Retrospective on microbial transformations of halogenated organics.

Prior to the 1960s, knowledge of biological transformations of highly halogenated aliphatic compounds was limited, except in mammalian organisms where enzymatic transformations occurred to rid the body of ingested harmful chemicals. Limited abiotic transformation of such compounds had also been observed, with half-lives varying from days to centuries. Commonly believed was that aerobic transformation might occur by cometabolism rather than to conserve energy for respiration, while anaerobic transformations were in general thought not to occur. However, in the late 1960s anaerobic transformation of chlorinated pesticides was noted, and then in the early 1980s, partial microbial dehalogenation of chlorinated solvents such as tetrachlorethene, trichloroethene, trichlorethane, and carbon tetrachloride was also found to occur. With only partial dechlorination, complete detoxification was not achieved. And at the time, dehalogenation reactions were not believed to yield energy for growth to the degrading microorganisms. However, in the 1990s bacteria began to be found that obtain energy from anaerobic transformations, often enabling complete dechlorination and detoxification. Since then such ability has been found among several bacterial species, many of which use molecular hydrogen as a donor substrate and halogenated organics as electron acceptors, thus conserving energy through reductive dehalogenation. Growth of knowledge in this field has grown rapidly since the 1960s. Broad usages of such microorganisms are now underway to rid contaminated groundwater of hazardous halogenated chemicals.

MicroNiche: an R package for assessing microbial niche breadth and overlap from amplicon sequencing data.

Niche is a fundamental concept in ecology. It integrates the sum of biotic and abiotic environmental requirements that determines a taxon's distribution. Microbiologists currently lack quantitative approaches to address niche-related hypotheses. We tested four approaches for the quantification of niche breadth and overlap of taxa in amplicon sequencing datasets, with the goal of determining generalists, specialists and environmental-dependent distributions of community members. We applied these indices to in silico training datasets first, and then to real human gut and desert biological soil crust (biocrust) case studies, assessing the agreement of the indices with previous findings. Implementation of each approach successfully identified a priori conditions within in silico training data, and we found that by including a limit of quantification based on species rank, one could identify taxa falsely classified as specialists because of their low, sparse counts. Analysis of the human gut study offered quantitative support for Bacilli, Gammaproteobacteria and Fusobacteria specialists enriched after bariatric surgery. We could quantitatively characterise differential niche distributions of cyanobacterial taxa with respect to precipitation gradients in biocrusts. We conclude that these approaches, made publicly available as an R package (MicroNiche), represent useful tools to assess microbial environment-taxon and taxon-taxon relationships in a quantitative manner.

Bioaugmentation as a soil bioremediation approach.

The debate over the efficacy of bioaugmentation rages on, with research continuing to demonstrate that its advantages for soil bioremediation are difficult to predict; however, when it works, the results are often very encouraging. The difficulties arise from, among others, the diversity of the microorganisms used, environmental heterogeneity, and variations in the influence of critical parameters (e.g. humidity, microbial predation and "bioavailability') which, unfortunately, are not even always identified.

Dechlorination of 2,3,5,6-tetrachlorobiphenyl by a phototrophic enrichment culture.

A phototrophic enrichment culture, using acetate as carbon source, reductively dechlorinated 2,3,5,6-tetrachlorobiphenyl. ortho chlorines were removed preferentially over meta chlorines. Tri- and dichlorobiphenyls were the major products. During 14 months incubation, chlorine was removed from 58% of the target molecules; 19% of the total chlorines were removed. Dechlorination did not occur in a control culture incubated in the dark.

Effects of electron acceptors and donors on transformation of tetrachloromethane by Shewanella putrefaciens MR-1.

Transformation of chlorinated aliphatic compounds was examined in Shewanella putrefaciens strain MR-1, an obligately respiring facultative anaerobe. Under anaerobic conditions, MR-1 has been shown to transform tetrachloromethane to trichloromethane (24%), CO2 (7%), cell-bound material (50%) and unidentified nonvolatile products (4%). The highest rate and extent of transformation were observed with MR-1 cells grown under iron(III)-respiring conditions. Lactate, formate and hydrogen were the most effective electron donors. Tetrachloromethane was not degraded in the presence of oxygen. Transformation of other chlorinated methanes and ethenes was not observed.

Selective occurrence of Rhizobiales in frost flowers on the surface of young sea ice near Barrow, Alaska and distribution in the polar marine rare biosphere.

