Jan Gerritse

Desulfitobacterium sp. strain PCE1, an anaerobic bacterium that can grow by reductive dechlorination of tetrachloroethene or ortho-chlorinated phenols

Abstract A strictly anaerobic bacterium, strain PCE1, was isolated from a tetrachloroethene-dechlorinating enrichment culture. Cells of the bacterium were motile curved rods, with approximately four lateral flagella. They possessed a gram-positive type of cell wall and contained cytochrome c. Optimum growth occurred at pH 7.2–7.8 and 34–38° C. The organism grew with l-lactate, pyruvate, butyrate, formate, succinate, or ethanol as electron donors, using either tetrachloroethene, 2-chlorophenol, 2,4,6-trichlorophenol, 3-chloro-4-hydroxy-phenylacetate, sulfite, thiosulfate, or fumarate as electron acceptors. Strain PCE1 also grew fermentatively with pyruvate as the sole substrate. l-Lactate and pyruvate were oxidized to acetate. Tetrachloroethene was reductively dechlorinated to trichloroethene and small amounts (< 5%) of cis-1,2-dichloroethene and trans-1,2-dichloroethene. Chlorinated phenolic compounds were dechlorinated specifically at the ortho-position. On the basis of 16S rRNA sequence analysis, the organism was identified as a species within the genus Desulfitobacterium, which until now only contained the chlorophenol-dechlorinating bacterium, Desulfitobacterium dehalogenans.

Isolation and Characterization of Alicycliphilus denitrificans Strain BC, Which Grows on Benzene with Chlorate as the Electron Acceptor

ABSTRACT A bacterium, strain BC, was isolated from a benzene-degrading chlorate-reducing enrichment culture. Strain BC degrades benzene in conjunction with chlorate reduction. Cells of strain BC are short rods that are 0.6 μm wide and 1 to 2 μm long, are motile, and stain gram negative. Strain BC grows on benzene and some other aromatic compounds with oxygen or in the absence of oxygen with chlorate as the electron acceptor. Strain BC is a denitrifying bacterium, but it is not able to grow on benzene with nitrate. The closest cultured relative is Alicycliphilus denitrificans type strain K601, a cyclohexanol-degrading nitrate-reducing betaproteobacterium. Chlorate reductase (0.4 U/mg protein) and chlorite dismutase (5.7 U/mg protein) activities in cell extracts of strain BC were determined. Gene sequences encoding a known chlorite dismutase (cld) were not detected in strain BC by using the PCR primers described in previous studies. As physiological and biochemical data indicated that there was oxygenation of benzene during growth with chlorate, a strategy was developed to detect genes encoding monooxygenase and dioxygenase enzymes potentially involved in benzene degradation in strain BC. Using primer sets designed to amplify members of distinct evolutionary branches in the catabolic families involved in benzene biodegradation, two oxygenase genes putatively encoding the enzymes performing the initial successive monooxygenations (BC-BMOa) and the cleavage of catechol (BC-C23O) were detected. Our findings suggest that oxygen formed by dismutation of chlorite can be used to attack organic molecules by means of oxygenases, as exemplified with benzene. Thus, aerobic pathways can be employed under conditions in which no external oxygen is supplied.

Correlation of Dehalococcoides 16S rRNA and Chloroethene-Reductive Dehalogenase Genes with Geochemical Conditions in Chloroethene-Contaminated Groundwater

