Free nitrous acid selectively inhibits and eliminates nitrite oxidizers from nitrifying sequencing batch reactor

In a complete nitrification sequencing batch reactor (CNSBR), where ammonium containing wastewater (200–1,000 mg N/L) is completely oxidized to nitrate up to 2.4 kg NH₄ ⁺–N/m³ d, both ammonia oxidizers and nitrite oxidizers were enriched in the sludge granules. Quantitative fluorescence in situ hybridization analyses of the sludge granules of the CNSBR showed that ammonia oxidizers and nitrite oxidizers occupied 31 and 4.2% of total bacteria, respectively. Most of the nitrite oxidizers were Nitrobacter species (95% of the nitrite oxidizers) and the remainder was Nitrospira species. The population of nitrite oxidizers was significantly higher than that of partial nitrification SBR (PNSBR) where most of the ammonium was oxidized to nitrite. The PNSBR had 37% (ammonia oxidizers) and 0.4% (nitrite oxidizers) of total bacteria. Comparative study with CNSBR and PNSBR revealed that free nitrous acid, rather than free ammonia, played a critical inhibition role to wash out nitrite oxidizers from the reactor. The concentrations of free ammonia and nitrite as well as free nitrous acid in the CNSBR selected Nitrobacter as the dominant nitrite oxidizers rather than Nitrospira.     

Analysis and succession of nitrifying bacteria community structure in sequencing biofilm batch reactor

To reveal the succession procedure of ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) community structure in sequencing biofilm batch reactor (SBBR), the molecular biological techniques of denaturing gradient gel electrophoresis (DGGE), cloning, and real-time PCR were applied. DGGE showed that the structural diversity of the bacterial community increased during the biofilm formation period, and some kinds of populations had been highly preponderant consistently. The results of cloning and sequencing revealed that Nitrosomonas was the dominant species. The real-time PCR analysis indicated that the amount of the AOB increased significantly after the cultivation period, and the NOB gradually decreased. The AOB content on the 25th day was 17 times that of the 6th day. It also showed the biofilm formed successfully with accumulating nitrite and prepared to achieve the achievement of simultaneous nitrification and denitrification in SBBR. Furthermore, the ammonia-oxidizing rate was in correspondence with the NH₄ ⁺-N removal efficiency.     

污水短程硝化体系氨氧化菌与亚硝酸盐氧化菌的生态学研究进展

短程硝化(partial nitrification, PN)是一种绿色低碳的生物脱氮创新技术,伴随厌氧氨氧化(anaerobic ammonia oxidation, Anammox)污水脱氮技术的进一步推广,短程硝化作为提供其电子受体的重要环节,已成为了污水脱氮领域的研究热点。氨氧化菌(ammonia-oxidizing bacteria,AOB)和亚硝酸盐氧化菌(nitrite-oxidizing bacteria, NOB)是该技术的核心竞争微生物,掌握这两类微生物的生态学特征,借助生态学理论和手段调控AOB淘汰NOB,提高种群的可预测性,对于实现稳定高效的短程硝化具有重要意义。本文基于生态学角度介绍了AOB和NOB基础分类、生理性能及生态位分离,重点综述了短程硝化系统中AOB和NOB的生长动力学、群落构建、环境因素和相互作用,最后对这两类微生物的未来研究重点和研究方法进行了展望,为短程硝化工艺的快速启动和稳定运行提供理论指导。     

Activity and population structure of nitrifying bacteria in an activated-sludge reactor containing polymer beads

