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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 reduction of (per)chlorate and nitrate in (per)chlorate-reducing bacteria shows similarities and differences. (Per)chlorate reductase and nitrate reductase both belong to the type II DMSO family of enzymes and have a common bis(molybdopterin guanine dinucleotide)molybdenum cofactor. There are two types of dissimilatory nitrate reductases. With respect to their localization, (per)chlorate reductase is more similar to the dissimilatory periplasmic nitrate reductase. However, the periplasmic, unlike the membrane-bound, respiratory nitrate reductase, is not able to use chlorate. Structurally, (per)chlorate reductase is more similar to respiratory nitrate reductase, since these reductases have analogous subunits encoded by analogous genes. Both periplasmic (per)chlorate reductase and membrane-bound nitrate reductase activities are induced under anoxic conditions in the presence of (per)chlorate and nitrate respectively. During microbial (per)chlorate reduction, molecular oxygen is generated. This is not the case for nitrate reduction, although an atypical reaction in nitrite reduction linked to oxygen formation has been described recently. Microbial oxygen production during reduction of oxyanions may enhance biodegradation of pollutants under anoxic conditions.

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.

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.

The biological anaerobic reductive dechlorination of beta-hexachlorocyclohexane under methanogenic conditions was tested in a number of contaminated soil samples from two locations in the Netherlands. Soils from a heavily polluted location showed rapid dechlorination of beta-hexachlorocyclohexane to benzene and chlorobenzene with lactate as electron donor. Soils from an adjacent slightly polluted location did not show substantial dechlorination of beta-hexachlorocyclohexane within 4 months. A heavily polluted sample was selected to optimise the dechlorination. All tested hexachlorocyclohexane isomers (alpha-, beta-, gamma-, and delta-), either added separately or simultaneously, were dechlorinated in this soil sample. The most rapid dechlorination was observed at a temperature of 30 degrees C. Dechlorination of beta-hexachlorocyclohexane was observed with acetate, propionate, lactate, methanol, H2, yeast extract and landfill leachate as electron donors. In a soil percolation column, packed with a selected heavily polluted soil sample, the presence of 10 mM sulphate in the influent led to simultaneous dechlorination of beta-hexachlorocyclohexane and sulphate reduction. When the column was fed with 10 mM nitrate instead of sulphate, dechlorination ceased immediately. After omitting nitrate from the influent, dechlorination activity recovered in about 1 month. Also in a separate column, the addition of nitrate from the start of the experiment did not result in dechlorination of beta-HCH. The significance of these experiments for in situ bioremediation of polluted soils is discussed.

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.

Halorespiring microorganisms are not only able to oxidize organic electron donors such as formate, acetate, pyruvate and lactate, but also H(2). Because these microorganisms have a high affinity for H(2), this may be the most important electron donor for halorespiration in the environment. We have studied the role of H(2)-threshold concentrations in pure halorespiring cultures and compared them with mixed cultures and field data. We have found H(2)-threshold values between 0.05 and 0.08 nM for Sulfurospirillum halorespirans, S. multivorans and Dehalobacter restrictus under PCE-reducing and nitrate-reducing conditions. The reduction of PCE and TCE can proceed at H(2) concentrations of below 1 nM at a polluted site. However, for the reduction of lower chlorinated ethenes a higher H(2) concentration is required. This indicates that the measured H(2) concentration in situ can be an indicator of the extent of anaerobic reductive dechlorination.

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.

The ability of granular methanogenic sludge to dechlorinate chloroethenes was investigated with unadapted sludge from an upflow anaerobic sludge blanket (UASB) reactor fed with methanol. The sludge degraded chlorinated ethenes, but the degradation rates were low. The addition of primary substrate was necessary to sustain dechlorination. The dechlorinating activity seemed to be constitutively present in the anaerobic bacteria. Usually, one chlorine atom was removed via reductive hydrogenolysis. Only trichloroethene (TCE) was converted to substantial amounts of vinylchloride (VC). 1,1-Dichloroethene (1,1DCE) was observed to be an important intermediate in the dechlorination by unadapted granular sludge, although previously this compound had not been commonly observed. Furthermore, the dechlorination of 1,1DCE was faster than the dechlorination of the other chloroethenes. Copyright ? 2001 Elsevier Science Ltd. The ability of granular methanogenic sludge to dechlorinate chloroethenes was investigated with unadapted sludge from an upflow anaerobic sludge blanket (UASB) reactor fed with methanol. The sludge degraded chlorinated ethenes, but the degradation rates were low. The addition of primary substrate was necessary to sustain dechlorination. The dechlorinating activity seemed to be constitutively present in the anaerobic bacteria. Usually, one chlorine atom was removed via reductive hydrogenolysis. Only trichloroethene (TCE) was converted to substantial amounts of vinylchloride (VC). 1,1-Dichloroethene (1,1DCE) was observed to be an important intermediate in the dechlorination by unadapted granular sludge, although previously this compound had not been commonly observed. Furthermore, the dechlorination of 1,1DCE was faster than the dechlorination of the other chloroethenes.

