Comparative Genomics Provides Insights into the Taxonomy of Azoarcus and Reveals Separate Origins of Nif Genes in the Proposed Azoarcus and Aromatoleum Genera.

Among other attributes, the Betaproteobacterial genus Azoarcus has biotechnological importance for plant growth-promotion and remediation of petroleum waste-polluted water and soils. It comprises at least two phylogenetically distinct groups. The "plant-associated" group includes strains that are isolated from the rhizosphere or root interior of the C4 plant Kallar Grass, but also strains from soil and/or water; all are considered to be obligate aerobes and all are diazotrophic. The other group (now partly incorporated into the new genus Aromatoleum) comprises a diverse range of species and strains that live in water or soil that is contaminated with petroleum and/or aromatic compounds; all are facultative or obligate anaerobes. Some are diazotrophs. A comparative genome analysis of 32 genomes from 30 Azoarcus-Aromatoleum strains was performed in order to delineate generic boundaries more precisely than the single gene, 16S rRNA, that has been commonly used in bacterial taxonomy. The origin of diazotrophy in Azoarcus-Aromatoleum was also investigated by comparing full-length sequences of nif genes, and by physiological measurements of nitrogenase activity using the acetylene reduction assay. Based on average nucleotide identity (ANI) and whole genome analyses, three major groups could be discerned: (i) Azoarcus comprising Az. communis, Az. indigens and Az. olearius, and two unnamed species complexes, (ii) Aromatoleum Group 1 comprising Ar. anaerobium, Ar. aromaticum, Ar. bremense, and Ar. buckelii, and (iii) Aromatoleum Group 2 comprising Ar. diolicum, Ar. evansii, Ar. petrolei, Ar. toluclasticum, Ar. tolulyticum, Ar. toluolicum, and Ar. toluvorans. Single strain lineages such as Azoarcus sp. KH32C, Az. pumilus, and Az. taiwanensis were also revealed. Full length sequences of nif-cluster genes revealed two groups of diazotrophs in Azoarcus-Aromatoleum with nif being derived from Dechloromonas in Azoarcus sensu stricto (and two Thauera strains) and from Azospira in Aromatoleum Group 2. Diazotrophy was confirmed in several strains, and for the first time in Az. communis LMG5514, Azoarcus sp. TTM-91 and Ar. toluolicum TT. In terms of ecology, with the exception of a few plant-associated strains in Azoarcus (s.s.), across the group, most strains/species are found in soil and water (often contaminated with petroleum or related aromatic compounds), sewage sludge, and seawater. The possession of nar, nap, nir, nor, and nos genes by most Azoarcus-Aromatoleum strains suggests that they have the potential to derive energy through anaerobic nitrate respiration, so this ability cannot be usefully used as a phenotypic marker to distinguish genera. However, the possession of bzd genes indicating the ability to degrade benzoate anaerobically plus the type of diazotrophy (aerobic vs. anaerobic) could, after confirmation of their functionality, be considered as distinguishing phenotypes in any new generic delineations. The taxonomy of the Azoarcus-Aromatoleum group should be revisited; retaining the generic name Azoarcus for its entirety, or creating additional genera are both possible outcomes.

Interaction of "Candidatus Accumulibacter" and nitrifying bacteria to achieve energy-efficient denitrifying phosphorus removal via nitrite pathway from sewage.

To achieve energy-efficient denitrifying phosphorus removal via nitrite pathway from sewage, interaction of "Candidatus Accumulibacter" and nitrifying bacteria was investigated in a continuous-flow process. When nitrite in returned sludge of secondary settler was above 13mg/L, nitrite inhibition on anaerobic P-release of poly-phosphate organisms (PAOs) occurred. Clades IIC and IID were dominant, reaching 3.1%-11.9% of total bacteria. Clade IIC was sensitive to nitrite. Under low concentration of nitrite (<8mg/L), clade IIC primarily contributed to anoxic P-uptake. Clade IID had a strong tolerance to nitrite exposure. At high nitrite level (above 16mg/L), anoxic P-uptake was mainly performed by clade IID due to its strong tolerance to nitrite exposure. Ammonia oxidizing bacteria (AOB), nitrite oxidizing bacteria (NOB) and Accumulibacter interacted through variations of nitrite accumulation. High AOB abundance coupled with inhibition of NOB favored denitrifying phosphorus removal by clade IID. All Accumulibacter lineages were sorted into four clades of Type II. The most dominant ppk1 gene homologs were affiliated with clade IID, accounting for 69% of ppk1 clone library, and thus played an important role in denitrifying phosphorus removal via nitrite pathway.

Progress toward understanding the distribution of Accumulibacter among full-scale enhanced biological phosphorus removal systems.