Frost flowers are highly saline ice structures that grow on the surface of young sea ice, a spatially extensive environment of increasing importance in the Arctic Ocean. In a previous study, we reported organic components of frost flowers in the form of elevated levels of bacteria and exopolymers relative to underlying ice. Here, DNA was extracted from frost flowers and young sea ice, collected in springtime from a frozen lead offshore of Barrow, Alaska, to identify bacteria in these understudied environments. Evaluation of the distribution of 16S rRNA genes via four methods (microarray analysis, T-RFLP, clone library and shotgun metagenomic sequencing) indicated distinctive bacterial assemblages between the two environments, with frost flowers appearing to select for Rhizobiales. A phylogenetic placement approach, used to evaluate the distribution of similar Rhizobiales sequences in other polar marine studies, indicated that some of the observed strains represent widely distributed members of the marine rare biosphere in both the Arctic and Antarctic.

Comparison of substrate utilization and growth kinetics between immobilized and suspended Pseudomonas cells.

A methodology is described for measurement if immobilized and suspended cell growth and substrate utilization kinetics parameters. Substrate utilization and growth kinetics were compared between immobilized and suspended cells for toluene degrading Pseudomonas strains K3-2 and 2,4-dichlorophenoxyacetic acid (2,4-D) degrading strain DBO131(pR0101), respectively. Kinetic parameters were estimated using nonlinear parameter estimation methods and compared between the immobilized and suspended Pseudomonas cells to determine the effect of immobilization on cellular growth and substrate utilization. Factors influencing the experimental design included calculated oxygen flux rates, primary carbon substrate flux rates, and shear stresses on the immobilize cell. Statistical interpretation of the cellular reaction rate parameters indicates that only the growth kinetics of the toluene system were significantly altered upon immobilization. Substrate utilization kinetics remained unchanged upon immobilization. The substrate growth associated half-saturation constant (K(g)) for the toluene system increased by 30-fold and the maximum specific growth rate (micro(max)) decreased by 2-fold upon immobilization. Implication of these results for experimental determination of cellular kinetic parameters and for immobilization cell bioreactors design are discussed.

Identification of the proton source for the microbial reductive dechlorination of 2,3,4,5,6-pentachlorobiphenyl.

Microbially mediated reductive dechlorination of polychlorinated biphenyls (PCBs) in anaerobic sediments has been observed during laboratory experiments. Reductive dechlorination is a two-electron transfer reaction which involves the release of chlorine as a chloride ion and its replacement on the aromatic ring by hydrogen. The exact mechanism of the electron transfer for PCBs is unknown; however, this work shows that the source of the hydrogen atom is the proton (H) from water.

Incorporation of Oxygen from Water into Toluene and Benzene during Anaerobic Fermentative Transformation.

Toluene and benzene were anaerobically transformed and eventually mineralized in mixed methanogenic cultures. However, the source of oxygen for the initial oxidation step had been unknown, owing to the presence of both methanol and water. No exogenous electron acceptors other than carbon dioxide, toluene, and benzene were present in the defined mineral medium. Through the use of O-labeled water, the oxygen incorporated into the monoaromatic compounds was shown to come from water. The cresol from the toluene and the phenol from the benzene contained up to 8% O label after incubation in 9% O-labeled medium. Gas chromatography-mass spectrometry was used to detect the O-labeled aromatic metabolites.

Complete degradation of polychlorinated hydrocarbons by a two-stage biofilm reactor.

A two-stage anaerobic-aerobic biofilm reactor successfully degraded a mixture of chlorinated organic compounds to water-soluble metabolic intermediates and carbon dioxide. Reductive dechlorination of hexachlorobenzene (HCB), tetrachloroethylene (PCE), and chloroform (CF) occurred on all tested primary carbon sources such as glucose, methanol, and acetate. However, the extent of dechlorination was maximum when the anaerobic biofilm column was fed acetate as a primary carbon source. HCB, PCE, and CF were dechlorinated to the levels of tri- and dichlorinated products (99, 80, and 32%, respectively) with acetate in the feed. This is important, since these less-chlorinated compounds can be metabolized by the aerobic biofilm. The effluent from the anaerobic biofilm column was fed directly into the aerobic column. After both columns, the total amount transformed into nonvolatile intermediates and carbon dioxide was 94, 96, and 83% for [14C]HCB, [14C]trichloroethylene, and [14C]CF, respectively. This research shows the potential application of this novel two-stage bioreactor system for treating groundwaters and industrial effluents composed of highly chlorinated aliphatic and aromatic hydrocarbons.

