Metagenomic analysis of Fe(II)-oxidizing bacteria for Fe(III) mineral formation and carbon assimilation under microoxic conditions in paddy soil.

Microbially mediated Fe(II) oxidation is prevalent and thought to be central to many biogeochemical processes in paddy soils. However, we have limited insights into the Fe(II) oxidation process in paddy fields, considered the world's largest engineered wetland, where microoxic conditions are ubiquitous. In this study, microaerophilic Fe(II) oxidizing bacteria (FeOB) from paddy soil were enriched in gradient tubes with FeS, FeCO3, and Fe3(PO4)2 as iron sources to investigate their capacity for Fe(II) oxidation and carbon assimilation. Results showed that the highest rate of Fe(II) oxidation (k = 0.836 mM d-1) was obtained in the FeCO3 tubes, and cells grown in the Fe3(PO4)2 tubes yielded maximum assimilation amounts of 13C-NaHCO3 of 1.74% on Day 15. Amorphous Fe(III) oxides were found in all the cell bands with iron substrates as a result of microbial Fe(II) oxidation. Metagenomics analysis of the enriched microbes targeted genes encoding iron oxidase Cyc2, oxygen-reducing terminal oxidase, and ribulose-bisphosphate carboxylase, with results indicated that the potential Fe(II) oxidizers include nitrate-reducing FeOB (Dechloromonas and Thiobacillus), Curvibacter, and Magnetospirillum. By combining cultivation-dependent and metagenomic approaches, our results found a number of FeOB from paddy soil under microoxic conditions, which provide insight into the complex biogeochemical interactions of iron and carbon within paddy fields. The contribution of the FeOB to the element cycling in rice-growing regions deserves further investigation.

Uncovering microbial responses to sharp geochemical gradients in a terrace contaminated by acid mine drainage.

Acid mine drainage (AMD) is harmful to the environment and human health. Microorganisms-mineral interactions are responsible for AMD generation but can also remediate AMD contamination. Understanding the microbial response to AMD irrigation will reveal microbial survival strategies and provide approaches for AMD remediation. A terrace with sharp geochemical gradients caused by AMD flooding were selected to study the microbial response to changes in environmental parameters related to AMD contamination. AMD intrusion reduced soil microbial community diversity and further changed phylogenetic clustering patterns along the terrace gradient. We observed several genera seldom reported in AMD-related environments (i.e., Corynebacterium, Ochrobactrum, Natronomonas), suggesting flexible survival strategies such as nitrogen fixation, despite the poor nutritional environment. A co-occurrence network of heavily-contaminated fields was densely connected. The phyla Proteobacteria, Acidobacteria, Chloroflexi, and Euryarchaeota were all highly interconnected members, which may affect the formation of AMD. Detailed microbial response to different soil characterizations were highlighted by random forest model. Results revealed the top three parameters influencing the microbial diversity and interactions were pH, Fe(III), and sulfate. Various acidophilic Fe- and S-metabolizing bacteria were enriched in the lower fields, which were heavily contaminated by AMD, and more neutrophiles prevailed in the less-contaminated upper fields. Many indicator species in the lower fields were identified, including Desulfosporosinus, Thermogymnomonas, Corynebacterium, Shewanella, Acidiphilium, Ochrobactrum, Leptospirillum, and Allobaculum, representing acid-tolerant bacteria community in relevant environment. The detection of one known sulfate-reducing bacteria (i.e., Desulfosporosinus) suggested that biotic sulfate reduction may occur in acidic samples, which offers multiple advantages to AMD contamination treatment. Collectively, results suggested that the geochemical gradients substantially altered the soil microbiota and enriched the relevant microorganisms adapted to the different conditions. These findings provide mechanistic insights into the effects of contamination on the soil microbiota and establish a basis for in situ AMD bioremediation strategies.

Bacterial response to sharp geochemical gradients caused by acid mine drainage intrusion in a terrace: Relevance of C, N, and S cycling and metal resistance.