The current knowledge on microbial reductive dechlorination of chlorinated ethenes (CEs) and its application are discussed. Physiological studies on CEs dechlorinating microorganisms indicate that ...

Multivariate analyses of a large set of physical, chemical, and biological measurements indicated possible effects that specific surface water properties have on biotransformation rates of pesticides. In this study, we investigated the roles of Mg, Mn, and P, which were identified as relevant, discriminating environmental variables in the overall biotransformation process as it occurred under nitrifying conditions. Nematicide aldicarb, herbicides simazine and MCPA were selected as their chemical group representatives. Series of mechanically aerated incubation vessels were set up to test nutrient enrichment effects on microbial development and subsequent pesticide transformation. Accounting for alterations in system conditions, relevant (physico)chemical characteristics were monitored. An increase in total microbial population was observed in surface water samples in the presence of both aldicarb and simazine. No increase was observed when MCPA was added, which was probably cometabolized. Large phosphorus concentrations not only favored bacterial growth, but also increased the residence time of dissolved Mn which under certain conditions promoted biotransformation. Furthermore, PO4 enrichment decreases aldicarb's aerobic metabolites' concentrations. Simazine was persistent over a period of at least 80 days, except for a short period coinciding with the nitrification period in which NH dissipates and NO2 and NO are formed. Selective bacterial growth was observed on simazine's transformation product DES. Relationships of Mg/Mn concentrations to MCPA transformation rates, and of PO4/PT concentrations to aldicarb transformation rates, are presented. These relationships are assessed as environmental indicators for potential biotransformation, but only under conditions warranting development and growth of a degrading population over a prolonged period. ©1999 John Wiley & Sons, Inc. Environ Toxicol 14: 329–338, 1999

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 dechlorinating activity of a methanogenic granular sludge from a methanol-fed upflow anaerobic sludge blanket reactor was investigated with chlorinated ethanes. This unadapted methanogenic consortium degraded all chloroethanes tested. The product formation rates decreased with the number of chlorine substituents. The more highly chlorinated ethanes were also converted, although at a lower rate, in the presence of autoclaved (dead) sludge, indicating the involvement of reduced heat-stable cofactors like vitamin B12 and F430. Direct chemical dechlorination of hexa-, penta- and tetrachloroethanes was also observed in medium without sludge, although at a much lower rate. The results show the importance of cometabolic and abiotic (chemical) conversions for the transformation of chlorinated ethanes by the methanogenic consortium. The types of reaction and the products formed were correlated with the Gibbs free-energy change (ΔG(0')). Reductive hydrogenolysis and dichloroelimination were important dechlorinating mechanisms. Generally, these reactions have a higher ΔG(0')) value than dehydrochlorination reactions, which occurred less frequently during the transformation of chloroethanes by the methanogenic granular sludge.

β-Hexachlorocyclohexane (β-HCH) was microbiologically transformed under anaerobic conditions by methanogenic granular sludges from upflow anaerobic sludge blanket (UASB) reactors fed with methanol, volatile fatty acids (VFA), or sucrose as substrates. These sludges, which had not priorily been exposed to β-HCH transformed β-HCH to benzene and chlorobenzene. Usually 2-3 times as much benzene as chlorobenzene was formed. The transformation rates ranged from 0.37 to 0.46 β-HCH (g of volatile suspended solids (VSS)) -1 day -1 at 30°C. β-HCH was not transformed by autoclaved (sterile) sludge, indicating the biotic nature of the reaction. β- and also α-HCH present in contaminated soil were both found to be converted to chlorobenzene and benzene upon incubation of the soil under anaerobic conditions. Equal amounts of benzene and chlorobenzene were formed. The results show that β-HCH transforming bacteria are present in different anaerobic environments. This finding may be of importance for the application of anaerobic bioremediation on sites contaminated with HCH isomers.

