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.

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.

A stable and sediment‐free, benzene mineralizing, sulfate‐reducing culture that resisted repeated attempts at isolation was examined using molecular approaches such as traditional cloning and sequencing and a direct PCR fingerprinting method for 16S rRNA genes. Despite the culture's long exposure to benzene as the only carbon and energy source (over 3 years) and repeated dilutions of the original enrichment, this consortium has remained relatively complex. Cloning and sequence analysis identified 12 unique small subunit rRNA genes. The 16S rRNA genes belong to different eubacterial phyla, including Proteobacteria, Cytophagales and Gram‐positives. There is one deeply branching clone which is not closely related to any known, sequenced, bacterium. A different clone, however, is closely related to a known sulfidogenic, aromatic hydrocarbon degrader. To assess 16S rRNA gene cloning efficiency, a fingerprinting method based on fluorescent, end‐labeling of PCR product (16S rRNA genes) and screening by restriction length polymorphism analysis (RFLP) was employed. The data obtained indicated that we had cloned and characterized nearly all of the eubacterial 16S rRNA genes amplified from the consortia.

A stable and sediment-free, benzene mineralizing, sulfate-reducing culture that resisted repeated attempts at isolation was examined using molecular approaches such as traditional cloning and sequencing and a direct PCR fingerprinting method for 16S rRNA genes. Despite the culture's long exposure to benzene as the only carbon and energy source (over 3 years) and repeated dilutions of the original enrichment, this consortium has remained relatively complex. Cloning and sequence analysis identified 12 unique small subunit rRNA genes. The 16S rRNA genes belong to different eubacterial phyla, including Proteobacteria, Cytophagales and Gram-positives. There is one deeply branching clone which is not closely related to any known, sequenced, bacterium. A different clone, however, is closely related to a known sulfidogenic, aromatic hydrocarbon degrader. To assess 16S rRNA gene cloning efficiency, a fingerprinting method based on fluorescent, end-labeling of PCR product (16S rRNA genes) and screening by restriction length polymorphism analysis (RFLP) was employed. The data obtained indicated that we had cloned and characterized nearly all of the eubacterial 16S rRNA genes amplified from the consortia. z

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.