A unique terrace with sharp gradient of environmental conditions was selected to study the microbial response and survival strategies to the extreme environments introduced by acid mine drainage (AMD) contamination. A combination of geochemical analyses, metagenomic sequencing, ex-situ microcosm setups, and statistical analyses were used to investigate the environment-microbe interactions. The microbial communities and metabolic potentials along the terrace were studied by focusing on the genes associated with important biogeochemical processes (i.e., C, N, S cycling and metal resistance). Results show that the variations of geochemical parameters substantially shaped the indigenous microbial communities. Sharp environmental gradients also impacted the microbial metabolic potentials, especially for C, N, and S cycling. Although the relative abundances of carbon fixing genes did not significantly vary along the environmental gradients, the taxa for carbon fixation varied significantly in more contaminated fields versus less contaminated fields, indicating the effects of AMD contamination on the autotrophic microbial communities. AMD input also influenced the N cycling, especially for nitrogen fixation and dissimilatory nitrate reduction to ammonium (DNRA). In addition, ex situ experiments were undertaken to evaluate the effects of AMD contamination on nitrogen fixation rates. Random Forest (RF) analysis indicated that nitrate, pH, total N, TOC exhibited positive correlations with the rates of nitrogen fixation while total Fe, Fe(III), and sulfate showed negative effects. Two co-occurrence networks at taxonomic and genomic levels indicated that geochemical parameters such as pH, TOC, total N, total S, and total Fe substantially influenced the innate microbial communities and their metabolic potentials. The current study provides an understanding for microbial response to AMD contamination and lays the foundation for future potential AMD bioremediation.

Microbial transformation of widely used pharmaceutical and personal care product compounds.

Pharmaceutical and personal care products (PPCPs) are commonly used chemicals that are increasingly detected in urban-impacted environments, particularly those receiving treated wastewater. PPCPs may have toxicological effects on the macrofauna that are exposed through contaminated water; thus, there is interest in microbially mediated transformations that may degrade PPCPs. This review discusses specific examples of PPCP transformations that may occur in anoxic environments, including O-methylation and O-demethylation.

Humic Substances Facilitate Arsenic Reduction and Release in Flooded Paddy Soil.

Organic matter is important for controlling arsenic reduction and release under anoxic conditions. Humic substances (HS) represent an important fraction of natural organic matter, yet the manner in which HS affect arsenic transformation in flooded paddy soil has not been thoroughly elucidated. In this study, anaerobic microcosms were established with arsenic-contaminated paddy soil and amended with three extracted humic fractions (fulvic acid, FA; humic acid, HA; and humin, HM). The HS substantially enhanced the extent of arsenic reduction and release in the order FA > HA > HM. It was confirmed that microbially reduced HS acted as an electron shuttle to promote arsenate reduction. HS, particularly FA, provided labile carbon to stimulate microbial activity and increase the relative abundances of Azoarcus, Anaeromyxobacter, and Pseudomonas, all of which may be involved in the reduction of HS, Fe(III), and arsenate. HS also increased the abundance of transcripts for an arsenate-respiring gene ( arrA) and overall transcription in arsenate-respiring Geobacter spp. The increase in both abundances lagged behind the increases in dissolved arsenate levels. These results help to elucidate the pathways of arsenic reduction and release in the presence of HS in flooded paddy soil.

Pharmaceuticals and Personal Care Products Can Be Transformed by Anaerobic Microbiomes in the Environment and in Waste-Treatment Processes.

Pharmaceuticals and personal care products (PPCPs) are emerging environmental contaminants that can be transformed by anaerobic microorganisms in anoxic environments. The present study examined 2 consortia, enriched under methanogenic and sulfate-rich conditions, that demethylate the phenylmethyl ether anti-inflammatory drug naproxen to 6-O-desmethylnaproxen. Both enriched consortia were also able to demethylate a range of phenylmethyl ether compounds of plant-based origin or used as PPCPs. Results from 16S rRNA gene sequencing showed that the 2 communities were very different despite sharing the same PPCP metabolism. In most cases, the demethylated metabolite was not further degraded but rather accumulated in the culture medium. For the expectorant guaifenesin, this resulted in a novel microbial metabolite. Furthermore, to our knowledge, this is the first report of methylparaben metabolism under methanogenic conditions. The wide range of phenylmethyl ether substrates that underwent O-demethylation in both methanogenic and sulfate-rich conditions suggests that there are potentially bioactive transformation products in the environment that have not yet been quantified. Environ Toxicol Chem 2019;38:1585-1593. © 2019 SETAC.