Substrate interactions of benzene, toluene, and para-xylene during microbial degradation by pure cultures and mixed culture aquifer slurries.

Benzene, toluene, and p-xylene (BTX) were degraded by indigenous mixed cultures in sandy aquifer material and by two pure cultures isolated from the same site. Although BTX compounds have a similar chemical structure, the fate of individual BTX compounds differed when the compounds were fed to each pure culture and mixed culture aquifer slurries. The identification of substrate interactions aided the understanding of this behavior. Beneficial substrate interactions included enhanced degradation of benzene and p-xylene by the presence of toluene in Pseudomonas sp. strain CFS-215 incubations, as well as benzene-dependent degradation of toluene and p-xylene by Arthrobacter sp. strain HCB. Detrimental substrate interactions included retardation in benzene and toluene degradation by the presence of p-xylene in both aquifer slurries and Pseudomonas incubations. The catabolic diversity of microbes in the environment precludes generalizations about the capacity of individual BTX compounds to enhance or inhibit the degradation of other BTX compounds.

Biotransformation of tetrachloroethylene to trichloroethylene, dichloroethylene, vinyl chloride, and carbon dioxide under methanogenic conditions.

Tetrachloroethylene (PCE) and trichloroethylene (TCE), common industrial solvents, are among the most frequent contaminants found in groundwater supplies. Due to the potential toxicity and carcinogenicity of chlorinated ethylenes, knowledge about their transformation potential is important in evaluating their environmental fate. The results of this study confirm that PCE can be transformed by reductive dehalogenation to TCE, dichloroethylene, and vinyl chloride (VC) under anaerobic conditions. In addition, [14C]PCE was at least partially mineralized to CO2. Mineralization of 24% of the PCE occurred in a continuous-flow fixed-film methanogenic column with a liquid detention time of 4 days. TCE was the major intermediate formed, but traces of dichloroethylene isomers and VC were also found. In other column studies under a different set of methanogenic conditions, nearly quantitative conversion of PCE to VC was found. These studies clearly demonstrate that TCE and VC are major intermediates in PCE biotransformation under anaerobic conditions and suggest that potential exists for the complete mineralization of PCE to CO2 in soil and aquifer systems and in biological treatment processes.

Transformation of toluene and benzene by mixed methanogenic cultures.

The aromatic hydrocarbons toluene and benzene were anaerobically transformed by mixed methanogenic cultures derived from ferulic acid-degrading sewage sludge enrichments. In most experiments, toluene or benzene was the only semicontinuously supplied carbon and energy source in the defined mineral medium. No exogenous electron acceptors other than CO2 were present. The cultures were fed 1.5 to 30 mM unlabeled or 14C-labeled aromatic substrates (ring-labeled toluene and benzene or methyl-labeled toluene). Gas production from unlabeled substrates and 14C activity distribution in products from the labeled substrates were monitored over a period of 60 days. At least 50% of the substrates were converted to CO2 and methane (greater than 60%). A high percentage of 14CO2 was recovered from the methyl group-labeled toluene, suggesting nearly complete conversion of the methyl group to CO2 and not to methane. However, a low percentage of 14CO2 was produced from ring-labeled toluene or from benzene, indicating incomplete conversion of the ring carbon to CO2. Anaerobic transformation pathways for unlabeled toluene and benzene were studied with the help of gas chromatography-mass spectrometry. The intermediates detected are consistent with both toluene and benzene degradation via initial oxidation by ring hydroxylation or methyl oxidation (toluene), which would result in the production of phenol, cresols, or aromatic alcohol. Additional reactions, such as demethylation and ring reduction, are also possible. Tentative transformation sequences based upon the intermediates detected are discussed.

Effects of organic substrates on dechlorination of aroclor 1242 in anaerobic sediments.

The effects of different organic substrates on the abilities of anaerobic sediment enrichments to reductively dechlorinate polychlorinated biphenyls (PCBs) were studied. Sediments collected from a site previously contaminated with PCBs were dosed with additional PCBs (Aroclor 1242; approximately 300 ppm [300 mug/g], sediment dry weight) and incubated anaerobically with acetate, acetone, methanol, or glucose. The pattern of dechlorination was similar for each substrate-fed batch; however, the extents and rates of dechlorination were different. Significant dechlorination over time was observed, with the relative rates and extents of dechlorination being greatest for methanol-, glucose-, and acetone-fed batches and least for acetate-fed batches. Dechlorination occurred primarily on the meta- and para- positions of the highly chlorinated congeners, resulting in the accumulation of less-chlorinated, primarily ortho-substituted products. No significant dechlorination was observed in incubation batches receiving no additional organic substrate, even though identical inorganic nutrients were added to all incubation batches. In addition, dechlorination was not observed in autoclaved controls that received substrate and nutrients.

