Lily Y. Young

Summary: 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 A s( V ) as the sole electron acceptor. Following growth and the respiratory reduction of A s( V ) to A s( III ), a yellow precipitate formed in active cultures, while no precipitate was observed in autoclaved controls, or in uninoculated media supplemented with A s( III ). The precipitate was identified by X ‐ray diffraction as alacranite, As ₈ S ₉ , a mineral previously only identified in hydrothermal environments. Sequencing of the 16S rRNA gene indicated that strain MPA ‐ C3 is a member of the D eferribacteres family, with relatively low (90%) identity to D enitrovibrio acetiphilus DSM 12809. The arsenate respiratory reductase gene, arrA , was sequenced, showing high homology to the arrA gene of D esulfitobacterium halfniense . In addition to A s( V ), strain MPA ‐ C3 utilizes NO ₃ ⁻ , S e( VI ), S e( IV ), fumarate and F e( 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 .

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 chemicals. Due to the prevalence of aromatic compounds in the environment, microorganisms have evolved mechanisms to metabolize that available carbon. While anaerobic monoaromatic degradation can be initiated in a number of different ways, the signature central metabolite for these pathways is benzoyl-CoA. 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, however the ring opening hydrolase, encoded by the bamA gene, is ideal because it is detected under a range of respiratory conditions, including denitrifying, iron-reducing, sulfate-reducing, and fermentative conditions. In this work we evaluated a number of DNA samples from diverse environments for the presence of the bamA gene, and had positive results for every sample. We further explored the bamA gene diversity in six of these sites as compared to published genome sequences and found that our clones were distributed throughout the dendrogram, although there were clone sequences from two sites that formed a unique clade. When we used a functional operational taxonomic unit based clustering analysis to compare the diversity of our sites to several locations reported in the literature, we found that there were two clusters of sites, and benzene contaminated sites were present in both clusters. Given the number of potential upstream inputs from natural and manmade monoaromatic compounds, the benzoyl-CoA pathway and the bamA gene play an important role in the global carbon cycle that has thus far been understudied.

Aim: To characterize the microbial community structure and bamA gene diversity involved in anaerobic degradation of toluene and benzoate under denitrifying conditions. Methods and Results: Nitrate-reducing enrichment cultures were established on either toluene, benzoate or without additional substrate. Bacterial community structures were characterized by 16S rRNA gene–based PCR-DGGE analysis. bamA gene diversity was analysed using DGGE and cloning methods. The results showed that bamA gene related to bamA of Thauera chlorobenzoica was dominant in toluene and benzoate cultures. However, a greater diversity of sequences was obtained in benzoate cultures. Low diversity of bamA gene was observed, and some similarities of bamA were also found between active cultures and backgrounds, suggesting that potential natural attenuation of aromatic compounds might occur. Conclusions: The combined analysis of 16S rRNA and bamA genes suggests that the species related to genera Thauera dominated toluene- and benzoate-degrading cultures. The combination of multiple methods (DGGE and cloning) provides a more complete picture of bamA gene diversity. Significance and Impact of the Study: To our knowledge, this is the first report of bamA gene in denitrifying enrichments using DGGE and cloning analysis.

This study reports that ongoing in situ anaerobic hydrocarbon biodegradation at a manufactured gas plant impacted site is occurring, 9 years after the initial investigation. Groundwater samples from the site monitoring wells (MW) were analyzed for biomarkers by GC-MS, end-point PCR, and quantitative PCR (qPCR). Metabolic biomarkers included specific intermediates of anaerobic naphthalene and/or 2-methylnaphthalene degradation: 2-naphthoic acid (2-NA); 5,6,7,8-tetrahydro-2-NA (TH-2-NA); hexahydro-2-NA (HH-2-NA); and carboxylated-2-methylnaphthalene (MNA). The analogues of gene bssA, encoding alpha subunit of enzyme benzylsuccinate synthase, were used as a genetic biomarker. Results indicate 1−2 orders of magnitude higher abundance of total bacteria in the impacted wells than in the unimpacted wells. End-point PCR analysis of bssA gene, with degenerate primers, indicated the presence of hydrocarbon degrading bacteria within the plume. In qPCR analysis, using primers based on toluene-degrading denitrifying or sulfate-reducing/methanogenic bacteria, bssA genes were detected only in MW-24, located downstream from the source. Metabolic biomarkers were detected in multiple wells. The highest abundance of 2-NA (6.7 μg/L), TH-2-NA (2.6 μg/L), HH-2-NA, and MNA was also detected in MW-24. The distribution of two independent biomarkers indicates that the site is enriched for anaerobic hydrocarbon biodegradation and provides strong evidence in support of natural attenuation.