Diastereoisomer-Specific Biotransformation of Hexabromocyclododecanes by a Mixed Culture Containing Dehalococcoides mccartyi Strain 195.

Hexabromocyclododecane (HBCD) stereoisomers may exhibit substantial differences in physicochemical, biological, and toxicological properties. However, there remains a lack of knowledge about stereoisomer-specific toxicity, metabolism, and environmental fate of HBCD. In this study, the biotransformation of (±)α-, (±)ß-, and (±)γ-HBCD contained in technical HBCD by a mixed culture containing the organohalide-respiring bacterium Dehalococcoides mccartyi strain 195 was investigated. Results showed that the mixed culture was able to efficiently biotransform the technical HBCD mixture, with 75% of the initial HBCD (∼12 µM) in the growth medium being removed within 42 days. Based on the metabolites analysis, HBCD might be sequentially debrominated via dibromo elimination reaction to form tetrabromocyclododecene, dibromocyclododecadiene, and 1,5,9-cyclododecatriene. The biotransformation of the technical HBCD was likely diastereoisomer-specific. The transformation rates of α-, ß-, and γ-HBCD were in the following order: α-HBCD > ß-HBCD > γ-HBCD. The enantiomer fractions of (±)α-, (±)ß-, and (±)γ-HBCD were maintained at about 0.5 during the 28 days of incubation, indicating a lack of enantioselective biotransformation of these diastereoisomers. Additionally, the amendment of another halogenated substrate tetrachloroethene (PCE), which supports the growth of strain 195, had a negligible impact on the transformation patterns of HBCD diastereoisomers and enantiomers. This study provided new insights into the stereoisomer-specific transformation patterns of HBCD by anaerobic microbes and has important implications for microbial remediation of anoxic environments contaminated by HBCD using the mixed culture containing Dehalococcoides.

Sulfate-Reducing Naphthalene Degraders Are Picky Eaters.

Polycyclic aromatic hydrocarbons (PAHs) are common organic contaminants found in anoxic environments. The capacity for PAH biodegradation in unimpacted environments, however, has been understudied. Here we investigate the enrichment, selection, and sustainability of a microbial community from a pristine environment on naphthalene as the only amended carbon source. Pristine coastal sediments were obtained from the Jacques Cousteau National Estuarine Research Reserve in Tuckerton, New Jersey, an ecological reserve which has no direct input or source of hydrocarbons. After an initial exposure to naphthalene, primary anaerobic transfer cultures completely degraded 500 µM naphthalene within 139 days. Subsequent transfer cultures mineralized naphthalene within 21 days with stoichiometric sulfate loss. Enriched cultures efficiently utilized only naphthalene and 2-methylnaphthalene from the hydrocarbon mixtures in crude oil. To determine the microorganisms responsible for naphthalene degradation, stable isotope probing was utilized on cultures amended with fully labeled 13C-naphthalene as substrate. Three organisms were found to unambiguously synthesize 13C-DNA from 13C-naphthalene within 7 days. Phylogenetic analysis revealed that 16S rRNA genes from two of these organisms are closely related to the known naphthalene degrading isolates NaphS2 and NaphS3 from PAH-contaminated sites. A third 16S rRNA gene was only distantly related to its closest relative and may represent a novel naphthalene degrading microbe from this environment.

Draft genome sequence of Bosea sp. WAO an arsenite and sulfide oxidizer isolated from a pyrite rock outcrop in New Jersey.