Development of pure culture biofilms of P. putida on solid supports.

Pseudomonas putida biofilms were developed on and biofilm accumulation rate data were obtained for the following two classes of support materials: charged surfaces and noncharged hydrophobic and hydrophilic surfaces. The effects of surface roughness and porosity on the rate of microbial attachment were also examined. Materials bearing a net positive or negative surface charge supported the greatest biofilm accumulation and the highest biofilm accumulation rate. Uncharged hydrophobic materials achieved the next greatest biofilm accumulation, averaging approximately 50% of the total biomass which was accumulated on the charged surface materials after 16 days. Uncharged hydrophilic materials supported very little biofilm development. In general, biofilm accumulation increased with decreased surface roughness. The effect of pore size on biofilm accumulation was not conclusive.The biofilm accumulation kinetics showed an exponential accumulation rate for the charged surfaces and an approximately linear accumulation rate for the hydrophobic materials. This difference in accumulation kinetics is consistent with proposed differences in the physicochemical mechanism governing attachment to these two types of surface materials.

Rhodanobacter lindaniclasticus gen. nov., sp. nov., a lindane-degrading bacterium.

Lindane-degrading activity under aerobic conditions has been observed in two bacterial strains: UT26, phenotypically identified as Sphingomonas paucimobilis, and a new single unidentified isolate named RP5557T. The rrs (16S rDNA) sequences for both strains and the phenotypic characteristics for the unidentified isolate RP5557T were determined. RP5557T does not have high identity (less than 90% in all cases) with any sequence in the GenBank or RDP databases. A phylogenetic analysis based on rrs sequences indicated that RP5557T belongs to the gamma-Proteobacteria in a coherent phylum that includes the genera Xanthomonas and Xylella (100% bootstrap), whereas UT26 is clearly separate from the Xanthomonas cluster. Based on the phylogenetic analyses and on the phenotypic characteristics, a new genus, Rhodanobacter, containing a single species, Rhodanobacter lindaniclasticus, is proposed for strain RP5557T (= LMG 18385T), which becomes the type strain.

Opportunistic colonization of Ralstonia solanacearum-infected plants by Acinetobacter sp. and its natural competence development.

The behavior of the soil bacterium Acinetobacter sp. BD413 was monitored in Ralstonia solanacearum-infected and non-infected tomato plants after direct injection into the stem or natural infection by roots. In healthy plants, Acinetobacter sp. BD413 failed to colonize plant tissue. In plants infected simultaneously by the pathogen R. solanacearum,the Acinetobacter population increased linearly to about 3.1 x 10(7) cells per gram plant material and was maintained at a high level until the death of the plant. Moreover, Acinetobacter sp. BD413 was found to develop a competent state when multiplying in planta, indicating it could possibly be transformed by bacterial or plant DNA.

Kinetics of aerobic biodegradation of benzene and toluene in sandy aquifer material.

Monod's equation adequately described aerobic biodegradation rates of benzene and toluene by the microbial population of a sandy aquifer when these compounds were initially present at concentrations lower than 100 mg/l each. Concentrations higher than 100 mg/l were inhibitory, and no benzene or toluene degradation was observed when these compounds were initially present at 250 mg/l each. The Monod coefficients were calculated as k = 8.3 g-benzene/g-cells/day and Ks = 12.2 mg/l for benzene, and k = 9.9 g-toluene/g-cells/day and Ks = 17.4 mg/l for toluene. Specific first-order coefficients would be 0.68 l/mg.day for benzene and 0.57 l.mg.day for toluene.

Reductive dechlorination of a polychlorinated biphenyl congener and hexachlorobenzene by vitamin B12.

The polychlorinated biphenyl congener 2,3,4,5,6-pentachlorobiphenyl and hexachlorobenzene were reductively dechlorinated in an aqueous biomimetic model system containing vitamin B12. The products of 2,3,4,5,6-pentachlorobiphenyl dechlorination were 2,3,5,6- and 2,3,4,6-tetrachlorobiphenyl. Hexachlorobenzene dechlorinated to pentachlorobenzene and a mixture of 1,2,4,5- and 1,2,3,5-tetrachlorobenzene. The proton from water was shown to be the source of the hydrogen atom used for the replacement of chlorine on the biphenyl ring.