Nitrate-reducing enrichments, amended with n -hexadecane, were established with petroleum-contaminated sediment from Onondaga Lake. Cultures were serially diluted to yield a sediment-free consortium. Clone libraries and denaturing gradient gel electrophoresis analysis of 16S rRNA gene community PCR products indicated the presence of uncultured alpha- and betaproteobacteria similar to those detected in contaminated, denitrifying environments. Cultures were incubated with H ₃₄ -hexadecane, fully deuterated hexadecane ( d ₃₄ -hexadecane), or H ₃₄ -hexadecane and NaH ¹³ CO ₃ . Gas chromatography-mass spectrometry analysis of silylated metabolites resulted in the identification of [H ₂₉ ]pentadecanoic acid, [H ₂₅ ]tridecanoic acid, [1- ¹³ C]pentadecanoic acid, [3- ¹³ C]heptadecanoic acid, [3- ¹³ C]10-methylheptadecanoic acid, and d ₂₇ -pentadecanoic, d ₂₅ -, and d _{2 4} -tridecanoic acids. The identification of these metabolites suggests a carbon addition at the C-3 position of hexadecane, with subsequent β-oxidation and transformation reactions (chain elongation and C-10 methylation) that predominantly produce fatty acids with odd numbers of carbons. Mineralization of [1- ¹⁴ C]hexadecane was demonstrated based on the recovery of ¹⁴ CO ₂ in active cultures.

Arsenic oxidation is recognized as being mediated by both heterotrophic and chemoautotrophic microorganisms. Enrichment cultures were established to d

Recent studies have demonstrated that fumarate addition and carboxylation are two possible mechanisms of anaerobic alkane degradation. In the present study, we surveyed metabolites formed during growth on hexadecane by the sulfate-reducing isolates AK-01 and Hxd3 and by a mixed sulfate-reducing consortium. The cultures were incubated with either protonated or fully deuterated hexadecane; the sulfate-reducing consortium was also incubated with [1,2- ¹³ C ₂ ]hexadecane. All cultures were extracted, silylated, and analyzed by gas chromatography-mass spectrometry. We detected a suite of metabolites that support a fumarate addition mechanism for hexadecane degradation by AK-01, including methylpentadecylsuccinic acid, 4-methyloctadecanoic acid, 4-methyloctadec-2,3-enoic acid, 2-methylhexadecanoic acid, and tetradecanoic acid. By using d ₃₄ -hexadecane, mass spectral evidence strongly supporting a carbon skeleton rearrangement of the first intermediate, methylpentadecylsuccinic acid, was demonstrated for AK-01. Evidence indicating hexadecane carboxylation was not found in AK-01 extracts but was observed in Hxd3 extracts. In the mixed sulfate-reducing culture, however, metabolites consistent with both fumarate addition and carboxylation mechanisms of hexadecane degradation were detected, which demonstrates that multiple alkane degradation pathways can occur simultaneously within distinct anaerobic communities. Collectively, these findings underscore that fumarate addition and carboxylation are important alkane degradation mechanisms that may be widespread among phylogenetically and/or physiologically distinct microorganisms.

