Daisuke Hira

Anammox organism KSU-1 expresses a NirK-type copper-containing nitrite reductase instead of a NirS-type with cytochrome cd1.

Anaerobic ammonium oxidation (anammox) and denitrification are two distinct microbial reactions relevant to the global nitrogen cycle. The proposed initial step of the anammox reactions, reduction of nitrite to nitric oxide, has been postulated to be identical to that in denitrification catalyzed by the dissimilatory nitrite reductase of the cytochrome cd 1‐type. Here, we characterized the copper‐containing nitrite reductase homolog encoded by nirK detected in the genome of an anammox bacterium strain KSU‐1. We hypothesize that this NirK‐type nitrite reductase, rather than a nitrite reductase of the cytochrome cd 1‐type (NirS), is likely to catalyze nitrite reduction in anammox organism KSU‐1.

Physiological characterization of anaerobic ammonium oxidizing bacterium ‘Candidatus Jettenia caeni’

To date, six candidate genera of anaerobic ammonium-oxidizing (anammox) bacteria have been identified, and numerous studies have been conducted to understand their ecophysiology. In this study, we examined the physiological characteristics of an anammox bacterium in the genus Candidatusu2005Jettenia. Planctomycete KSU-1 was found to be a mesophilic (20-42.5°C) and neutrophilic (pH 6.5-8.5) bacterium with a maximum growth rate of 0.0020u2009h(-1) . Planctomycete KSU-1 cells showed typical physiological and structural features of anammox bacteria; i.e. (29) N2 gas production by coupling of (15) NH4 (+) and (14) NO2 (-) , accumulation of hydrazine with the consumption of hydroxylamine and the presence of anammoxosome. In addition, the cells were capable of respiratory ammonification with oxidation of acetate. Notably, the cells contained menaquinone-7 as a dominant respiratory quinone. Proteomic analysis was performed to examine underlying core metabolisms, and high expressions of hydrazine synthase, hydrazine dehydrogenase, hydroxylamine dehydrogenase, nitrite/nitrate oxidoreductase and carbon monoxide dehydrogenase/acetyl-CoA synthase were detected. These proteins require iron or copper as a metal cofactor, and both were dominant in planctomycete KSU-1 cells. On the basis of these experimental results, we proposed the name Ca. Jettenia caeni sp. nov. for the bacterial clade of the planctomycete KSU-1.

Enhancement of anammox performance in a novel non-woven fabric membrane bioreactor (nMBR)

To reduce operating costs and membrane fouling of conventional membrane bioreactors (cMBR), a novel MBR using a non-woven fabric membrane (nMBR) was constructed and the performance of the two MBRs was compared for anaerobic ammonium oxidation (anammox) cultivation. The results showed that the start-up period for the nMBR (44 days) was notably shorter than that for the cMBR (56 days), meanwhile the nMBR achieved a 2-times higher nitrogen removal rate (231.5 mg N per L per d) compared to the cMBR (112.3 mg N per L per d). Illumina MiSeq sequencing showed that Candidatus Kuenenia and Candidatus Jettenia were the main distinguished anammox bacteria. FISH analysis revealed that anammox bacteria predominated in both reactors, especially in the nMBR (58%) corresponding to a qPCR analysis of 1.07 × 109 copies per mL (day 120). N2O emission analysis confirmed the advantage of the nMBR in N2O reduction to reduce the influence of greenhouse gas emission while treating identical nitrogen. These results clearly demonstrated that nMBRs could be a prospective choice for anammox start-up and performance enhancement.

Reduction of nitric oxide catalyzed by hydroxylamine oxidoreductase from an anammox bacterium.

The hydroxylamine oxidoreductase (HAO) from the anammox bacterium, Candidatus Kuenenia stuttgartiensis has been reported to catalyze the oxidation of hydroxylamine (NH2OH) to nitric oxide (NO) by using bovine cytochrome c as an oxidant. In contrast, we investigated whether the HAO from anammox bacterium strain KSU-1 could catalyze the reduction of NO with reduced benzyl viologen (BVred) and the NO-releasing reagent, NOC 7. The reduction proceeded, resulting in the formation of NH2OH as a product. The oxidation rate of BVred was proportional to the concentration of BVred itself for a short period in each experiment, a situation that was termed quasi-steady state. The analyses of the states at various concentrations of HAO allowed us to determine the rate constant for the catalytic reaction, (2.85 ± 0.19) × 10(5) M(-1) s(-1), governing NO reduction by BVred and HAO, which was comparable to that reported for the HAO from the ammonium oxidizer, Nitrosomonas with reduced methyl viologen. These results suggest that the anammox HAO functions to adjust anammox by inter-conversion of NO and NH2OH depending on the redox potential of the physiological electron transfer protein in anammox bacteria.