ABSTRACT Quantitative analysis of genes that code for Dehalococcoides 16S rRNA and chloroethene-reductive dehalogenases TceA, VcrA, and BvcA was done on groundwater sampled from 150 monitoring wells spread over 11 chlorinated ethene polluted European locations. Redundancy analysis was used to relate molecular data to geochemical conditions. Dehalococcoides 16S rRNA- and vinyl chloride (VC)-reductase genes were present at all tested locations in concentrations up to 106 gene copies per ml of groundwater. However, differences between and also within locations were observed. Variation in Dehalococcoides 16S rRNA gene copy numbers were most strongly correlated to dissolved organic carbon concentration in groundwater and to conditions appropriate for biodegradation of chlorinated ethenes (U.S. Environmental Protection Agency score). In contrast, vcrA gene copy numbers correlated most significantly to VC and chlorinated ethene concentrations. Interestingly, bvcA and especially tceA were more correlated with oxidizing conditions. In groundwater microcosms, dechlorination of 1 mM VC was correlated to an increase of vcrA and/or bvcA gene copies by 2 to 4 orders of magnitude. Interestingly, in 34% of the monitoring wells and in 40% of the active microcosms, the amount of individual VC-reductase gene copies exceeded that of Dehalococcoides 16S rRNA gene copies. It is concluded that the geographical distribution of the genes was not homogeneous, depending on the geochemical conditions, whereby tceA and bvcA correlated to more oxidized conditions than Dehalococcoides 16S rRNA and vcrA. Because the variation in VC-reductase gene numbers was not directly correlated to variation in Dehalococcoides spp., VC-reductase genes are better monitoring parameters for VC dechlorination capacity than Dehalococcoides spp.

COMPLETE DEGRADATION OF TETRACHLOROETHENE BY COMBINING ANAEROBIC DECHLORINATING AND AEROBIC METHANOTROPHIC ENRICHMENT CULTURES

Degradation of tetrachloroethene (perchloroethylene, PCE) was investigated by combining the metabolic abilities of anaerobic bacteria, capable of reductive dechlorination of PCE, with those of aerobic methanotrophic bacteria, capable of co-metabolic degradation of the less-chlorinated ethenes formed by reductive dechlorination of PCE. Anaerobic communities reductively dechlorinating PCE, trichloroethene (TCE) and dichloroethenes were enriched from various sources. The maximum rates of dechlorination observed for various chloroethenes in these batch enrichments were: PCE to TCE (341 μmol 1−1 day−1), TCE tocis-dichloroethene (159 μmol 1−1 day−1),cis-dichloroethene ethene to chloroethene (99 μmol 1−1 day−1) andtrans-dichloroethene to chloroethene (22 μmol 1−1 day−1). A mixture of these enrichments was inoculated into an anoxic fixed-bed upflow column. In this colum PCE was converted mainly intocis-1,2-dichloroethene, small amounts of TCE and chloroethene, and chloride. Enrichments of aerobic methanotrophic bacteria were grown in an oxic fixed-bed downflow column. Less-chlorinated ethenes, formed in the anoxic column, were further metabolized in this oxic methanotrophic column. On the basis of analysis of chloride production and the disappearance of chlorinated ethenes it was demonstrated that complete degradation of PCE was possible by combining these two columns. Operation of the two-column system under various process conditions indicated that the sensitivity of the methanotrophic bacteria to chlorinated intermediates respresented the bottle-neck in the sequential anoxic/oxic degradation process of PCE.

Anaerobic benzene degradation under denitrifying conditions: Peptococcaceae as dominant benzene degraders and evidence for a syntrophic process

An anaerobic microbial community was enriched in a chemostat that was operated for more than 8 years with benzene and nitrate as electron acceptor. The coexistence of multiple species in the chemostat and the presence of a biofilm, led to the hypothesis that benzene-degrading species coexist in a syntrophic interaction, and that benzene can be degraded in syntrophy by consortia with various electron acceptors in the same culture. The benzene-degrading microorganisms were identified by DNA-stable isotope probing with [U-(13) C]-labelled benzene, and the effect of different electron donors and acceptors on benzene degradation was investigated. The degradation rate constant of benzene with nitrate (0.7 day(-1) ) was higher than reported previously. In the absence of nitrate, the microbial community was able to use sulfate, chlorate or ferric iron as electron acceptor. Bacteria belonging to the Peptococcaceae were identified as dominant benzene consumers, but also those related to Rhodocyclaceae and Burkholderiaceae were found to be associated with the anaerobic benzene degradation process. The benzene degradation activity in the chemostat was associated with microbial growth in biofilms. This, together with the inhibiting effect of hydrogen and the ability to degrade benzene with different electron acceptors, suggests that benzene was degraded via a syntrophic processAn anaerobic microbial community was enriched in a chemostat that was operated for more than 8 years with benzene and nitrate as electron acceptor. The coexistence of multiple species in the chemostat and the presence of a biofilm, led to the hypothesis that benzene-degrading species coexist in a syntrophic interaction, and that benzene can be degraded in syntrophy by consortia with various electron acceptors in the same culture. The benzene-degrading microorganisms were identified by DNA-stable isotope probing with [U-(13) C]-labelled benzene, and the effect of different electron donors and acceptors on benzene degradation was investigated. The degradation rate constant of benzene with nitrate (0.7 day(-1) ) was higher than reported previously. In the absence of nitrate, the microbial community was able to use sulfate, chlorate or ferric iron as electron acceptor. Bacteria belonging to the Peptococcaceae were identified as dominant benzene consumers, but also those related to Rhodocyclaceae and Burkholderiaceae were found to be associated with the anaerobic benzene degradation process. The benzene degradation activity in the chemostat was associated with microbial growth in biofilms. This, together with the inhibiting effect of hydrogen and the ability to degrade benzene with different electron acceptors, suggests that benzene was degraded via a syntrophic process.

