Azospira inquinata sp. nov., a nitrate-reducing bacterium of the family Rhodocyclaceae isolated from contaminated groundwater.

Two novel Gram-stain-negative bacterial strains, Azo-3T and Azo-2, were isolated from a toluene-producing enrichment culture that originated from contaminated groundwater at a site in southeast Louisiana (USA). Cells are non-spore forming straight to curved rods with single polar flagella. Strains Azo-3T and Azo-2 are oxidase-positive, catalase-negative, use nitrate and nitrite as electron acceptors, and are able to fix nitrogen. Poly-ß-hydroxybutyrate storage granules are produced. Dominant fatty acids when grown in R2A medium at 37 °C are C16:0, summed feature 3 (C16:1 ω7c and/or C15:0 iso 2OH), C17:0 cyclo and C18:1 ω7c. 16S rRNA gene sequence based phylogenetic analysis indicated that the strains cluster within the family Rhodocyclaceae, class Betaproteobacteria, most closely related to but distinct from type strains of the species Azospira oryzae (96.94% similarity) and Azospira restricta (95.10% similarity). Complete genome sequences determined for strains Azo-3T and Azo-2 revealed DNA G+C content of 62.70 mol%. Genome-wide comparisons based on average nucleotide identity by orthology and estimated DNA-DNA hybridization values combined with phenotypic and chemotaxonomic traits and phylogenetic analysis indicate that strains Azo-3T and Azo-2 represent a novel species within the genus Azospira for which the name Azospira inquinata sp. nov. is proposed. The type strain of Azospira inquinata is Azo-3T (=NRRL B-65590T=DSM 112046T).

Genome Sequence of the Type Strain Azospira restricta SUA2 (DSM 18626).

Azospira restricta SUA2T (DSM 18626) is a Gram-negative-staining bacterium able to fix nitrogen and accumulate polyhydroxybutyrate storage granules. Here, we report the complete genome sequence (3,975,213 bp with 68.64 mol% G+C content), which may prove useful in future efforts to assess the role of Azospira in nutrient cycling.

Mo2N nanobelt cathodes for efficient hydrogen production in microbial electrolysis cells with shaped biofilm microbiome.

High cost platinum (Pt) catalysts limit the application of microbial electrolysis cells (MECs) for hydrogen (H2) production. Here, inexpensive and efficient Mo2N nanobelt cathodes were prepared using an ethanol method with minimized catalyst and binder loadings. The chronopotentiometry tests demonstrated that the Mo2N nanobelt cathodes had similar catalytic activities for H2 evolution compared to that of Pt/C (10 wt%). The H2 production rates (0.39 vs. 0.37 m3-H2/m3/d), coulombic efficiencies (90% vs. 77%), and overall hydrogen recovery (74% vs. 70%) of MECs with the Mo2N nanobelt cathodes were also comparable to those with Pt/C cathodes. However, the cost of Mo2N nanobelt catalyst ($ 31/m2) was much less than that of Pt/C catalysts ($ 1930/m2). Furthermore, the biofilm microbiomes at electrodes were studied using the PacBio sequencing of full-length 16S rRNA gene. It indicated Stenotrophomonas nitritireducens as a putative electroactive bacterium dominating the anode biofilm microbiomes. The majority of dominant species in the Mo2N and Pt/C cathode communities belonged to Stenotrophomonas nitritireducens, Stenotrophomonas maltophilia, and Comamonas testosterone. The dominant populations in the cathode biofilms were shaped by the cathode materials. This study demonstrated Mo2N nanobelt catalyst as an alternative to Pt catalyst for H2 production in MECs.

Bioremediation Strategies Aimed at Stimulating Chlorinated Solvent Dehalogenation Can Lead to Microbially-Mediated Toluene Biogenesis.

