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.

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

We examined nitrate-dependent Fe(2+) oxidation mediated by anaerobic ammonium oxidation (anammox) bacteria. Enrichment cultures of "Candidatus Brocadia sinica" anaerobically oxidized Fe(2+) 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 (15)N tracer experiment demonstrated that coupling of nitrate-dependent Fe(2+) oxidation and the anammox reaction was responsible for producing nitrogen gas from NO3(-) by "Ca. Brocadia sinica." The activities of nitrate-dependent Fe(2+) 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 Fe(2+) oxidation was further demonstrated by another anammox bacterium, "Candidatus Scalindua sp.," whose rates of Fe(2+) 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 Fe(2+) 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.

Rapid quantification of polyhydroxyalkanoates (PHA) concentration in activated sludge with the fluorescent dye Nile blue A.

The present study was conducted (1) to develop a rapid quantification method of polyhydroxyalkanoates (PHA) concentration in activated sludge by Nile blue A staining and fluorescence measurement and (2) to perform on-line monitoring of PHA concentrations in activated sludge. Activated sludge samples collected from laboratory scale sequencing batch reactors and full-scale wastewater treatment plants were stained with Nile blue A and their fluorescence intensities were determined. There was a high correlation (R2 > 0.97) between the fluorescence intensities of Nile blue A and PHA concentrations in activated sludge determined by gas chromatography. The Nile blue A staining and fluorescence measurement method allows us to determine PHA concentrations in activated sludge within only five minutes and up to 96 samples can be measured at once by using microplate reader. On-line monitoring of PHA concentrations in activated sludge was achieved by using a fluorometer equipped with a flow cell and the time point at which PHA concentration in activated sludge reached the maximum level could be identified. In addition, we examined the influence of pH, floc size and co-existing chemicals in activated sludge suspension on the fluorescence intensities of Nile blue A.

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).