This genome report describes the draft genome and physiological characteristics of Bosea sp. WAO (=DSM 102914), a novel strain of the genus Bosea in the family Bradyrhizobiaceae. Bosea sp. WAO was isolated from pulverized pyritic shale containing elevated levels of arsenic. This aerobic, gram negative microorganism is capable of facultative chemolithoautotrophic growth under aerobic conditions by oxidizing the electron donors arsenite, elemental sulfur, thiosulfate, polysulfide, and amorphous sulfur. The draft genome is of a single circular chromosome 6,125,776 bp long consisting of 21 scaffolds with a G + C content of 66.84%. A total 5727 genes were predicted of which 5665 or 98.92% are protein-coding genes and 62 RNA genes. We identified the genes aioA and aioB, which encode the large and small subunits of the arsenic oxidase respectively. We also identified the genes for the complete sulfur oxidation pathway sox which is used to oxidize thiosulfate to sulfate.

The antihistamine diphenhydramine is demethylated by anaerobic wastewater microorganisms.

While emerging pharmaceutical contaminants are monitored in wastewater treatment and the environment, there is little information concerning their microbial metabolites. The transformation of diphenhydramine by microorganisms in anaerobic digester sludge was investigated using anaerobic cultures amended with 1 mM diphenhydramine as the sole carbon source. Complete transformation of the parent compound to a persistent metabolite occurred within 191 days. Using GC/MS analysis, the metabolite was identified as N-desmethyl diphenhydramine. Loss of the parent compound diphenhydramine followed a first order rate constant of 0.013 day-1. There was no observed decrease in metabolite concentration even after a further 12 months of incubation, suggesting that the metabolite resists further degradation during wastewater treatment. Bacterial community diversity in the diphenhydramine transforming assay cultures showed enrichment in Comamonadaceae, Symbiobacteriaceae, Anaerolineaceae, and Prevotellaceae relative to unamended background controls. An anaerobic toxicity assay demonstrated that diphenhydramine has an inhibitory effect on both fermentative bacteria and methanogenic archaea in the wastewater community. In contrast, the metabolite N-desmethyl diphenhydramine partially suppressed methanogens but did not impact the fermenting community. To our knowledge, this is the first report of diphenhydramine metabolism by a bacterial community. The limited transformation of diphenhydramine by wastewater microorganisms indicates that N-desmethyl diphenhydramine will enter the environment along with unmetabolized diphenhydramine.

Naproxen Is Transformed Via Acetogenesis and Syntrophic Acetate Oxidation by a Methanogenic Wastewater Consortium.

Over-the-counter pharmaceutical compounds can serve as microbial substrates in wastewater treatment processes as well as in the environment. The metabolic pathways and intermediates produced during their degradation, however, are poorly understood. In this study, we investigate an anaerobic wastewater community that metabolizes naproxen via demethylation. Enriched cultures, established from anaerobic digester inocula receiving naproxen as the sole carbon source, transformed naproxen to 6-O-desmethylnaproxen (DMN) within 22 days. Continual enrichment and culture transfer resulted in consistent demethylation of naproxen with no loss of DMN observed. Methane was generated at 0.83 mmol per 1 mmol transformed naproxen. In addition to naproxen, the consortium readily demethylated syringic acid and vanillic acid. DNA analysis revealed a community of acetogenic bacteria and syntrophic acetate oxidizing archaea. Combined with the biotransformation data, this suggests the enriched consortium performs aromatic O-demethylation through a syntrophic relationship between specific acetogens, acetate oxidizers, and methanogens. The proposed model of carbon transfer through the anaerobic food web highlights the significance of linked community interactions in the anaerobic transformation of aromatic O-methyl compounds such as naproxen.

Transcriptional Activity of Arsenic-Reducing Bacteria and Genes Regulated by Lactate and Biochar during Arsenic Transformation in Flooded Paddy Soil.

