Mamoru Oshiki

N2O emission from a partial nitrification-anammox process and identification of a key biological process of N2O emission from anammox granules.

Emission of nitrous oxide (N(2)O) during biological wastewater treatment is of growing concern. The emission of N(2)O from a lab-scale two-reactor partial nitrification (PN)-anammox reactor was therefore determined in this study. The average emission of N(2)O from the PN and anammox process was 4.0±1.5% (9.6±3.2% of the removed nitrogen) and 0.1±0.07% (0.14±0.09% of the removed nitrogen) of the incoming nitrogen load, respectively. Thus, a larger part (97.5%) of N(2)O was emitted from the PN reactor. The total amount of N(2)O emission from the PN reactor was correlated to nitrite (NO(2)(-)) concentration in the PN effluent rather than DO concentration. In addition, further studies were performed to indentify a key biological process that is responsible for N(2)O emission from the anammox process (i.e., granules). In order to characterize N(2)O emission from the anammox granules, the in situ N(2)O production rate was determined by using microelectrodes for the first time, which was related to the spatial organization of microbial community of the granule as determined by fluorescence in situ hybridization (FISH). Microelectrode measurement revealed that the active N(2)O production zone was located in the inner part of the anammox granule, whereas the active ammonium consumption zone was located above the N(2)O production zone. Anammox bacteria were present throughout the granule, whereas ammonium-oxidizing bacteria (AOB) were restricted to only the granule surface. In addition, addition of penicillin G that inhibits most of the heterotrophic denitrifiers and AOB completely inhibited N(2)O production in batch experiments. Based on these results obtained, denitrification by putative heterotrophic denitrifiers present in the inner part of the granule was considered the most probable cause of N(2)O emission from the anammox reactor (i.e., granules).

Nitrate-dependent ferrous iron oxidation by anaerobic ammonium oxidation (anammox) bacteria.

ABSTRACT We examined nitrate-dependent Fe2+ oxidation mediated by anaerobic ammonium oxidation (anammox) bacteria. Enrichment cultures of “Candidatus Brocadia sinica” anaerobically oxidized Fe2+ and reduced NO3 − to nitrogen gas at rates of 3.7 ± 0.2 and 1.3 ± 0.1 (mean ± standard deviation [SD]) nmol mg protein−1 min−1, respectively (37°C and pH 7.3). This nitrate reduction rate is an order of magnitude lower than the anammox activity of “Ca. Brocadia sinica” (10 to 75 nmol NH4 + mg protein−1 min−1). A 15N tracer experiment demonstrated that coupling of nitrate-dependent Fe2+ oxidation and the anammox reaction was responsible for producing nitrogen gas from NO3 − by “Ca. Brocadia sinica.” The activities of nitrate-dependent Fe2+ oxidation were dependent on temperature and pH, and the highest activities were seen at temperatures of 30 to 45°C and pHs ranging from 5.9 to 9.8. The mean half-saturation constant for NO3 − ± SD of “Ca. Brocadia sinica” was determined to be 51 ± 21 μM. Nitrate-dependent Fe2+ oxidation was further demonstrated by another anammox bacterium, “Candidatus Scalindua sp.,” whose rates of Fe2+ oxidation and NO3 − reduction were 4.7 ± 0.59 and 1.45 ± 0.05 nmol mg protein−1 min−1, respectively (20°C and pH 7.3). Co-occurrence of nitrate-dependent Fe2+ oxidation and the anammox reaction decreased the molar ratios of consumed NO2 − to consumed NH4 + (ΔNO2 −/ΔNH4 +) and produced NO3 − to consumed NH4 + (ΔNO3 −/ΔNH4 +). These reactions are preferable to the application of anammox processes for wastewater treatment.

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.