Photo-crosslinked polymer beads were introduced into a laboratory activated-sludge unit containing municipal sewage sludge and the effect on nitrifying capacity was examined. The ammonia load started at a nitrogen-loading rate of 0.02 kg m(-3) day(-1) and was increased stepwise. It was found that the bead-containing unit could almost completely oxidize ammonia (over 95%) up to a nitrogen-loading rate of 0.216 kg m(-3) day(-1), whereas the maximum loading rate of the control unit (without polymer beads) was 0.096 kg m(-3) day(-1). The nitrifying potential of suspended and bead-associated organisms in the bead-containing unit was measured under different loading conditions. It was found that the bead-associated organisms exhibited high specific activities under high loading conditions and that the contribution of the bead-associated organisms to nitrification was greater than that of the suspended solids under these conditions. The bacterial population dynamics in the suspended solids and bead-associated organisms were analysed by denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rRNA gene fragments and by fluorescence in situ hybridization with group-specific probes. Among the known nitrifying organisms, ammonia-oxidizing beta-proteobacteria and Nitrospira-related organisms were detected by these approaches. A comparison of the activity dynamics and population dynamics, however, suggested a possibility that other organisms may also have been involved in the nitrification process under high loading conditions.     

Decreased N2O reduction by low soil pH causes high N2O emissions in a riparian ecosystem

Quantification of harmful nitrous oxide (N(2)O) emissions from soils is essential for mitigation measures. An important N(2)O producing and reducing process in soils is denitrification, which shows deceased rates at low pH. No clear relationship between N(2)O emissions and soil pH has yet been established because also the relative contribution of N(2)O as the denitrification end product decreases with pH. Our aim was to show the net effect of soil pH on N(2)O production and emission. Therefore, experiments were designed to investigate the effects of pH on NO(3)(-) reduction, N(2)O production and reduction and N(2) production in incubations with pH values set between 4 and 7. Furthermore, field measurements of soil pH and N(2)O emissions were carried out. In incubations, NO(3)(-) reduction and N(2) production rates increased with pH and net N(2)O production rate was highest at pH 5. N(2)O reduction to N(2) was halted until NO(3)(-) was depleted at low pH values, resulting in a built up of N(2)O. As a consequence, N(2)O:N(2) production ratio decreased exponentially with pH. N(2)O reduction appeared therefore more important than N(2)O production in explaining net N(2)O production rates. In the field, a negative exponential relationship for soil pH against N(2)O emissions was observed. Soil pH could therefore be used as a predictive tool for average N(2)O emissions in the studied ecosystem. The occurrence of low pH spots may explain N(2)O emission hotspot occurrence. Future studies should focus on the mechanism behind small scale soil pH variability and the effect of manipulating the pH of soils.     

Sources and sinks of nitrous oxide (N2O) in deep lakes

As reported from marine systems, we found that also in15 prealpine lakes N₂O concentrations werestrongly correlated with O₂ concentrations. Inoxic waters below the mixed surface layer, N₂Oconcentrations usually increased with decreasingO₂ concentrations. N₂O is produced in oxicepilimnia, in oxic hypolimnia and at oxic-anoxicboundaries, either in the water or at the sediment-waterinterface. It is consumed, however, incompletely anoxic layers. Anoxic water layers weretherefore N₂O undersaturated. All studied lakeswere sources for atmospheric N₂O, including thosewith anoxic, N₂O undersaturated hypolimnia.However, compared to agriculture, lakes seem not tocontribute significantly to atmospheric N₂Oemissions.     

Influence of nutrient solution pH on N2O and N2 emissions from a soilless culture system

The influence of nutrient solution pH on the emission of N2O and N2 was investigated during cultivation of cucumbers in a closed-loop rockwool system. Between pH 4 and 7 these gaseous nitrogen losses increased from 1.6 to 21.1% of the N fertilizer input. This was equivalent to average flux rates of 0.06 and 0.85 kg nitrogen per hectare greenhouse area and day, respectively. The N2O/N2 ratio was inversely related to the total gaseous nitrogen losses. At neutral pH dinitrogen was the main emission product, whereas more acidic conditions favoured the emission of nitrous oxide. The pH effects were probably not indirectly affected by root respiration or exudation as much as by a direct inhibition of the activity of denitrifying microorganisms due to high H+ concentrations since similar results were obtained in unplanted nutrient solution systems with the addition of glucose as carbon source. Despite the low microbial denitrification activity under acidic conditions, nitrogen balance deficits of up to one-fifth of the N input still occurred. It is suggested these losses were predominantly caused by chemodenitrification.     