Organic substrates and biochar are important in controlling arsenic release from sediments and soils; however, little is known about their impact on arsenic-reducing bacteria and genes during arsenic transformation in flooded paddy soils. In this study, microcosm experiments were established to profile transcriptional activity of As(V)-respiring gene (arrA) and arsenic resistance gene (arsC) as well as the associated bacteria regulated by lactate and/or biochar in anaerobic arsenic-contaminated paddy soils. Chemical analyses revealed that lactate as the organic substrate stimulated microbial reduction of As(V) and Fe(III), which was simultaneously promoted by lactate+biochar, due to biochar's electron shuttle function that facilitates electron transfer from bacteria to As(V)/Fe(III). Sequencing and phylogenetic analyses demonstrated that both arrA closely associated with Geobacter (>60%, number of identical sequences/number of the total sequences) and arsC related to Enterobacteriaceae (>99%) were selected by lactate and lactate+biochar. Compared with the lactate microcosms, transcriptions of the bacterial 16S rRNA gene, Geobacter spp., and Geobacter arrA and arsC genes were increased in the lactate+biochar microcosms, where transcript abundances of Geobacter and Geobacter arrA closely tracked with dissolved As(V) concentrations. Our findings indicated that lactate and biochar in flooded paddy soils can stimulate the active As(V)-respiring bacteria Geobacter species for arsenic reduction and release, which probably increases arsenic bioavailability to rice plants.

Benzoyl-CoA, a universal biomarker for anaerobic degradation of aromatic compounds.

Aromatic compounds are a major component of the global carbon pool and include a diverse range of compounds such as humic acid, lignin, amino acids, and industrial contaminants. Due to the prevalence of aromatic compounds in the environment, aerobic and anaerobic microorganisms have evolved mechanisms by which to metabolize that available carbon. Less well understood are the anaerobic pathways. We now know that anaerobic metabolism of a variety of monoaromatic compounds can be initiated in a number of different ways, and a key metabolite for these pathways is benzoyl-CoA. Chemicals can have different upstream anaerobic degradation pathways yet can still be assessed by targeting the downstream benzoyl-CoA pathway. In this pathway, we propose that the ring opening hydrolase, encoded by the bamA gene, is especially useful because, in contrast to the benzoyl-CoA reductase, it is detected under a number of respiratory settings, including denitrifying, iron-reducing, sulfate-reducing, and fermentative conditions, and has a wide distribution in the environment. This review examines the bamA gene in enrichment cultures and environmental DNA extracts to consider whether it can be used as a biomarker for anaerobic aromatic degradation. Given the number of potential upstream inputs from natural and man-made monoaromatic compounds, the benzoyl-CoA pathway and the bamA gene in particular may play an important role in the global carbon cycle that has thus far been overlooked.

The bamA gene for anaerobic ring fission is widely distributed in the environment.

Benzoyl-CoA is the signature central metabolite associated with the anaerobic metabolism of a diverse range of compounds such as humic acid, lignin, amino acids, and industrial chemicals. Aromatic chemicals with different upstream degradation pathways all funnel into the downstream benzoyl-CoA pathway. Different genes encoding enzymes of the benzoyl-CoA pathway could be used as biomarkers for the anaerobic benzoyl-CoA pathway, however, the ring opening hydrolase, encoded by the bamA gene, is ideal because it is detected under a range of respiratory conditions, including under denitrifying, iron-reducing, sulfate-reducing, and fermentative conditions. This work evaluated DNA samples from six diverse environments for the presence of the bamA gene, and had positive results for every sample. Individual bamA gene clones from these sites were compared to published genome sequences. The clone sequences were distributed amongst the genome sequences, although there were clone sequences from two of the analyzed sites that formed a unique clade. Clone sequences were then grouped by site and analyzed with a functional operational taxonomic unit based clustering program to compare the bamA gene diversity of these sites to that of several locations reported in the literature. The results showed that the sequence diversity of the sites separated into two clusters, but there was no clear trend that could be related to the site characteristics. Interestingly, two pristine freshwater sites formed a subgroup within one of the larger clusters. Thus far the bamA gene has only been examined within the context of contaminated environments, however, this study demonstrates that the bamA gene is also detected in uncontaminated sites. The widespread presence of the bamA gene in diverse environments suggests that the anaerobic benzoyl-CoA pathway plays an important role in the global carbon cycle that has thus far been understudied.

Precipitation of alacranite (As8S9) by a novel As(V)-respiring anaerobe strain MPA-C3.