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.

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.

Biotransformation is controlled by the biochemical activity of microorganisms and the mass transfer of a chemical to the microorganisms. A generic mathematical concept for bioavailability is presented taking both factors into account. The combined effect of mass transfer of a substance to the cell and the intrinsic activity of the cell using the substance as primary substrate, is quantified in a bioavailability number (Bn). The concept can easily be extended to secondary substrates. The approach has been applied to explain the observed kinetics of the biotransformation of organic compounds in soil slurries and in percolation columns. The model allowed us to predict threshold concentrations below which no biotransformation is possible. Depending on the environmental system and the chemical involved, predicted threshold concentrations span a range of 11 orders of magnitude from nanograms to grams per liter and match with published experimental data. Mass transferand not the intrinsic microbial activityis in most cases the critical factor in bioremediation.

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.

Lindane (γ-HCH) is a pesticide that has mainly been used in agriculture. Lindane and the other HCH isomers are highly chlorinated hydrocarbons. The presence of a large number of electron withdrawing chlorine groups makes some of the HCH isomers rather recalcitrant in oxic environments. Especially β-HCH is poorly degraded by aerobic bacteria. The chlorine groups make HCH isomers more accessible for an initial reductive attack, a common mechanism in anoxic environments. Among the HCH isomers, γ-HCH is degraded most easily while β-HCH is most persistent. Little is known about the diversity of the microorganisms involved in anaerobic HCH degradation. Thus far, species within the genera Clostridium and Bacillus, two Desulfovibrio species, and one species each of Desulfococcus, Desulfobacter, Citrobacter and Dehalobacter have been found to metabolize lindane and other HCH isomers. Benzene and monochlorobenzene are the end products of anaerobic degradation, while in some studies pentachlorocyclohexane, tetrachlorocyclohexene, chlorobenzenes and chlorophenols have been detected as intermediates. Enzymes and coding genes involved in the reductive dechlorination of HCH isomers are largely unknown. Recently, a metagenomic analysis has indicated the presence of numerous putative reductive dehalogenase genes in the genome of β-HCH degrading Dehalobacter sp. High-throughput omics techniques can help to explore the key players and enzymes involved in the reductive dehalogenation of lindane and other HCH isomers.

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.

During the production of the pesticide lindane (γ-hexachlorocyclohexane; γ-HCH), large quantities of byproducts, like the α-, β-, and δ-HCH isomers, were discarded at dump sites. β-HCH was found to be extremely persistent in the environment under aerobic conditions. We studied the degradation of this isomer under methanogenic conditions in a flow-through column packed with polluted sediment. β-HCH was completely removed in this system. Chlorobenzene was detected in the effluent as a product. A β-HCH transforming anaerobic enrichment culture was obtained in batch cultures by using the column material as inoculum. 6-2,3,4,5-Tetrachlorocyclohexene is proposed as an intermediate during transformation, while benzene and chlorobenzene were formed as stable end products. The enrichment culture was also able to dechlorinate α-HCH at a comparable rate and y- and 6-HCH at lower rates. Dechlorination was inhibited by the addition of vancomycin, but not by the addition of bromoethanesulfonic acid. Pasteurization inhibited dechlorination completely. This is the first detailed description of the biodegradation of β-HCH, including intermediate and end product identification, under defined anaerobic conditions.

The biotransformation of toluene, benzene and naphthalene was examined in anaerobic sediment columns. Five columns filled with a mixture of sediments were operated in the presence of bicarbonate, sulfate, iron, manganese, or nitrate as electron acceptor. The columns were continuously percolated with a mixture of the three organic compounds (individual concentrations 25–200 μM) at 20°C. Toluene was transformed readily (within 1 to 2 months) under all redox conditions tested. Benzene was recalcitrant over the test period of 375–525 days in all five columns. Naphthalene was partly transformed in the column with nitrate or manganese as electron acceptor present; the addition of benzoate had a positive effect in the column with nitrate. In the column with sulfate, the majority of the added naphthalene disappeared. No effect was observed after adding and omitting an easier degradable substrate. [14C]naphthalene was used to confirm this disappearance to be the result of degradation; two third of the naphthalene was converted to CO2.