Differential distribution of ammonia- and nitrite-oxidising bacteria in flocs and granules from a nitrifying/denitrifying sequencing batch reactor

A lab-scale sequencing batch reactor was operated with alternating anoxic/aerobic conditions for nitrogen removal. Flocs and granules co-existed in the same reactor, with distinct aggregate structure and size, for over 180 days of reactor operation. Process data showed complete nitrogen removal, with temporary nitrite accumulation before full depletion of ammonia in the aerobic phase. Microbial quantification of the biomass by fluorescence in situ hybridisation showed that granules contained most of the nitrite-oxidising bacteria (NOB) whereas the ammonium-oxidising bacteria (AOB) seemed to be more abundant in the flocs. This was supported by microsensor measurements, which showed a higher potential of NO₂⁻ uptake than NH₄ uptake in the granules. The segregation is possibly linked to the different growth rates of the two types of nitrifiers and the reactor operational conditions, which produced different sludge retention time for flocs and granules. The apparent physical separation of AOB and NOB in two growth forms could potentially affect mass transfer of NO₂⁻ from AOB to NOB, but the data presented here shows that it did not impact negatively on the overall nitrogen removal.     

Ammonia-oxidizing bacteria dominates over ammonia-oxidizing archaea in a saline nitrification reactor under low DO and high nitrogen loading

A continuous nitrification reactor treating saline wastewater was operated for almost 1 year under low dissolved oxygen (DO) levels (0.15-0.5 mg/L) and high nitrogen loadings (0.26-0.52 kg-N/(m(3) day)) in four phases. The diversity and abundance of ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB) were analyzed by cloning, terminal restriction fragment length polymorphism (T-RFLP) and quantitative polymerase chain reaction (qPCR). The results showed that there were only one dominant AOA species and one dominant AOB species in the reactor in all of the four experimental phases. The amoA gene of the dominant AOA only had a similarity of 89.3% with the cultured AOA species Nitrosopumilus maritimus SCM1. All of the AOB species detected in the reactor belong to Nitrosomonas genus and it was found that the AOB populations changed with the ammonium loadings and DO levels. The abundance of AOB in the reactor was ～40 times larger than that of AOA, and the ratio of AOB to AOA increased significantly up to ～2,000 to ～4,000 with the increase of ammonium loading, indicating that AOB are much more competitive than AOA in high ammonium environments and probably AOA play a less important role than AOB in the nitrification reactors.     

Differential distribution of ammonia- and nitrite-oxidising bacteria in flocs and granules from a nitrifying/denitrifying sequencing batch reactor

A lab-scale sequencing batch reactor was operated with alternating anoxic/aerobic conditions for nitrogen removal. Flocs and granules co-existed in the same reactor, with distinct aggregate structure and size, for over 180 days of reactor operation. Process data showed complete nitrogen removal, with temporary nitrite accumulation before full depletion of ammonia in the aerobic phase. Microbial quantification of the biomass by fluorescence in situ hybridisation showed that granules contained most of the nitrite-oxidising bacteria (NOB) whereas the ammonium-oxidising bacteria (AOB) seemed to be more abundant in the flocs. This was supported by microsensor measurements, which showed a higher potential of NO₂⁻ uptake than NH₄ uptake in the granules. The segregation is possibly linked to the different growth rates of the two types of nitrifiers and the reactor operational conditions, which produced different sludge retention time for flocs and granules. The apparent physical separation of AOB and NOB in two growth forms could potentially affect mass transfer of NO₂⁻ from AOB to NOB, but the data presented here shows that it did not impact negatively on the overall nitrogen removal.     