Strain MPA-C3 was isolated by incubating arsenic-bearing sediments under anaerobic, mesophilic conditions in minimal media with acetate as the sole source of energy and carbon, and As(V) as the sole electron acceptor. Following growth and the respiratory reduction of As(V) to As(III), a yellow precipitate formed in active cultures, while no precipitate was observed in autoclaved controls, or in uninoculated media supplemented with As(III). The precipitate was identified by X-ray diffraction as alacranite, As8 S9 , a mineral previously only identified in hydrothermal environments. Sequencing of the 16S rRNA gene indicated that strain MPA-C3 is a member of the Deferribacteres family, with relatively low (90%) identity to Denitrovibrio acetiphilus DSM 12809. The arsenate respiratory reductase gene, arrA, was sequenced, showing high homology to the arrA gene of Desulfitobacterium halfniense. In addition to As(V), strain MPA-C3 utilizes NO3(-), Se(VI), Se(IV), fumarate and Fe(III) as electron acceptors, and acetate, pyruvate, fructose and benzoate as sources of carbon and energy. Analysis of a draft genome sequence revealed multiple pathways for respiration and carbon utilization. The results of this work demonstrate that alacranite, a mineral previously thought to be formed only chemically under hydrothermal conditions, is precipitated under mesophilic conditions by the metabolically versatile strain MPA-C3.

Impact of mercury on denitrification and denitrifying microbial communities in nitrate enrichments of subsurface sediments.

The contamination of groundwater with mercury (Hg) is an increasing problem worldwide. Yet, little is known about the interactions of Hg with microorganisms and their processes in subsurface environments. We tested the impact of Hg on denitrification in nitrate reducing enrichment cultures derived from subsurface sediments from the Oak Ridge Integrated Field Research Challenge site, where nitrate is a major contaminant and where bioremediation efforts are in progress. We observed an inverse relationship between Hg concentrations and onset and rates of denitrification in nitrate enrichment cultures containing between 53 and 1.1 µM of inorganic Hg; higher Hg concentrations increasingly extended the time to onset of denitrification and inhibited denitrification rates. Microbial community complexity, as indicated by terminal restriction fragment length polymorphism (tRFLP) analysis of the 16S rRNA genes, declined with increasing Hg concentrations; at the 312 nM Hg treatment, a single tRFLP peak was detected representing a culture of Bradyrhizobium sp. that possessed the merA gene indicating a potential for Hg reduction. A culture identified as Bradyrhizobium sp. strain FRC01 with an identical 16S rRNA sequence to that of the enriched peak in the tRFLP patterns, reduced Hg(II) to Hg(0) and carried merA whose amino acid sequence has 97 % identity to merA from the Proteobacteria and Firmicutes. This study demonstrates that in subsurface sediment incubations, Hg may inhibit denitrification and that inhibition may be alleviated when Hg resistant denitrifying Bradyrhizobium spp. detoxify Hg by its reduction to the volatile elemental form.

Pathways for arsenic from sediments to groundwater to streams: biogeochemical processes in the Inner Coastal Plain, New Jersey, USA.

The Cretaceous and Tertiary sediments that underlie the Inner Coastal Plain of New Jersey contain the arsenic-rich mineral glauconite. Streambed sediments in two Inner Coastal Plain streams (Crosswicks and Raccoon Creeks) that traverse these glauconitic deposits are enriched in arsenic (15-25mg/kg), and groundwater discharging to the streams contains elevated levels of arsenic (>80µg/L at a site on Crosswicks Creek) with arsenite generally the dominant species. Low dissolved oxygen, low or undetectable levels of nitrate and sulfate, detectable sulfide concentrations, and high concentrations of iron and dissolved organic carbon (DOC) in the groundwater indicate that reducing environments are present beneath the streambeds and that microbial activity, fueled by the DOC, is involved in releasing arsenic and iron from the geologic materials. In groundwater with the highest arsenic concentrations at Crosswicks Creek, arsenic respiratory reductase gene (arrA) indicated the presence of arsenic-reducing microbes. From extracted DNA, 16s rRNA gene sequences indicate the microbial community may include arsenic-reducing bacteria that have not yet been described. Once in the stream, iron is oxidized and precipitates as hydroxide coatings on the sediments. Arsenite also is oxidized and co-precipitates with or is sorbed to the iron hydroxides. Consequently, dissolved arsenic concentrations are lower in streamwater than in the groundwater, but the arsenic contributed by groundwater becomes part of the arsenic load in the stream when sediments are suspended during high flow. A strong positive relation between concentrations of arsenic and DOC in the groundwater samples indicates that any process-natural or anthropogenic-that increases the organic carbon concentration in the groundwater could stimulate microbial activity and thus increase the amount of arsenic that is released from the geologic materials.

