Craig. D. Phelps

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.

Stable isotope probing (SIP) was used to identify the active members in a benzene-degrading sulfidogenic consortium. SIP-terminal restriction fragment length polymorphism analysis indicated that a 270-bp peak incorporated the majority of the¹³C label and is a sequence closely related to that of clone SB-21 (GenBank accession no. AF029045). This target may be an important biomarker for anaerobic benzene degradation in the field.

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.

Naturally occurring arsenic is found predominantly as arsenate [As(V)] or arsenite [As(III)], and can be readily oxidized or reduced by microorganisms. Given the health risks associated with arsenic in groundwater and the interest in arsenic-active microorganisms, we hypothesized that environmental microorganisms could mediate a redox cycling of arsenic that is linked to their metabolism. This hypothesis was tested using an As(V) respiring reducer (strain Y5) and an aerobic chemoautotrophic As(III) oxidizer (strain OL1) both isolated from a Superfund site, Onondaga Lake, in Syracuse, NY. Strains were grown separately and together in sealed serum bottles, and the oxic/anoxic condition was the only parameter changed. Initially, under anoxic conditions when both isolates were grown together, 2 mM As(V) was stoichiometrically reduced to As(III) within 14 days. Following complete reduction, sterile ambient air was added and within 24 h As(III) was completely oxidized to As(V). The anoxic−oxic cycle was repeated, and sterile controls showed no abiotic transformation within the 28-day incubation period. These results demonstrate that microorganisms can cycle arsenic in response to dynamic environmental conditions, thereby affecting the speciation, and hence mobility and toxicity of arsenic in the environment.

The active bacterial community able to utilize benzoate under denitrifying conditions was elucidated in two coastal sediments using stable-isotope probing (SIP) and nosZ gene amplification. The SIP method employed samples from Norfolk Harbor, Virginia, and a Long-Term Ecosystem Observatory (no. 15) off the coast of Tuckerton, New Jersey. The SIP method was modified by use of archaeal carrier DNA in the density gradient separation. The carrier DNA significantly reduced the incubation time necessary to detect the ¹³ C-labeled bacterial DNA from weeks to hours in the coastal enrichments. No denitrifier DNA was found to contaminate the archaeal ¹³ C-carrier when [ ¹² C]benzoate was used as a substrate in the sediment enrichments. Shifts in the activity of the benzoate-utilizing denitrifying population could be detected throughout a 21-day incubation. These results suggest that temporal analysis using SIP can be used to illustrate the initial biodegrader(s) in a bacterial population and to document the cross-feeding microbial community.

During the past 15 years researchers have made great strides in understanding the metabolism of hydrocarbons by anaerobic bacteria. Organisms capable of utilizing benzene, toluene, ethylbenzene, xylenes, alkanes, and polycyclic aromatic hydrocarbons have ...

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.

Summary: During the anaerobic biodegradation of naphthalene and methylnaphthalene, unique metabolites are formed by specific microbial carboxylation and ring‐reduction reactions. Groundwater samples from an anoxic, shallow aquifer contaminated with gasoline were examined for the presence of these metabolites by extraction, derivatization and gas chromatography coupled to mass spectroscopy. Several metabolites [2‐naphthoic acid (2‐NA), tetrahydro‐2‐naphthoic acid (TH‐2‐NA), hexahydro‐2‐naphthoic acid (HH‐2‐NA) and methylnaphthoic acid (MNA)] were found to be present in the groundwater samples. The concentration of 2‐NA at each monitoring well was quantified and correlated to the zones of contamination. The presence of the other metabolites in the same wells as 2‐NA was used as confirmation that the anaerobic pathway was indeed active. The distribution of metabolites at this site shows that they can be used as biomarkers for demonstrating in situ biodegradation.

An enriched sulphidogenic consortium capable of mineralizing benzene was used to study the metabolic pathway of anaerobic benzene degradation. Benzoate was detected in active cultures and benzene was confirmed to be the source of this benzoate by the addition of deuterated benzene (D6) and subsequent detection of deuterated benzoate (D5) in active cultures but not in autoclaved controls. Benzoate was utilized by this culture at 1/12 the rate of benzene, while its presence did not inhibit benzene utilization. The benzene utilization rate was reduced, however, in the presence of 2‐fluorobenzoate. When the culture was supplemented with [ ¹³ C]‐bicarbonate, the carboxyl group on benzoate was not labelled with [ ¹³ C]‐carbon, suggesting that this transformation relies on a more complex set of reactions than simple addition of carbonate.