminated aquifers contain large volumes that are dominated by anaerobic processes it is apparent that anaerobic microorganisms play an important role in the degradation of contaminants. The tendency of low-chlorinated hydrocarbons to undergo oxidative dehalogenation increases when the number of chlorine substituents decreases. Whether low-chlorinated hydrocarbons are more likely to be degraded by oxidation than by reduction processes under anoxic conditions depends primarily on environmental conditions (e.g. redox conditions, availability of electron acceptors/donors, present microorganisms).The potential for anaerobic oxidation of different pollutants was studied by performing both up-flow soil columns and batch experiments, inoculated with contaminated soil and supplied with a mixture of different (chlorinated) hydrocarbons. They were operated in the absence of oxygen under nitrate-,iron(III)- or manganese(IV)-reducing conditions or in the absence of an externally supplied electron acceptor.It was found that 1,2-dichloroethane (DCA), 2-chloroethanol (CE), vinyl chloride (VC), monochlorobenzene (MCB) and octane could be removed in the absence of oxygen.Fromaenrichment culture that showed DCA degradation with nitrate as sole electron acceptor, Pseudomonas stutzeri stain JJ was isolated. This bacterium is the first isolate known that can grow anaerobically with CE as sole carbon and energy source and nitrate as electron acceptor. Strain JJ was however not able to grow on DCA. The degradation pathway of CE under denitrifying conditions by strain JJ seems to be the same as found in aerobic bacteria that degrade CE . According to this pathway, CE is first oxidized to chloroacetaldehyde, then chloroacetaldehyde is oxidized to chloroacetate and subsequently chloroacetate is dehalogenated to glycolate, which enters the central metabolic pathways.MCB removal was studied in a nitrate-reducing up-flow soil column. Different observations and calculationssuggeststhat MCB was oxidized in the absence of oxygen with nitrate as electron acceptor.These results underline the potential importance of anaerobic oxidation processes for the clean-up of sites polluted with low-chlorinated hydrocarbons as pointed out by others. Anaerobic oxidation pathways can be of particular importance when plumes contaminated with chlorinated compounds spread from reduced (e.g. methanogenic, sulfate-reducing) to more oxidized (e.g. iron-reducing and denitrifying) redox zones. These redox situations frequently occur at polluted sites in Western Europe.

Chlorinated ethenes are widespread soil and groundwater pollutants. Over the last 2 decades a lot of effort has been made to understand the degradation mechanisms for these pollutants. In the early eighties reduction of tetrachloroethene (PCE) was observed in anaerobic soil samples, which was shown to be mediated by microorganisms. The first microorganism able to couple the anaerobic reduction of PCE to growth in a process called halorespiration (alternative terms are chlororespiration, chloridogenesis or dehalorespiration) was isolated in 1993. Since then, about 15 bacteria able to reduce PCE metabolically have been isolated. This thesis describes research on different aspects influencing the reductive dechlorination of chlorinated ethenes by anaerobic halorespiring bacteria.A new halorespiring bacterium is described in chapter 1. This bacterium, Sulfurospirillum halorespirans PCE-M2, was isolated from a polluted soil near Rotterdam harbor. Strain PCE-M2 is a metabolically versatile bacterium able to use a variety of electron acceptors and electron donors. This new strain is closely related to Dehalospirillum multivorans, but more detailed studies indicated that strain PCE-M2 belongs to the genus Sulfurospirillum, It also appeared that Dehalospirillum multivorans had to be included in this genus. Consequently, it was reclassified to Sulfurospirillum multivorans.Members of the genus Sulfurospirillum were originally known for their sulphur, selenate and arsenate respiring properties. Therefore, we screened a number of halorespiring and related bacteria for their metal reducing properties (Chapter 2). It was shown that the reduction of metals such as ferric iron, manganese, selenate and arsenate is a common property amongst halorespiring bacteria. We also investigated the quinone reducing and oxidizing abilities. AU tested bacteria are able to reduce AQDS7 a quinone-bearing humic acid analogue. Some of the tested bacteria (Desulfitobacterium hafniense DP7, Sulfurospirillum barnesii, S. deleyianum and S. arsenophilum) are also able to oxidize AEbQDS coupled to nitrate reduction.The influence of some alternative electron acceptors on the reductive dechlorination is discussed in chapter 3. Sulfurospirillum halorespirans preferably reduces nitrate (to ammonium) and then PCE. In contrast, Sulfurospirillum multivorans reduces nitrate only to nitrite, and PCE reduction is blocked irreversibly in the presence of nitrate. In Desulfitobacterium frappieri TCEl, PCE and nitrate are reduced simultaneously in excess of electron donor. Under electron donor limitation PCE reduction was inhibited (Gerritse Bt al., AppI. Environ. Microbiol. 1999, 65, 5212-5221). The influence of nitrate on the reduction of chlorinated ethenes by halorespiring bacteria differs between species and may also depend on the availability of electron donor. Sulphate, which is not used as electron acceptor by chlorinated ethenes respiring bacteria is often found at polluted sites. We have tested the influence of sulphate on halorespiring bacteria (Chapter 3). It appeared that sulphate does not influence these microorganisms. Sulphite however, a possible electron acceptor for Desulfitobacterium species, inhibits the reduction of PCE. This inhibition may be the result of a chemical interaction between sulphite and cobalamine containing dehalogenases. We also studied the adaptation of Sulfurospirillum halorespirans PCE-M2 to different alternative electron acceptors (Chapter 3). Both nitrate and arsenate are reduced by cells pre-grown on PCE, nitrate, arsenate and selenate. This indicates that the enzymes responsible for the reduction of nitrate and arsenate are constitutiveiy present in S, halorespirans. In contrast, PCE and selenate are only reduced by cells pre-grown on PCE or selenate respectively.Halorespiring bacteria have a high affinity for hydrogen (H2). H2 may even be the most important electron donor for these organisms in natural environments. We have studied H2^thresh0ld concentrations in pure cultures of halorespiring bacteria (Chapter 4). H2-threshold values between 0.05 and 0.08 nM under PCE-reducing and nitrate-reducing conditions were measured. Furthermore, we measured H2 concentrations at a field site polluted with chlorinated ethenes. PCE and trichloroethene (TCE) reduction can occur at H2 concentrations below 1 nM. However, for the reduction of lower chlorinated ethenes a higher H2 concentration seems to be required.Accumulation of cis-l,2-dichloroethene (cis-l,2-DCE) and vinyl chloride (VC) under anaerobic conditions is often observed. The enrichment of two cultures (DCE-I and DCE-2) able to reduce VC at relative high rates is described in chapter 5. Cis-l,2-DCE is reduced at approximately 20-30 fold lower rates than VC. Our results suggest that these two enrichment cultures are able to gain energy from the reduction of lower chlorinated ethenes. When we performed these studies, no microorganisms had been isolated able to grow by the reduction of VC. However, recently He et al. (Nature. 2003, 424, 62-65) isolated Dehalococcoides strain BAVl, which is able to couple the reduction of DCE and VC to ethene to growth.Finally, the results obtained are combined with available literature data to obtain a state-of-the-art on chlorinated ethenes respiring microorganisms, the influence of alternative electron acceptors on these microorganisms and the role of H3 and H?-threshold values in halorespiration.