Characterization of the microbial community from the marine sediment of the Venice lagoon capable of reductive dechlorination of coplanar polychlorinated biphenyls (PCBs).

The native microbial community of a contaminated sediment from Brentella Canal (Venice Lagoon, Italy) was enriched in slurry microcosms consisting of sterile sediment suspended in sterile site water in the presence of 3,3',4,4'-tetrachlorobiphenyl, 3,3',4,4',5- and 2,3',4,4',5-pentachlorobiphenyls, 3,3',4,4',5,5'- and 2,3,3',4,4',5-hexachlorobiphenyls. The enrichment cultures were characterized at each subculturing step by 16S rRNA gene Terminal-Restriction Fragment Length Polymorphism (T-RFLP) and Denaturing Gradient Gel Electrophoresis (DGGE) analysis. About 90% of spiked polychlorinated biphenyls (PCBs) were stoichiometrically converted into di- and tri-chlorinated congeners by each enriched culture via dechlorination of flanked para chlorines and ortho-flanked meta chlorines. A 2-fold increase in PCB-dechlorination rate, the disappearance of lag phase, as well as a remarkable increase of sulfate consumption and a decline of methanogenic activity, were observed throughout subculturing. A reduction of complexity of the archaeal community, which was composed by Methanomicrobiales and Methanosarcinales, was also observed as a result of culture enrichment. The bacterial community included members of the Alpha, Gamma, Delta and Epsilon divisions of Proteobacteria, Firmicutes and Chloroflexi. Two sequence phylotypes related to the genus Sulforovum and the species Desulfococcus multivorans and two Chloroflexi enriched throughout subculturing, thus suggesting that these bacteria were involved in PCB dechlorination in the marine sediments of Brentella canal.

Detection of 2,4,6-trinitrotoluene-utilizing anaerobic bacteria by 15N and 13C incorporation.

2,4,6-Trinitrotoluene ((15)N or (13)C labeled) was added to Norfolk Harbor sediments to test whether anaerobic bacteria use TNT for growth. Stable-isotope probing (SIP)-terminal restriction fragment length polymorphism (TRFLP) detected peaks in the [(15)N]TNT cultures (60, 163, and 168 bp). The 60-bp peak was also present in the [(13)C]TNT cultures and was related to Lysobacter taiwanensis.

Novel autotrophic arsenite-oxidizing bacteria isolated from soil and sediments.

Arsenic oxidation is recognized as being mediated by both heterotrophic and chemoautotrophic microorganisms. Enrichment cultures were established to determine whether chemoautotrophic microorganisms capable of oxidizing arsenite As(III) to arsenate As(V) are present in selected contaminated but nonextreme environments. Three new organisms, designated as strains OL-1, S-1 and CL-3, were isolated and found to oxidize 10 mM arsenite to arsenate under aerobic conditions using CO2-bicarbonate (CO2/HCO3-) as a carbon source. Based on 16S rRNA gene sequence analyses, strain OL-1 was 99% most closely related to the genus Ancylobacter, strain S-1 was 99% related to Thiobacillus and strain CL-3 was 98% related to the genus Hydrogenophaga. The isolates are facultative autotrophs and growth of isolated strains on different inorganic electron donors other than arsenite showed that all three had a strong preference for several sulfur species, while CL-3 was also able to grow on ammonium and nitrite. The RuBisCO Type I (cbbL) gene was positively amplified and sequenced in strain CL-3, and the Type II (cbbM) gene was detected in strains OL-1 and S-1, supporting the autotrophic nature of the organisms.