Summary: Autotrophic microorganisms have been isolated that are able to derive energy from the oxidation of arsenite [As(III)] to arsenate [As(V)] under aerobic conditions. Based on chemical energetics, microbial oxidation of As(III) can occur in the absence of oxygen, and may be relevant in some environments. Enrichment cultures were established from an arsenic contaminated industrial soil amended with As(III) as the electron donor, inorganic C as the carbon source and nitrate as the electron acceptor. In the active enrichment cultures, oxidation of As(III) was stoichiometrically coupled to the reduction of NO ₃ ^– . Two autotrophic As(III)‐oxidizing strains were isolated that completely oxidized 5 mM As(III) within 7 days under denitrifying conditions. Based on 16S rRNA gene sequencing results, strain DAO1 was 99% related to Azoarcus and strain DAO10 was most closely related to a Sinorhizobium . The nitrous oxide reductase ( nosZ ) and the RuBisCO Type II ( cbbM ) genes were successfully amplified from both isolates underscoring their ability to denitrify and fix CO ₂ while coupled to As(III) oxidation. Although limited work has been done to examine the diversity of anaerobic autotrophic oxidizers of As(III), this process may be an important component in the biological cycling of arsenic within the environment.

A novel anaerobic bacterium was isolated from the sediment of Onondaga Lake (Syracuse, NY), which can use arsenate [As(V)] as a respiratory electron acceptor. The isolate, designated strain Y5 is a spore-forming, motile rod, with lateral flagella. It is Gram-negative though it phylogenetically falls within the low G + C Gram-positive organisms. In addition to the more usual electron donors such as lactate and succinate, strain Y5 also can use H₂+ CO₂ chemoautotrophically and metabolize aromatic compounds such as syringic acid, ferulic acid, phenol, benzoate and toluene, coupled to arsenate reduction. Aside from As(V), nitrate, sulfate, thiosulfate and Fe(III) can also serve as electron acceptors. Based on 16S rDNA phylogeny and its physiological characteristics, strain Y5 was identified as most closely related to the genus Desulfosporosinus. The ability of microorganisms to reduce arsenate for respiration appears to be widely distributed and may be relevant in the biogeochemical cycling of arsenic in environments containing mixed contaminants.

Strain Hxd3, an alkane-degrading sulfate reducer previously isolated and described by Aeckersberg et al. (F. Aeckersberg, F. Bak, and F. Widdel, Arch. Microbiol. 156:5-14, 1991), was studied for its alkane degradation mechanism by using deuterium and ¹³ C-labeled compounds. Deuterated fatty acids with even numbers of C atoms (C-even) and ¹³ C-labeled fatty acids with odd numbers of C atoms (C-odd) were recovered from cultures of Hxd3 grown on perdeuterated pentadecane and [1,2- ¹³ C ₂ ]hexadecane, respectively, underscoring evidence that C-odd alkanes are transformed to C-even fatty acids and vice versa. When Hxd3 was grown on unlabeled hexadecane in the presence of [ ¹³ C]bicarbonate, the resulting 15:0 fatty acid, which was one carbon shorter than the alkane, incorporated a ¹³ C label to form its carboxyl group. The same results were observed when tetradecane, pentadecane, and perdeuterated pentadecane were used as the substrates. These observations indicate that the initial attack of alkanes includes both carboxylation with inorganic bicarbonate and the removal of two carbon atoms from the alkane chain terminus, resulting in a fatty acid one carbon shorter than the original alkane. The removal of two terminal carbon atoms is further evidenced by the observation that the [1,2- ¹³ C ₂ ]hexadecane-derived fatty acids contained either two ¹³ C labels located exclusively at their acyl chain termini or none at all. Furthermore, when perdeuterated pentadecane was used as the substrate, the 14:0 and 16:0 fatty acids formed both carried the same numbers of deuterium labels, while the latter was not deuterated at its carboxyl end. These observations provide further evidence that the 14:0 fatty acid was initially formed from perdeuterated pentadecane, while the 16:0 fatty acid was produced after chain elongation of the former fatty acid with nondeuterated carbon atoms. We propose that strain Hxd3 anaerobically transforms an alkane to a fatty acid through a mechanism which includes subterminal carboxylation at the C-3 position of the alkane and elimination of the two adjacent terminal carbon atoms.