Settleability and kinetics of a nitrifying sludge in a sequencing batch reactor

A physiological study of a nitrifying sludge was carried out in a sequencing batch reactor (SBR). Pseudo steady-state nitrification conditions were obtained with an ammonium removal efficiency of 99% +/- 1% and 98% +/- 2% conversion of NH4+-N to NO3 - -N. The rate of biomass production was negligible (1.3 +/- 0.1 mg microbial protein-N.L(-1).d(-1)). The sludge presented good settling properties with sludge volume index values lower than 20 mL.g(-1) and an exopolymeric protein/carbohydrate ratio of 0.53 +/- 0.34. Kinetic results indicated that the nitrifying behavior of the sludge changed with the number of cycles. After 22 cycles, a decrease in the specific rate of NO3- -N production coupled with an increase in the NO2- -N accumulation were observed. These results showed that the activity of the nitrite oxidizing bacteria decreased at a longer operation time. Ammonia oxidizing bacteria were found to exhibit the best stability. After 4 months of operation, the specific rates of NH4+-N consumption and NO3- -N production were 1.72 NH4+-N per microbial protein-N per hour (g.g(-1).h(-1)) and 0.54 NO3- -N per microbial protein-N per hour (g.g(-1).h(-1)), respectively.     

Microenvironments and distribution of nitrifying bacteria in a membrane-bound biofilm

The distribution of nitrifying bacteria of the genera Nitrosomonas, Nitrosospira, Nitrobacter and Nitrospira was investigated in a membrane-bound biofilm system with opposed supply of oxygen and ammonium. Gradients of oxygen, pH, nitrite and nitrate were determined by means of microsensors while the nitrifying populations along these gradients were identified and quantified using fluorescence in situ hybridization (FISH) in combination with confocal laser scanning microscopy. The oxic part of the biofilm which was subjected to high ammonium and nitrite concentrations was dominated by Nitrosomonas europaea -like ammonia oxidizers and by members of the genus Nitrobacter. Cell numbers of Nitrosospira sp. were 1–2 orders of magnitude lower than those of N. europaea . Nitrospira sp. were virtually absent in this part of the biofilm, whereas they were most abundant at the oxic–anoxic interface. In the totally anoxic part of the biofilm, cell numbers of all nitrifiers were relatively low. These observations support the hypothesis that N. europaea and Nitrobacter sp. can out-compete Nitrosospira and Nitrospira spp. at high substrate and oxygen concentrations. Additionally, they suggest microaerophilic behaviour of yet uncultured Nitrospira sp. as a factor of its environmental competitiveness.     

Mediating N2O emissions from municipal solid waste landfills: Impacts of landfill operating conditions on community structure of ammonia-oxidizing bacteria in cover soils

Chemolithotrophic ammonia-oxidizing bacteria (AOB) can produce N₂O, a highly potent greenhouse gas. Denaturing gradient gel electrophoresis (DGGE) analyses of the ammonia monooxygenase structural gene (amoA) and 16S rDNA gene were used to investigate the AOB community structure in the cover soils of municipal solid waste (MSW) landfills under three operating conditions: (a) MSW with soil cover, (b) MSW with soil cover, irrigation piping and vegetation, and (c) MSW covered with high-density polyethylene (HDPE) liner, soil cover, irrigation piping and vegetation. AOB species in MSW cover soils were significantly distinguished by the operation of HDPE liner isolation. The community structures of the Nitrosomonas europaea-like AOB species dominated in soils without HDPE liner isolation, whether vegetation and irrigation with landfill leachate existed or not, whereas Nitrospira-like AOB species dominated in soils with HDPE liner isolation. Lower N₂O flux from the soils with HDPE liner isolation would be partially related to these special community structures.     

Formation and long-term stability of nitrifying granules in a sequencing batch reactor

The formation and long-term stability of nitrifying granules in a sequencing batch reactor was investigated in this study. The results showed that nitrifying granules with a size of 240 microm and SVI of 40 ml g(-1) were formed on day 21 at a settling time of 10 min. Maintaining settling time at 15 min from day 57 to 183 did not affect the physical characteristics of sludge and the fraction of suspended floc in the sludge. In addition, nitrifying granules could tolerate the fluctuations of nitrogen loading rate from 0.72 to 1.8 g l(-1)d(-1) during 2 months without the change of physical characteristics. However, it was observed that complete nitrification to nitrate and partial nitrification to nitrite by sludge converted each other corresponding to the change of the influent NH4+-N concentration. Thus, an appropriate method is needed to maintain a stable complete nitrification or partial nitrification under the conditions with changing influent NH4+-N concentrations and nitrogen loading rates.     