Development of long-term stable partial nitrification and subsequent anammox process

The partial nitrification reactor was successfully started up and operated stably for more than 250 days with a maximum nitrite production rate of 1.12 kg-Nm(-3)day(-1). The important factors for successful partial nitrification were high ammonium loading rate (>1.0 kg-Nm(-3)day(-1)) and relatively high pH (ca. 8.0), giving high free ammonia concentrations (>10mg NH(3)-NL(-1)). In addition, the air flow rate must be controlled at the ratio of air flow rate to ammonium loading rate below 0.1 (m(air)(3)day(-1))/(kg-Nm(-3)day(-1)). After the establishment of stable partial nitrification, the effluent NO(2)(-)-N/NH(4)(+)-N ratio and effluent NO(3)(-)-N concentration were 1.20 ± 0.33 and 1.2 ± 1.0mg-NL(-1), respectively, which was then fed into an granular-sludge anammox reactor. Consistent nitrogen removal was achieved for more than 250 days with a maximum nitrogen removal rate of 15.0 kg-TNm(-3)day(-1).

Source identification of nitrous oxide on autotrophic partial nitrification in a granular sludge reactor

Emission of nitrous oxide (N2O) during biological wastewater treatment is of growing concern since N2O is a major stratospheric ozone-depleting substance and an important greenhouse gas. The emission of N2O from a lab-scale granular sequencing batch reactor (SBR) for partial nitrification (PN) treating synthetic wastewater without organic carbon was therefore determined in this study, because PN process is known to produce more N2O than conventional nitrification processes. The average N2O emission rate from the SBR was 0.32 ± 0.17 mg-N L(-1) h(-1), corresponding to the average emission of N2O of 0.8 ± 0.4% of the incoming nitrogen load (1.5 ± 0.8% of the converted NH4(+)). Analysis of dynamic concentration profiles during one cycle of the SBR operation demonstrated that N2O concentration in off-gas was the highest just after starting aeration whereas N2O concentration in effluent was gradually increased in the initial 40 min of the aeration period and was decreased thereafter. Isotopomer analysis was conducted to identify the main N2O production pathway in the reactor during one cycle. The hydroxylamine (NH2OH) oxidation pathway accounted for 65% of the total N2O production in the initial phase during one cycle, whereas contribution of the NO2(-) reduction pathway to N2O production was comparable with that of the NH2OH oxidation pathway in the latter phase. In addition, spatial distributions of bacteria and their activities in single microbial granules taken from the reactor were determined with microsensors and by in situ hybridization. Partial nitrification occurred mainly in the oxic surface layer of the granules and ammonia-oxidizing bacteria were abundant in this layer. N2O production was also found mainly in the oxic surface layer. Based on these results, although N2O was produced mainly via NH2OH oxidation pathway in the autotrophic partial nitrification reactor, N2O production mechanisms were complex and could involve multiple N2O production pathways.

Draft Genome Sequence of an Anaerobic Ammonium-Oxidizing Bacterium, “Candidatus Brocadia sinica”

ABSTRACT A draft genome sequence of an anaerobic ammonium-oxidizing (anammox) bacterium, “Candidatus Brocadia sinica,” was determined by pyrosequencing and by screening a fosmid library. A 4.07-Mb genome sequence comprising 3 contigs was assembled, in which 3,912 gene-coding regions, 47 tRNAs, and a single rrn operon were annotated.

PHA-accumulating microorganisms in full-scale wastewater treatment plants.

A study was conducted to clarify phylogenetic affiliations of polyhydroxyalkanoate (PHA)-accumulating microorganisms in full-scale activated sludge processes. Activated sludge samples obtained from three full-scale activated sludge processes were aerobically incubated with excess acetate to increase their PHA content. The buoyant density separation method was applied to selectively collect PHA-accumulating cells, which were then analysed by the group-level FISH and the PCR-DGGE-sequencing methods, and possible PHA-accumulating microbial groups were screened. A set of oligonucleotide probes targeting the microbial groups suspected to accumulate PHA was introduced, and seven oligonucleotide probes were newly designed for this purpose. PHA accumulation of probe-positive cells was confirmed by the post-FISH PHA staining method, wherein PHA staining with Nile Blue A (NBA) was applied after FISH. As a result, the following seven bacterial groups were found to have PHA: Dechloromonas, Accumulibacter, Thauera, Zoogloea, Comamonas, Competibacter and a novel cluster in Beta-proteobacteria. Based on the results of the post-FISH PHA staining method, these seven bacterial groups were estimated to account for around four-tenths to two-thirds of total PHA-accumulating microorganisms.

