Takanori Awata

Physiological Characterization of an Anaerobic Ammonium-Oxidizing Bacterium Belonging to the “Candidatus Scalindua” Group

ABSTRACT The phylogenetic affiliation and physiological characteristics (e.g., Ks and maximum specific growth rate [μmax]) of an anaerobic ammonium oxidation (anammox) bacterium, “Candidatus Scalindua sp.,” enriched from the marine sediment of Hiroshima Bay, Japan, were investigated. “Candidatus Scalindua sp.” exhibits higher affinity for nitrite and a lower growth rate and yield than the known anammox species.

Influence of temperature and salinity on microbial structure of marine anammox bacteria

Anaerobic ammonium oxidation (anammox) is a type of biological oxidation mediated by a group of Planctomycete-like bacteria. Members of the genus Candidatus Scalindua are mainly found in marine environments, but not exclusively. This group is cultured using different inoculums and conditions; however, its optimal growth conditions are not clear. Additionally, little information is known about the factors that influence the activity and the selection of a population of marine anammox bacteria. This study was conducted to investigate the influence of temperature and salinity on the marine anammox community. To accomplish this, an up-flow fixed-bed column reactor was operated, and quantitative fluorescence in situ hybridization (FISH) with probes specific to dominant marine anammox bacteria was conducted. Anammox activity was observed at 20 and 30 °C, but not at 10 °C. A nitrogen removal rate of 0.32 kg TN m(-3) day(-1) was obtained at 20 °C. These results suggest that temperature affects the activity (nitrogen removal rate) of anammox bacteria, while salinity does not affect the activity in the marine anammox biofilm.

Enrichment of marine anammox bacteria in Hiroshima Bay sediments

Anaerobic ammonium oxidation (anammox) involves the microbiological oxidation of ammonium with nitrite as the electron acceptor and dinitrogen gas as the main product. The Scalindua species, an anammox genus that dominates natural habitats, plays an important role in catalysing the loss of nitrogen from marine environments. Until now, a few Scalindua species have been reported to be enriched from sea sediments. The objective of this study is to enrich marine anammox bacteria with coastal sediments in Hiroshima Bay as the inocula. The enrichment was achieved using a continuous upflow column reactor with synthetic sea water containing ammonium and nitrite. After 48 days of incubation, a simultaneous decrease in ammonium and nitrite was observed. A total nitrogen removal rate of 1.16 kg-N m(-3) day(-1) was attained after 306 days of incubation when the nitrogen loading rate was 1.32 kg-N m(-3) day(-1). Phylogenetic analysis revealed that the sequence similarity between the marine anammox-like bacteria in this reactor and the unidentified Candidatus Scalindua sp. was 96-98%. We successfully enriched marine anammox bacteria in the sediments of Hiroshima Bay by using synthetic sea water. Further studies are needed to investigate the characteristics of marine anammox bacteria, including optimal pH, temperature, and nitrogen loading rate.

Nitrogen removal using an anammox membrane bioreactor at low temperature

Membrane bioreactors (MBRs) have the ability to completely retain biomass and are thus suitable for slowly growing anammox bacteria. In the present study, an anammox MBR was operated to investigate whether the anammox activity would remain stable at low temperature, without anammox biomass washout. The maximum nitrogen removal rates were 6.7 and 1.1 g-N L⁻¹ day⁻¹ at 35 °C and 15 °C, respectively. Fluorescence in situ hybridization and 16S rRNA-based phylogenetic analysis revealed no change in the predominant anammox species with temperature because of the complete retention of anammox biomass in the MBR. These results indicate that the predominant anammox bacteria in the MBR cannot adapt to a low temperature during short-term operation. Conversely, anammox activity recovered rapidly after restoring the temperature from the lower value to the optimal temperature (35 °C). The rapid recovery of anammox activity is a distinct advantage of using an MBR anammox reactor.

Biomass Yield Efficiency of the Marine Anammox Bacterium, “Candidatus Scalindua sp.,” is Affected by Salinity

The growth rate and biomass yield efficiency of anaerobic ammonium oxidation (anammox) bacteria are markedly lower than those of most other autotrophic bacteria. Among the anammox bacterial genera, the growth rate and biomass yield of the marine anammox bacterium “Candidatus Scalindua sp.” is still lower than those of other anammox bacteria enriched from freshwater environments. The activity and growth of marine anammox bacteria are generally considered to be affected by the presence of salinity and organic compounds. Therefore, in the present study, the effects of salinity and volatile fatty acids (VFAs) on the anammox activity, inorganic carbon uptake, and biomass yield efficiency of “Ca. Scalindua sp.” enriched from the marine sediments of Hiroshima Bay, Japan, were investigated in batch experiments. Differences in VFA concentrations (0–10 mM) were observed under varying salinities (0.5%–4%). Anammox activity was high at 0.5%–3.5% salinity, but was 30% lower at 4% salinity. In addition, carbon uptake was higher at 1.5%–3.5% salinity. The results of the present study clearly demonstrated that the biomass yield efficiency of the marine anammox bacterium “Ca. Scalindua sp.” was significantly affected by salinity. On the other hand, the presence of VFAs up to 10 mM did not affect anammox activity, carbon uptake, or biomass yield efficiency.

Polyphasic characterization of an anaerobic hexachlorobenzene-dechlorinating microbial consortium with a wide dechlorination spectrum for chlorobenzenes

An anaerobic consortium that was capable of reductively dechlorinating hexachlorobenzene (HCB) to benzene was enriched from contaminated sediment. The consortium was capable of dechlorinating all chlorobenzene isomers except 1,4-dichlorobenzene. Singly and doubly flanked chlorines, as well as unflanked meta-substituted chlorines, were dechlorinated, although doubly flanked chlorines were preferred. Formate, acetate and lactate (but not ethanol) could be utilized as optimum electron donors for reductive dechlorination. Alternative electron acceptors, including nitrate and sulfate, completely inhibited HCB degradation, whereas amorphous iron oxide (FeOOH) did not suppress dechlorination activity. No degradation was found in chloramphenicol-treated consortium; however, vancomycin, molybdate, and 2-bromoethanesulfonate did not inhibit HCB dechlorination. The results of inhibitory treatments suggested that the dechlorinators were non-sulfate-reducing gram-negative or vancomycin resistant gram-positive bacteria. In addition to physiological characterization, analyses of 16S rRNA gene library of the consortium and quantitative PCR of 16S rRNA genes suggested that Dehalococcoides sp. was involved in the reductive dechlorination of HCB, and Geobacter sp. may serve as a dechlorinating candidate.

Characterization of the in situ ecophysiology of novel phylotypes in nutrient removal activated sludge treatment plants

An in depth understanding of the ecology of activated sludge nutrient removal wastewater treatment systems requires detailed knowledge of the community composition and metabolic activities of individual members. Recent 16S rRNA gene amplicon surveys of activated sludge wastewater treatment plants with nutrient removal indicate the presence of a core set of bacterial genera. These organisms are likely responsible for the bulk of nutrient transformations underpinning the functions of these plants. While the basic activities of some of these genera in situ are known, there is little to no information for the majority. This study applied microautoradiography coupled with fluorescence in situ hybridization (MAR-FISH) for the in situ characterization of selected genus-level-phylotypes for which limited physiological information is available. These included Sulfuritalea and A21b, both within the class Betaproteobacteria, as well as Kaga01, within sub-group 10 of the phylum Acidobacteria. While the Sulfuritalea spp. were observed to be metabolically versatile, the A21b and Kaga01 phylotypes appeared to be highly specialized.