Properties of a trichlorodibenzo‐p‐dioxin‐dechlorinating mixed culture with a Dehalococcoides as putative dechlorinating species

An anaerobic mixed culture enriched over 16 transfers (1/10) from Saale river sediment reductively dehalogenated 1,2,4- and 1,2,3-trichlorodibenzo-p-dioxin (TrCDD) to di- and monochlorinated congeners in the presence of pyruvate (or lactate) and fumarate as cosubstrates. Besides TrCDD, tetrachloroethene and 1,2,3-trichlorobenzene were dechlorinated. Dioxin dehalogenation was sensitive to pasteurization, but was not remarkably influenced by inhibitors of methanogens, sulfate-reducing bacteria or Gram-positive bacteria. The rate of 1,3-dichlorodibenzo-p-dioxin formation increased with rising initial concentrations of 1,2,4-TrCDD (1-250 microM) from 0.05 to 5.4 micromol l(-1) day(-1). Two isolates, belonging to Sulfurospirillum and Trichococcus, did not show reductive dehalogenation. 16S rDNA-targeted methods further revealed the presence of Acetobacterium, Desulfitobacterium, Desulfuromonas and Dehalococcoides. Nested polymerase chain reaction (PCR) indicated the presence of Dehalococcoides in highest most probable number (MPN) dilutions that were positive for dioxin dechlorination.

Two-membered mixed cultures of methanogenic and aerobic bacteria in O2-limited chemostats

SUMMARY: Three different co-cultures composed of a methanogenic and a strictly aerobic bacterium were grown under O2-limitation in continuous cultures. The combinations used were (1) Methanobacterium formicicum with the aerobic heterotroph Comamonas testosteroni; (2) M. formicicum with a methanotrophic Methylocystis species; and (3) Methanosavcina barkeri with C. testosteroni. Although true steady-states were not obtained, growth and metabolic activity of the methanogenic and aerobic organisms occurred during O2-limited growth of these mixed cultures over extended periods of time. Co-cultures with C. testosteroni were considerably more stable than those with Methylocystis. Co-cultures with M. barkeri were less O2-sensitive than those with M. formicicum. C. testosteroni exhibited a higher O2-affinity than Methylocystis, resulting in a lower dissolved oxygen tension and a superior protection of the methanogenic bacteria against O2-poisoning than in mixed cultures with Methylocystis. The dissolved O2-concentrations in the mixed cultures were below the detection limit of the O2-probes used (0.2 μM). Calculations based on growth properties of pure cultures of C. testosteroni, M. barkeri and M. formicicum suggested that the dissolved O2-concentrations in the mixed cultures, as well as the O2-inhibition constants (apparent K 1 o2) of the methanogens were in the nanomolar range.