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.

Laboratory column experiments were performed to evaluate the fate of a series of chlorinated and nonchlorinated organic contaminants in Rhine sediment and in sediment from the infiltration area of the Municipal Water Works of Amsterdam, near Zandvoort, The Netherlands. Columns were operated under aerobic, denitrifying, the methanogenic conditions. All nonchlorinated and few chlorinated compounds were aerobically transformed. Of the compounds tested under denitrifying conditions, only 1,2-dichloro-4-nitrobenzene was partially transformed. Methanogenic conditions favored the transformation of chlorinated substances by reductive dechlorination. Toluene was the only nonhalogenated compound that was transformed under methanogenic conditions. Steady-state effluent concentrations after biotransformation were at least 10 times lower than the drinking water limit of 1 {micro}g/l, except in the case of 1,2,4-trichlorobenzene which had a steady-state effluent concentration of 2.6 {micro}g/l. Steady-state effluent concentrations did not depend on the influent concentration applied. Most transformations proceeded at the same steady-state rates at a temperature of 4 C, although the process of reductive dechlorination was slower at 4 C than at 20 C. Hydrological calculations revealed that the combined action of hydrology and sorption to organic matter in the infiltration system can reduce the concentrations of 2 weeks pulses of polar and nonpolar contaminants by at leastmore » 80 and 95%, respectively. There was a good qualitative agreement between removals observed in column experiments and in the dune infiltration area.« less

Toxic aromatic pollutants, concentrated in industrial wastes and contaminated sites, can potentially be eliminated by low cost bioremediation systems. Most commonly, the goal of these treatment systems is directed at providing optimum environmental conditions for the mineralization of the pollutants by naturally occurring microflora. Electrophilic aromatic pollutants with multiple chloro, nitro and azo groups have proven to be persistent to biodegradation by aerobic bacteria. These compounds are readily reduced by anaerobic consortia to lower chlorinated aromatics or aromatic amines but are not mineralized further. The reduction increases the susceptibility of the aromatic molecule for oxygenolytic attack. Sequencing anaerobic and and aerobic biotreatment steps provide enhanced mineralization of many electrophilic aromatic pollutants. The combined activity of anaerobic and aerobic bacteria can also be obtained in a single treatment step if the bacteria are immobilized in particulate matrices (e.g. biofilm, soil aggregate, etc.). Due to the rapid uptake of oxygen by aerobes and facultative bacteria compared to the slow diffusion of oxygen, oxygen penetration into active biofilms seldom exceeds several hundred micrometers. The anaerobic microniches established inside the biofilms can be applied to the reduction of electron withdrawing functional groups in order to prepare recalcitrant aromatic compounds for further mineralization in the aerobic outer layer of the biofilm. Aside from mineralization, polyhydroxylated and chlorinated phenols as well as nitroaromatics and aromatic amines are susceptible to polymerization in aerobic environments. Consequently an alternative approach for bioremediation systems can be directed towards incorporating these aromatic pollutants into detoxified humic-like substances. The activation of aromatic pollutants for polymerization can potentially be encouraged by an anaerobic pretreatment step prior to oxidation. Anaerobic bacteria can modify aromatic pollutants by demethylating methoxy groups and reducing nitro groups. The resulting phenols and aromatic amines are readily polymerized in a subsequent aerobic step.

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.

Transformations of hexachloro-1,3-butadiene were studied in columns packed with Rhine River sediment and in batch incubations containing titanium(III) citrate and hydroxocobalamin. Columns were operated under various redox conditions. Transformation was observed in a methanogenic column at influent concentrations of 4 and 400 nmol/L but not in columns where oxygen or nitrate were fed as terminal electron acceptors. Hexachloro-1,3-butadiene was reductively dechlorinated to (E,E)-1,2,3,4-tetrachlorobutadiene (>90%) and traces of a trichloro-1,3-butadiene isomer (

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)

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)

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.