This study investigated the role of Accumulibacter-related bacterial populations and factors influencing their distribution in enhanced biological phosphorus removal (EBPR) systems in the USA. For this purpose, five full-scale wastewater treatment facilities performing EBPR were surveyed. The facilities had different configurations but were all treating primarily domestic wastewater. Two facilities had history of poor EBPR performance. Batch-scale acetate uptake and inorganic phosphate (P(i)) release and uptake experiments were conducted to evaluate the EBPR activity of each sludge. Typical P(i) and acetate profiles were observed, and EBPR activity was found to be positively correlated to polyphosphate (polyP)-accumulating organism (PAO) abundance, as determined by staining intracellular polyP. The abundance of Accumulibacter-related organisms was investigated using fluorescent in situ hybridization. Accumulibacter-related organisms were present in all full-scale EBPR facilities, at levels ranging from 9 to 24% of total cells. More than 80% of Accumulibacter-related organisms were estimated to have high polyP content, confirming their involvement in EBPR in these five facilities. However, Accumulibacter-related PAOs were only a fraction (40-69%) of the total PAO population. The variation of Accumulibacter-related PAO abundance among these EBPR systems suggests that multiple interacting factors such as wastewater characteristics and operational conditions are structuring PAO communities.

An Azospira oryzae (syn Dechlorosoma suillum) strain that reduces selenate and selenite to elemental red selenium.

A bacterium that reduces the soluble selenium oxyanions, selenate and selenite, to insoluble elemental red selenium (Se(0)) was isolated from a laboratory reactor developed to remove selenate from groundwater. Gene sequence alignment of the 16S rRNA allowed identification of the isolate as Azospira oryzae. Biochemical and morphologic characterization confirm the identification. The isolate reduces selenate and selenite to Se(0) under microaerophilic and denitrifying conditions but not under aerobic conditions. It does not use selenate or selenite as terminal e(-) donors. Se oxyanion reduction causes the formation of Se nanospheres that are 0.25 +/- 0.04 microm in diameter. Nanospheres may be associated with the cells or free in the medium. The enzymatic activity associated with the reduction of selenate has a molecular mass of approximately 500 kD, and the enzymatic activity associated with the reduction of selenite has a mass of approximately 55 kD. Selenite reduction was inhibited by tungsten. The molecular masses of these activities were different from those associated with the reduction of dimethylsulfoxide, sulfate, and nitrite. This bacterium, or perhaps its enzymes or DNA, might be useful for the remediation of waters contaminated with Se oxyanions.

Ecophysiology of a group of uncultured Gammaproteobacterial glycogen-accumulating organisms in full-scale enhanced biological phosphorus removal wastewater treatment plants.

The presence of glycogen-accumulating organisms (GAOs) in enhanced biological phosphorus removal (EBPR) plants can seriously deteriorate the biological P-removal by out-competing the polyphosphate-accumulating organisms (PAOs). In this study, uncultured putative GAOs (the GB group, belonging to the Gammaproteobacteria) were investigated in detail in 12 full-scale EBPR plants. Fluorescence in situ hybridization (FISH) revealed that the biovolume of the GB bacteria constituted 2-6% of total bacterial biovolume. At least six different subgroups of the GB bacteria were found, and the number of dominant subgroups present in each plant varied between one and five. Ecophysiological investigations using microautoradiography in combination with FISH showed that, under aerobic or anaerobic conditions, all subgroups of the GB bacteria could take up acetate, pyruvate, propionate and some amino acids, while some subgroups in addition could take up formate and thymidine. Glucose, ethanol, butyrate and several other organic substrates were not taken up. Glycolysis was essential for the anaerobic uptake of organic substrates. Polyhydroxyalkanoates (PHA) but not polyphosphate (polyP) granules were detected in all GB bacterial cells. Polyhydroxyalkanoate formation after anaerobic uptake of acetate was confirmed by measuring the increase in fluorescence intensity of PHA granules inside GB bacterial cells after Nile blue staining. One GB subgroup was possibly able to denitrify, and several others were able to reduce nitrate to nitrite. PAOs were also enumerated by FISH in the same treatment plants. Rhodocyclus-related PAOs and Actinobacteria-related PAOs constituted up to 7% and 29% of total bacterial biovolume respectively. Rhodocyclus-related PAOs always coexisted with the GB bacteria and showed many physiological similarities. Factors of importance for the competition between the three groups of important bacteria in EBPR plants are discussed.

Petrobacter succinatimandens gen. nov., sp. nov., a moderately thermophilic, nitrate-reducing bacterium isolated from an Australian oil well.

A novel Gram-negative, aerobic and moderately thermophilic bacterium, strain 4BON(T), was isolated from a non-water-flooded Australian terrestrial oil reservoir. Cells were non-spore-forming straight rods, which were motile by means of a polar flagellum. The optimum growth conditions were 55 degrees C, pH 6.9 and 0.5 % NaCl. Strain 4BON(T) was oxidase- and catalase-positive; it grew on fumarate, pyruvate, succinate, formate, ethanol and yeast extract in the presence of oxygen or nitrate as terminal electron acceptor. Nitrate was reduced to nitrous oxide. The DNA G+C content of the strain was 58.6 mol%. The closest phylogenetic relative of strain 4BON(T) was Hydrogenophilus thermoluteolus (similarity of 91.8 %), of the beta-Proteobacteria. As strain 4BON(T) is physiologically and phylogenetically different from H. thermoluteolus, it is proposed that it be assigned to a novel species of a novel genus, Petrobacter succinatimandens gen. nov., sp. nov. The type strain is 4BON(T) (=DSM 15512(T)=CIP 107790(T)).