Ammonia Removal Using Nitrification and Anammox in a Single Reactor

In this work we evaluated the combination of nitrification and anammox bacteria in a single tank to remove ammonia by deammonification process. The deammonification process is a completely autotrophic nitrogen removal approach that eliminates the carbon needs for denitrification. Thus, it can be a promising approach for the biological removal of ammonia (NH4+) from anaerobic digester effluents that are low in carbon and high in ammonia concentration. A high performance nitrifying sludge, HPNS (NRRL B-50298), was mixed with anammox bacterial sludge, Brocadia caroliniensis (NRRL B-50286), in a single reactor. The reactor was an aerated vessel operated under continuous flow. It contained biofilm plastic carriers that were fluidized by the aeration. The process water temperature was 22oC and DO <0.5 mg/L. It was tested using inorganic synthetic wastewater and anaerobically digested swine wastewater. Ammonia removal rates of 1.2 kg N/m3-reactor/day were obtained with influent wastewater concentration of 440 mg/L NH4-N and a removal efficiency of 87%. The stoichiometry of the reaction obtained was consistent with deammonification process combining partial nitritation and anammox. Analyses of the 16S rRNA of the single tank bacterial community detected Brocadia caroliniensis and Nitrosomonas europaea, and a large number (54 to 63%) of Bacteroidetes. Results obtained with the deammonification process showed several advantages over nitrification/denitrification: 1) it reduced 56% of the oxygen requirements to remove the ammonia; 2) it did not require carbon; and 3) it removed nitrogen at higher rates in a single-tank, further reducing equipment costs. Therefore, deammonification can be a key technology for development of more economical and energy efficient biological ammonia removal systems in the near future.

Impact of aerobic acclimation on the nitrification performance and microbial community of landfill leachate sludge

Nitrogenous pollution of water is regarded as a global environmental problem, and nitrogen removal has become an important issue in wastewater treatment processes. Landfill leachate is a typical large source of nitrogenous wastewater. Although the characteristics of leachate vary according to the age of the landfill, leachates of mature landfill have high concentrations of nitrogenous compounds. Most nitrogen in these leachates is in the form of ammonium nitrogen. In this study, we investigated the bacterial community of sludge from a landfill leachate lagoon by pyrosequencing of the bacterial 16S rRNA gene. The sludge was acclimated in a laboratory-scale reactor with aeration using a mechanical stirrer to promote nitrification. On 149 days, nitrification was achieved and then the bacterial community was also analyzed. The bacterial community was also analyzed after nitrification was achieved. Pyrosequencing analyses revealed that the abundances of ammonia-oxidizing and nitrite-oxidizing bacteria were increased by acclimation and their total proportions increased to >15% of total biomass. Changes in the sulfate-reducing and sulfur-oxidizing bacteria were also observed during the acclimation process. The aerobic acclimation process enriched a nitrifying microbial community from the landfill leachate sludge. These results suggested that the aerobic acclimation is a processing method for the nitrification ammonium oxidizing throw the enrichment of nitrifiers. Improvement of this acclimation method would allow nitrogen removal from leachate by nitrification and sulfur denitrification.

Anammox Organism KSU-1 Expresses a Novel His/DOPA Ligated Cytochrome c.

Anammox is a bacterial energy metabolic process that forms N2 gas from nitrite and ammonium ions. The enzymatic mechanisms of anammox have been gradually revealed; however, the electron transport chain in anammox bacteria remains poorly understood. In the present study, we purified and characterized two low-molecular-weight c-type cytochromes from an enriched culture of the anammox bacterium strain, KSU-1. Their genes, KSU1_B0428 and KSU1_C0855, were identified in the KSU-1 genome, and their recombinant proteins were characterized. KSU1_B0428 is a typical c-type cytochrome with a His/Met coordinated heme, acting as an electron transfer protein. In contrast, KSU1_C0855 could not be assigned as a known cytochrome and its heme was suggested to have an uncommon axial ligand set. Crystal structural analyses of C0855 clearly showed that its heme iron is coordinated by His15 as a fifth ligand. Moreover, the sixth coordination site is occupied by the aromatic ring of Tyr60, and an unassignable electron density that is inseparable with that of aromatic carbon of Tyr60 was found. The additional electron density was assigned to an O atom by molecular mass analyses. Therefore, Tyr60 would be chemically modified to 3,4-dihydroxyphenylalanine and bound to the Fe atom. We revealed that an anammox bacterium strain KSU-1 expresses a novel cytochrome c having an unprecedented His/3,4-dihydroxyphenylalanine coordinating heme. The expression of the novel c-type cytochrome might be required for the redox reaction of the anammox process.