Exocellular electron transfer plays an important role in anaerobic microbial communities that degrade organic matter. Interspecies hydrogen transfer between microorganisms is the driving force for complete biodegradation in methanogenic environments. Many organic compounds are degraded by obligatory syntrophic consortia of proton-reducing acetogenic bacteria and hydrogen-consuming methanogenic archaea. Anaerobic microorganisms that use insoluble electron acceptors for growth, such as iron- and manganese-oxide as well as inert graphite electrodes in microbial fuel cells, also transfer electrons exocellularly. Soluble compounds, like humic substances, quinones, phenazines and riboflavin, can function as exocellular electron mediators enhancing this type of anaerobic respiration. However, direct electron transfer by cell-cell contact is important as well. This review addresses the mechanisms of exocellular electron transfer in anaerobic microbial communities. There are fundamental differences but also similarities between electron transfer to another microorganism or to an insoluble electron acceptor. The physical separation of the electron donor and electron acceptor metabolism allows energy conservation in compounds as methane and hydrogen or as electricity. Furthermore, this separation is essential in the donation or acceptance of electrons in some environmental technological processes, e.g. soil remediation, wastewater purification and corrosion.

The pathway of 2-chloroethanol degradation in the denitrifying Pseudomonas stutzeri strain JJ was investigated. In cell-free extracts, activities of a phenazine methosulfate (PMS)-dependent chloroethanol dehydrogenase, an NAD-dependent chloroacetaldehyde dehydrogenase, and a chloroacetate dehalogenase were detected. This suggested that the 2-chloroethanol degradation pathway in this denitrifying strain is the same as found in aerobic bacteria that degrade chloroethanol. Activity towards primary alcohols, secondary alcohols, diols, and other chlorinated alcohols could be measured in cell-free extracts with chloroethanol dehydrogenase (CE-DH) activity. PMS and phenazine ethosulfate (PES) were used as primary electron acceptors, but not NAD, NADP or ferricyanide. Cells of strain JJ cultured in a continuous culture under nitrate limitation exhibited chloroethanol dehydrogenase activity that was a 12 times higher than in cells grown in batch culture. However, under chloroethanol-limiting conditions, CE-DH activity was in the same range as in batch culture. Cells grown on ethanol did not exhibit CE-DH activity. Instead, NAD-dependent ethanol dehydrogenase (E-DH) activity and PMS-dependent E-DH activity were detected.

A bacterium that uses 2-chloroethanol as sole energy and carbon source coupled to denitrification was isolated from 1,2-dichloroethane-contaminated soil. Its 16 S rDNA sequence showed 98% similarity with the type strain of Pseudomonas stutzeri (DSM 5190) and the isolate was tentatively identified as Pseudomonas stutzeri strain JJ. Strain JJ oxidized 2-chloroethanol completely to CO(2) with NO(3)(- )or O(2) as electron acceptor, with a preference for O(2) if supplied in combination. Optimum growth on 2-chloroethanol with nitrate occurred at 30 degrees C with a mu(max) of 0.14 h(-1) and a yield of 4.4 g protein per mol 2-chloroethanol metabolized. Under aerobic conditions, the mu(max) was 0.31 h(-1). NO(2)(-) also served as electron acceptor, but reduction of Fe(OH)(3), MnO(2), SO(4)(2-), fumarate or ClO(3)(-) was not observed. Another chlorinated compound used as sole energy and carbon source under aerobic and denitrifying conditions was chloroacetate. Various different bacterial strains, including some closely related Pseudomonas stutzeri strains, were tested for their ability to grow on 2-chloroethanol as sole energy and carbon source under aerobic and denitrifying conditions, respectively. Only three strains, Pseudomonas stutzeri strain LMD 76.42, Pseudomonas putida US2 and Xanthobacter autotrophicus GJ10, grew aerobically on 2-chloroethanol. This is the first report of oxidation of 2-chloroethanol under denitrifying conditions by a pure bacterial culture.