In situ bioremediation practices that include subsurface addition of fermentable electron donors to stimulate reductive dechlorination by anaerobic bacteria have become widely employed to combat chlorinated solvent contamination in groundwater. At a contaminated site located near Baton Rouge, Louisiana (USA), toluene was transiently observed in groundwater at concentrations that sometimes far exceeded the US drinking water maximum contaminant level (MCL) of 1 mg/L after a fermentable substrate (agricultural feed grade cane molasses) was injected into the subsurface with the intent of providing electron donors for reductive dechlorination. Here, we present data that demonstrate that indigenous microorganisms can biologically produce toluene by converting phenylacetic acid, phenylalanine, phenyllactate, and phenylpyruvate to toluene. When grown in defined medium with phenylacetic acid at concentrations ≤350 mg/L, the molar ratio between toluene accumulated and phenylacetic acid supplied was highly correlated ( R2 ≥ 0.96) with a toluene yield exceeding 0.9:1. Experiments conducted using 13C labeled compounds (phenylacetic acid-2-13C and l-phenylalanine-3-13C) resulted in production of toluene-α-13C, confirming that toluene was synthesized from these precursors by two independently developed enrichment cultures. Results presented here suggest that monitoring of aromatic hydrocarbons is warranted during enhanced bioremediation activities where electron donors are introduced to stimulate anaerobic biotransformation of chlorinated solvents.

Biofilter treatment of gas phase ß-caryophyllene at an elevated temperature.

Experiments were carried out to test the capacity for a laboratory-scale biofilter operated at an elevated temperature level (∼50°C) to remove an air stream containing ß-caryophyllene, a naturally occurring sesquiterpene of environmental concern emitted from wood-related industrial facilities. A water jacket was used to maintain high temperatures in a laboratory-scale biofilter. Inocula, pollutant loading and nutrient supply rate effects were evaluated over 84 days of biofilter operation. The start-up process took over two months when citrus peels were used as inocula while a relatively short start-up period was achieved after introducing forest compost products. While using a sparged-gas bioreactor to cultivate an enrichment culture for 97 days, removal efficiencies in excess of 80% were observed after 18 days. At empty bed contact times of 50 s and at a pollutant loading rate of 3.05 mg C/L/hr, removal efficiency levels reached 90% and the elimination capacity level reached 2.29 mg C/L/hr, corresponding to an elimination capacity of 2.60 mg ß-caryophyllene/L/hr. Collectively, these results demonstrate that ß-caryophyllene can be successfully removed from contaminated air using a biofilter operated at a high temperature (∼50°C), expanding the temperature range within which biofilters are known to biodegrade sesquiterpenes.

Dehalogenimonas formicexedens sp. nov., a chlorinated alkane-respiring bacterium isolated from contaminated groundwater.

A strictly anaerobic, Gram-stain-negative, non-spore-forming bacterium designated NSZ-14T, isolated from contaminated groundwater in Louisiana (USA), was characterized using a polyphasic approach. Strain NSZ-14T reductively dehalogenated a variety of polychlorinated aliphatic alkanes, producing ethene from 1,2-dichloroethane, propene from 1,2-dichloropropane, a mixture of cis- and trans-1,2-dichloroethene from 1,1,2,2-tetrachloroethane, vinyl chloride from 1,1,2-trichloroethane and allyl chloride (3-chloro-1-propene) from 1,2,3-trichloropropane. Formate or hydrogen could both serve as electron donors. Dechlorination occurred between pH 5.5 and 7.5 and over a temperature range of 20-37 °C. Major cellular fatty acids included C18 : 1ω9c, C14 : 0 and C16 : 0. 16S rRNA gene sequence-based phylogenetic analysis indicated that the strain clusters within the class Dehalococcoidia of the phylum Chloroflexi, most closely related to but distinct from type strains of the species Dehalogenimonas alkenigignens (97.63 % similarity) and Dehalogenimonas lykanthroporepellens (95.05 %). A complete genome sequence determined for strain NSZ-14T revealed a DNA G+C content of 53.96 mol%, which was corroborated by HPLC (54.1±0.2 mol% G+C). Genome-wide comparisons based on average nucleotide identity by orthology and estimated DNA-DNA hybridization values combined with phenotypic and chemotaxonomic traits and phylogenetic analysis indicate that strain NSZ-14T represents a novel species within the genus Dehalogenimonas, for which the name Dehalogenimonas formicexedens sp. nov. is proposed. The type strain is NSZ-14T (=HAMBI 3672T=JCM 19277T=VKM B-3058T). An emended description of Dehalogenimonas alkenigignens is also provided.

Genome sequence of the organohalide-respiring Dehalogenimonas alkenigignens type strain (IP3-3(T)).

Dehalogenimonas alkenigignens IP3-3(T) is a strictly anaerobic, mesophilic, Gram negative staining bacterium that grows by organohalide respiration, coupling the oxidation of H2 to the reductive dehalogenation of polychlorinated alkanes. Growth has not been observed with any non-polyhalogenated alkane electron acceptors. Here we describe the features of strain IP3-3(T) together with genome sequence information and its annotation. The 1,849,792 bp high-quality-draft genome contains 1936 predicted protein coding genes, 47 tRNA genes, a single large subunit rRNA (23S-5S) locus, and a single, orphan, small unit rRNA (16S) locus. The genome contains 29 predicted reductive dehalogenase genes, a large majority of which lack cognate genes encoding membrane anchoring proteins.