The influence of pH on the mutagenicity in yeast of N-methylnitrosamides and nitrous acid

Summary In a reverse mutation system with the haploid, adenine requiring strain, ad_6–45, of Saccharomyces cerevisiae it could be demonstrated that N-methyl-nitrosamides are highly mutagenic down to ph 2. By chemical methods it could be shown that nitrosamides decompose into nitrous acid at ph 2.2–2.3. Moreover, in the case of NMG, NMH and NMU, deamination of adenine to hypoxanthine was found to occur at pH 2. These results led to the conclusion that N-methyl-nitrosamides at low pH possibly exert their mutagenicities via deamination by nitrous acid besides the alkylation by diazomethane probably prevailing at higher pH. Mere incubation of yeast cells in buffers of low pH was not mutagenic.     

Characterization and quantification of ammonia-oxidizing archaea (AOA) and bacteria (AOB) in a nitrogen-removing reactor using T-RFLP and qPCR

Using ammonia monooxygenase α-subunit (amoA) gene and 16S rRNA gene, the community structure and abundance of ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB) in a nitrogen-removing reactor, which was operated for five phases, were characterized and quantified by cloning, terminal restriction fragment length polymorphism (T-RFLP), and quantitative polymerase chain reaction (qPCR). The results suggested that the dominant AOB in the reactor fell to the genus Nitrosomonas, while the dominant AOA belonged to Crenarchaeotal Group I.1a in phylum Crenarchaeota. Real-time PCR results demonstrated that the levels of AOB amoA varied from 2.9 × 10³ to 2.3 × 10⁵ copies per nanogram DNA, greatly (about 60 times) higher than those of AOA, which ranged from 1.7 × 10² to 3.8 × 10³ copies per nanogram DNA. This indicated the possible leading role of AOB in the nitrification process in this study. T-RFLP results showed that the AOB community structure significantly shifted in different phases while AOA only showed one major peak for all the phases. The analyses also suggested that the AOB community was more sensitive than that of AOA to operational conditions, such as ammonia loading and dissolved oxygen.     

Effects of roxithromycin on ammonia-oxidizing bacteria and nitrite-oxidizing bacteria in the rhizosphere of wheat

In a pot-cultural experiment, the impact of the antibiotic roxithromycin (ROX) addition was assessed on the diversities of microbial structure and function communities, especially involved in ammonia and nitrite oxidation in wheat rhizosphere soil with and without the addition of earthworms. The abundances of ammonia-oxidizing bacteria (AOB), nitrite-oxidizing bacteria (NOB), and total bacteria were surveyed by the quantitative PCR. The quantities of total bacteria, AOB, and NOB with earthworms were higher than those without earthworms because of the synergistic effect. ROX inhibited the growth of AOB in all treatments, although the quantities of AOB were in a light increase in medium and heavy polluted treatments compared with that in the light polluted treatments. Different from AOB, the quantities of NOB were lowest in light polluted treatments, but the quantities of NOB were rapidly increased in medium and heavy polluted treatments compared with that in the control. These results indicated that the application of ROX principally had a negative effect on nitrification performance by affecting the abundances and relative ratios of both AOB and NOB in soil communities, which affected the N cycle in an agricultural ecosystem. According to the metabolic diversities evaluated by the biologic assay, the tendency of metabolic diversities was quite contrary to the quantities of NOB in all treatments and showed the contrast growing relation of autotrophic and heterotrophic bacteria under ROX pollution pressure in agricultural ecosystems.     