An anaerobic bacterium, strain DP7, was isolated from human feces in mineral medium with formate and 0.02% yeast extract as energy and carbon source. This rod-shaped motile bacterium used pyruvate, lactate, formate, hydrogen, butyrate, and ethanol as electron donor for sulfite reduction. Other electron accepters such as thiosulfate, nitrate and fumarate stimulated growth in the presence of 0.02% yeast extract and formate. Acetate was the only product during fermentative growth on pyruvate. Six mol of pyruvate were fermented to 7 mol of acetate. C-13-NMR labeling experiments showed homoacetogenic C-13-CO2 incorporation into acetate. The pH and temperature optimum of fermentative growth on pyruvate was 7.4 and 37 degreesC, respectively. The growth rate under these conditions was approximately 0.10 h(-1). Strain DP7 was identified as a new strain of Desulfitobacterium frappieri on the basis of 16S rRNA sequence analysis (99% similarity) and DNA-DNA hybridization (reassociation value of 83%) with Desulfitobacterium frappieri TCE1. In contrast to described Desulfitobacterium strains, the newly isolated strain has not been isolated from a polluted environment and did not use chloroethenes or chlorophenols as electron acceptor.

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.

Desulfitobacterium strain PCE1 is able to use tetrachloroethene and chloroaromatics as terminal electron acceptors for growth. Cell extracts of Desulfitobacterium strain PCE1 grown with tetrachloroethene as electron acceptor showed no dehalogenase activity with 3-chloro-4-hydroxyphenylacetate (Cl-OH-phenylacetate) and other ortho-chlorophenolic compounds in an in vitro assay. Extracts of cells that were grown with Cl-OH-phenylacetate as electron acceptor dechlorinated tetrachloroethene at 10% of the dechlorination rate of Cl-OH-phenylacetate. In both cell extracts dechlorination was inhibited by the addition of 1-iodopropane and dinitrogen oxide, inhibitors of cobalamin-containing enzymes. The enzymes responsible for tetrachloroethene and Cl-OH-phenylacetate dechlorination were partially purified. A 100-fold enriched fraction of chlorophenol reductive dehalogenase was obtained that mainly contained a protein with a subunit size of 48 kDa. The characteristics of this enzyme are similar to that of the chlorophenol reductive dehalogenase of D. dehalogenans. After partial purification of the tetrachloroethene reductive dehalogenase, a fraction was obtained that also contained a 48-kDa protein, but the N-terminal sequence showed no similarity with that of the chlorophenol reductive dehalogenase sequence or with the N-terminal amino acid sequence of tetra- and trichloroethene reductive dehalogenase of Desulfitobacterium strain TCE1. These results provide strong evidence that two different enzymes are responsible for tetrachloroethene and chlorophenol dechlorination in Desulfitobacterium strain PCE1. Furthermore, the characterization of partially purified tetrachloroethene reductive dehalogenase indicated that this enzyme is a novel type of reductive dehalogenase.

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.

Introduction The binding of hydrophobic organic compounds (HOC) on or into the soil matrix may reduce the potential for bioremediation. However, little is known about the mechanisms involved and the factors influencing ad- and desorption kinetics. Binding of organic pollutants shows a strong correlation with the soil organic matter fraction. In this study, humic acids act as model compound for the soil organic matter and pentachlorobenzene (QCB) is a model compound for HOC. Desorption kinetics of loaded humic acids have also been measured after flocculation by metal ions.

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.

Microbial transformation of chlorinated organic contaminants in columns, packed with sediment from a dune infiltration area and from River Rhine, has been investigated under different redox conditions. Low redox potentials favour reductive dechlorination of chlorinated ethylenes and benzenes to lower or non-chlorinated compounds. Under aerobic conditions transformation of 1,2- and 1,4 dichlorobenzene, but not of 1,3-dichlorobenzene took place. Biotransformation of chlorinated organic substances under denitrifying conditions was hardly observed. Only a slight removal of 1,2dichloro,4-nitrobenzene could be detected. Results indicate that biotransformations may also occur under conditions which approach a field situation (lowered temperature, presence of extra carbon source).

Concentrated cell suspensions of methanogenic bacteria reductively dechlorinated 1,2-dichloroethane via two reaction-mechanisms: a dihalo-elimination yielding ethylene and two hydrogenolysis reactions yielding chloroethane and ethane, consecutively. The transformation of chloroethane to ethane was inhibited by 1,2-dichloroethane. Stimulation of methanogenesis caused an increase in the amount of dechlorination products formed, whereas the opposite was found when methane formation was inhibited. Cells of Methanosarcina barkeri grown on H2/CO2 converted 1,2-dichloroethane and chloroethane at higher rates than acetate or methanol grown cells.