Reassessment of PCR primers targeting 16S rRNA genes of the organohalide-respiring genus Dehalogenimonas.

Representatives from the genus Dehalogenimonas have the metabolic capacity to anaerobically transform a variety of environmentally important polychlorinated aliphatic compounds. In light of the recent isolation of additional strains, description of a new species, and an expanded number of uncultured DNA sequences, PCR primers and protocols intended to uniquely target members of this organohalide-respiring genus were reevaluated. Nine of fourteen primer combinations reported previously as genus-specific failed to amplify 16S rRNA genes of recently isolated Dehalogenimonas strains. Use of alternative combinations or modified genus-specific primers, however, allowed detection of all presently known Dehalogenimonas strains. Use of a modified primer set in qPCR revealed an approximately two-order of magnitude increase in concentration of Dehalogenimonas 16S rRNA gene copies following subsurface injection of electron donors at a Louisiana Superfund site, demonstrating the utility of the newly developed protocol and suggesting that the genus Dehalogenimonas can respond to biostimulation remediation strategies in a manner similar to that previously reported for other dechlorinating genera such as Dehalococcoides.

Dehalogenimonas lykanthroporepellens BL-DC-9T simultaneously transcribes many rdhA genes during organohalide respiration with 1,2-DCA, 1,2-DCP, and 1,2,3-TCP as electron acceptors.

The genome sequence of the organohalide-respiring bacterium Dehalogenimonas lykanthroporepellensBL-DC-9(T) contains numerous loci annotated as reductive dehalogenase homologous (rdh) genes based on inferred protein sequence identity with functional dehalogenases of other bacterial species. Many of these genes are truncated, lack adjacent regulatory elements, or lack cognate genes coding for membrane-anchoring proteins typical of the functionally characterized active reductive dehalogenases of organohalide-respiring bacteria. To investigate the expression patterns of the rdh genes in D. lykanthroporepellensBL-DC-9(T), oligonucleotide primers were designed to uniquely target 25 rdh genes present in the genome as well as four putative regulatory genes. RNA extracts from cultures of strain BL-DC-9(T) actively dechlorinating three different electron acceptors, 1,2-dichloroethane, 1,2-dichloropropane, and 1,2,3-trichloropropane were reverse-transcribed and subjected to PCR amplification using rdh-specific primers. Nineteen rdh gene transcripts, including 13 full-length rdhA genes, six truncated rdhA genes, and five rdhA genes having cognate rdhB genes were consistently detected during the dechlorination of all three of the polychlorinated alkanes tested. Transcripts from all four of the putative regulatory genes were also consistently detected. Results reported here expand the diversity of bacteria known to simultaneously transcribe multiple rdh genes and provide insights into the transcription factors associated with rdh gene expression.

Substrate interactions in dehalogenation of 1,2-dichloroethane, 1,2-dichloropropane, and 1,1,2-trichloroethane mixtures by Dehalogenimonas spp.

When chlorinated alkanes are present as soil or groundwater pollutants, they often occur in mixtures. This study evaluated substrate interactions during the anaerobic reductive dehalogenation of chlorinated alkanes by the type strains of two Dehalogenimonas species, D. lykanthroporepellens and D. alkenigignens. Four contaminant mixtures comprised of combinations of the chlorinated solvents 1,2-dichloroethane (1,2-DCA), 1,2-dichloropropane (1,2-DCP), and 1,1,2-trichloroethane (1,1,2-TCA) were assessed for each species. Chlorinated solvent depletion and daughter product formation determined as a function of time following inoculation into anaerobic media revealed preferential dechlorination of 1,1,2-TCA over both 1,2-DCA and 1,2-DCP for both species. 1,2-DCA in particular was not dechlorinated until 1,1,2-TCA reached low concentrations. In contrast, both species concurrently dechlorinated 1,2-DCA and 1,2-DCP over a comparably large concentration range. This is the first report of substrate interactions during chlorinated alkane dehalogenation by pure cultures, and the results provide insights into the chlorinated alkane transformation processes that may be expected for contaminant mixtures in environments where Dehalogenimonas spp. are present.