Microbial Community Composition of Polyhydroxyalkanoate-Accumulating Organisms in Full-Scale Wastewater Treatment Plants Operated in Fully Aerobic Mode

The removal of biodegradable organic matter is one of the most important objectives in biological wastewater treatments. Polyhydroxyalkanoate (PHA)-accumulating organisms (PHAAOs) significantly contribute to the removal of biodegradable organic matter; however, their microbial community composition is mostly unknown. In the present study, the microbial community composition of PHAAOs was investigated at 8 full-scale wastewater treatment plants (WWTPs), operated in fully aerobic mode, by fluorescence in situ hybridization (FISH) analysis and post-FISH Nile blue A (NBA) staining techniques. Our results demonstrated that 1) PHAAOs were in the range of 11–18% in the total number of cells, and 2) the microbial community composition of PHAAOs was similar at the bacterial domain/phylum/class/order level among the 8 full-scale WWTPs, and dominant PHAAOs were members of the class Alphaproteobacteria and Betaproteobacteria. The microbial community composition of α- and β-proteobacterial PHAAOs was examined by 16S rRNA gene clone library analysis and further by applying a set of newly designed oligonucleotide probes targeting 16S rRNA gene sequences of α- or β-proteobacterial PHAAOs. The results demonstrated that the microbial community composition of PHAAOs differed in the class Alphaproteobacteria and Betaproteobacteria, which possibly resulted in a different PHA accumulation capacity among the WWTPs (8.5–38.2 mg-C g-VSS−1 h−1). The present study extended the knowledge of the microbial diversity of PHAAOs in full-scale WWTPs operated in fully aerobic mode.

Revealing microbial community structures in large- and small-scale activated sludge systems by barcoded pyrosequencing of 16S rRNA gene

The diversity of bacterial groups in activated sludge from large- and small-scale wastewater treatment plants was explored by barcoded pyrosequencing of 16S rRNA gene. Activated sludge samples (three small and 17 large scale) were collected from 12 wastewater treatment plants to clarify precise taxonomy and relative abundances. DNA was extracted, and amplified by 4 base barcoded 27f/519r primer set. The 454 Titanium (Roche) pyrosequences were obtained and analyses performed by Quantitative Insight Into Microbial Ecology (QIIME) with around 100,000 reads. Sequence statistics were computed, while constructing a phylogenetic tree and heatmap. Computed results explained total microbial diversity at phylum and class level and resolution was further extended to Operational Taxonomic Unit (OTU) based taxonomic assignment for investigating community distribution based on individual sample. Composition of sequence reads were compared and microbial community structures for large- and small-scale treatment plants were identified as major phyla (Proteobacteria and Bacteroidetes) and classes (Betaproteobacteria and Bacteroidetes). Also, family level breakdowns were explained and differences in family Nitrospiraceae and phylum Actinobacteria found at their species level were also illustrated. Thus, the pyrosequencing method provides high resolution insight into microbial community structures in activated sludge that might have been unnoticed with conventional approaches.

Acetate uptake by PHA-accumulating and non-PHA-accumulating organisms in activated sludge from an aerobic sequencing batch reactor fed with acetate

The present study was conducted to evaluate the specific acetate uptake rates of microorganisms with and without polyhydroxyalkanoates (PHA) accumulation. Activated sludge was aerobically incubated with 75 mgC L(-1) radiolabeled or non-labeled acetate, and acetate consumption and PHA accumulation were monitored. Microorganisms were quantified as follows: all microbial cells by DAPI staining, whole acetate utilizing organisms by microautoradiography, and PHA-accumulating organisms by staining with Nile blue A. The abundance of acetate-utilizing organisms without PHA accumulation was also calculated from the outcomes. The estimate of acetate utilized by PHAAOs included both the acetate converted to PHA and that used to supply reducing power and ATP. Acetate utilized by PHAAOs and non-PHAAOs were divided by their respective abundances to obtain their respective specific acetate uptake rates: PHAAOs ranged between 5.3 and 8.0 x 10(-10) mgC cell(-1) h(-1), and non-PHAAOs ranged between 2.8 and 4.2 x 10(-10) mgC cell(-1) h(-1).