Degradation of 1,2-dichloroethane by microbial communities from river sediment at various redox conditions

Insight into the pathways of biodegradation and external factors controlling their activity is essential in adequate environmental risk assessment of chlorinated aliphatic hydrocarbon pollution. This study focuses on biodegradation of 1,2-dichloroethane (1,2-DCA) in microcosms containing sediment sourced from the European rivers Ebro, Elbe and Danube. Biodegradation was studied under different redox conditions. Reductive dechlorination of 1,2-DCA was observed with Ebro and Danube sediment with chloroethane, or ethene, respectively, as the major dechlorination products. Different reductively dehalogenating micro-organisms (Dehalococcoides spp., Dehalobacter spp., Desulfitobacterium spp. and Sulfurospirillum spp.) were detected by 16S ribosomal RNA gene-targeted PCR and sequence analyses of 16S rRNA gene clone libraries showed that only 2-5 bacterial orders were represented in the microcosms. With Ebro and Danube sediment, indications for anaerobic oxidation of 1,2-DCA were obtained under denitrifying or iron-reducing conditions. No biodegradation of 1,2-DCA was observed in microcosms with Ebro sediment under the different tested redox conditions. This research shows that 1,2-DCA biodegradation capacity was present in different river sediments, but not in the water phase of the river systems and that biodegradation potential with associated microbial communities in river sediments varies with the geochemical properties of the sediments.

Complete degradation of tetrachloroethene in coupled anoxic and oxic chemostats.

Abstract Anaerobic tetrachloroethene(C2Cl4)-dechlorinating bacteria were enriched in slurries from chloroethene-contaminated soil. With methanol as electron donor, C2Cl4 and trichloroethene (C2HCl3) were reductively dechlorinated to cis-1,2-dichloroethene (cis-C2H2Cl2), whereas, with l-lactate or formate, complete dechlorination of C2Cl4 via C2HCl3, cis-C2H2Cl2 and chloroethene (C2H3Cl) to ethene was obtained. In oxic soil slurries with methane as a substrate, complete co-metabolic degradation of cis-C2H2Cl2 was obtained, whereas C2HCl3 was partially degraded. With toluene or phenol both of the above were readily co-metabolized. Complete degradation of C2Cl4 was obtained in sequentially coupled anoxic and oxic chemostats, which were inoculated with the slurry enrichments. Apparent steady states were obtained at various dilution rates (0.02–0.4 h−1) and influent C2Cl4-concentrations (100–1000 μM). In anoxic chemostats with a mixture␣of␣formate and glucose as the carbon and electron source, C2Cl4 was transformed at high rates (above␣140 μmol l−1 h−1, corresponding to 145 nmol Cl− min−1 mg protein−1) into cis-C2H2Cl2 and C2H3Cl. Reductive dechlorination was not affected by addition of 5 mM sulphate, but strongly inhibited after addition of 5 mM nitrate. Our results (high specific dechlorination rates and loss of dechlorination capacity in the absence of C2Cl4) suggest that C2Cl4-dechlorination in the anoxic chemostat was catalysed by specialized dechlorinating bacteria. The partially dechlorinated intermediates, cis-C2H2Cl2 and C2H3Cl, were further degraded by aerobic phenol-metabolizing bacteria. The maximum capacity for chloroethene (the sum of tri-, di- and monochloro derivatives removed) degradation in the oxic chemostat was 95 μmol l−1 h−1 (20 nmol min−1 mg protein−1), and that of the combined anoxic → oxic reactor system was 43.4 μmol l−1 h−1. This is significantly higher than reported thus far.

Genome Sequences of Alicycliphilus denitrificans Strains BC and K601T

Alicycliphilus denitrificans strain BC and A. denitrificans strain K601(T) degrade cyclic hydrocarbons. These strains have been isolated from a mixture of wastewater treatment plant material and benzene-polluted soil and from a wastewater treatment plant, respectively, suggesting their role in bioremediation of soil and water. Although the strains are phylogenetically closely related, there are some clear physiological differences. The hydrocarbon cyclohexanol, for example, can be degraded by strain K601(T) but not by strain BC. Furthermore, both strains can use nitrate and oxygen as an electron acceptor, but only strain BC can use chlorate as electron acceptor. To better understand the nitrate and chlorate reduction mechanisms coupled to the oxidation of cyclic compounds, the genomes of A. denitrificans strains BC and K601(T) were sequenced. Here, we report the complete genome sequences of A. denitrificans strains BC and K601(T).