Insights into the Effect of Soil pH on N2O and N2 Emissions and Denitrifier Community Size and Activity

The objective of this study was to investigate how changes in soil pH affect the N₂O and N₂ emissions, denitrification activity, and size of a denitrifier community. We established a field experiment, situated in a grassland area, which consisted of three treatments which were repeatedly amended with a KOH solution (alkaline soil), an H₂SO₄ solution (acidic soil), or water (natural pH soil) over 10 months. At the site, we determined field N₂O and N₂ emissions using the ¹⁵N gas flux method and collected soil samples for the measurement of potential denitrification activity and quantification of the size of the denitrifying community by quantitative PCR of the narG, napA, nirS, nirK, and nosZ denitrification genes. Overall, our results indicate that soil pH is of importance in determining the nature of denitrification end products. Thus, we found that the N₂O/(N₂O + N₂) ratio increased with decreasing pH due to changes in the total denitrification activity, while no changes in N₂O production were observed. Denitrification activity and N₂O emissions measured under laboratory conditions were correlated with N fluxes in situ and therefore reflected treatment differences in the field. The size of the denitrifying community was uncoupled from in situ N fluxes, but potential denitrification was correlated with the count of NirS denitrifiers. Significant relationships were observed between nirS, napA, and narG gene copy numbers and the N₂O/(N₂O + N₂) ratio, which are difficult to explain. However, this highlights the need for further studies combining analysis of denitrifier ecology and quantification of denitrification end products for a comprehensive understanding of the regulation of N fluxes by denitrification.Denitrification is the microbial reduction of NO₃⁻ via NO₂⁻ to gaseous NO, N₂O, and N₂, which are then lost into the atmosphere (36). It therefore results in considerable loss of nitrogen, one of the most limiting nutrients for crop production in agriculture (20). Denitrification is also of environmental concern since, together with nitrification, it is the main biological process responsible for N₂O emissions (7). N₂O is a potent greenhouse gas which has a global warming potential about 320 times greater than that of CO₂ and has a lifetime of approximately 120 years (32). In the stratosphere, N₂O can also react with O₂ to produce NO, which induces the destruction of stratospheric ozone (8). N₂O can be released into the atmosphere by incomplete denitrification due to the effect of environmental conditions on the regulation of the different denitrification reductases (14, 41, 51), but it has recently been suggested that it could also be due to lack of nitrous oxide reductase in some denitrifiers (19, 41). Since N₂O is an intermediate in the denitrification pathway, both the amount of N₂O produced and the N₂O/(N₂O + N₂) ratio are important in understanding and predicting N₂O fluxes from soils.The main environmental factors known to influence the N₂O/(N₂O + N₂) ratio are pH, organic carbon and NO₃⁻ availability, water content, and O₂ partial pressure (50). Soil pH is one of the most important factors influencing both denitrification and N₂O production (43). In general, the denitrification rate increases with increasing pH values (up to the optimum pH) while, in contrast, the N₂O/(N₂O + N₂) ratio decreases (50). This relationship has been characterized in laboratory experiments (9, 45), but it is not clear whether the same relationships exist in the field because of methodological limitations of in situ measurement of N₂ emissions (16). Nevertheless, ¹⁵N tracing experiments based on the addition of a labeled denitrification substrate to soil offer a useful tool to quantify emissions of both N₂O and N₂ in situ (47, 49). Soil pH is also an important factor influencing denitrifier community composition (35, 39), which can be an important driver of denitrification activity and N₂O emissions (5, 21). A recent study reported a negative relationship between the proportion of bacteria genetically capable of reducing N₂O within the total bacterial community and the N₂O/(N₂O + N₂) ratio, with both being strongly correlated with soil pH (38).The objective of the present study was to explore the effect of changes in soil pH on in situ N₂O and N₂ emissions, denitrifying enzyme activity (DEA), and potential N₂O production. In addition, we also investigated whether differences in N fluxes could be related to changes in the size of the microbial community possessing the different denitrification genes. A field experiment was conducted using replicated grassland plots in which the soil pH was modified by addition of either acid or hydroxide to the soil. A ¹⁵N tracer method was used to provide information on N emissions. In addition to measuring potential denitrification activity, the size of the denitrifier community was determined by real-time PCR quantification of the denitrification genes.