Nitrifying bacterial community structures and their nitrification performance under sufficient and limited inorganic carbon conditions

This study examined the hypothesis that different inorganic carbon (IC) conditions enrich different ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) populations by operating two laboratory-scale continuous-flow bioreactors fed with 15 and 100 mg IC/L, respectively. During this study, both bioreactors maintained satisfactory nitrification performance and stably oxidized 250 mg?N/L of influent ammonium without nitrite accumulation. Based on results of cloning/sequencing and terminal restriction fragment length polymorphism targeting on the ammonia monooxygenase subunit A (amoA) gene, Nitrosomonas nitrosa lineage was identified as the dominant AOB population in the high-IC bioreactor, while Nitrosomonas europaea and Nitrosomonas nitrosa lineage AOB were dominant in the low-IC bioreactor. Results of real-time polymerase chain reactions for Nitrobacter and Nitrospira 16S rRNA genes indicated that Nitrospira was the predominant NOB population in the high-IC bioreactor, while Nitrobacter was the dominant NOB in the low-IC bioreactor. Furthermore, batch experiment results suggest that N. europaea and Nitrobacter populations are proliferated in the low-IC bioreactor due to their higher rates under low IC conditions despite the fact that these two populations have been identified as weak competitors, compared with N. nitrosa and Nitrospira, under low ammonium/nitrite environments. This study revealed that in addition to ammonium/nitrite concentrations, limited IC conditions may also be important in selecting dominant AOB/NOB communities of nitrifying bioreactors.     

Shifts between Nitrospira‐ and Nitrobacter‐like nitrite oxidizers underlie the response of soil potential nitrite oxidation to changes in tillage practices

Despite their role in soil functioning, the ecology of nitrite‐oxidizing bacteria, NOB, and their response to disturbances such as those generated by agricultural practices are scarcely known. Over the course of 17 months, we surveyed the potential nitrite oxidation, PNO, the abundance of the Nitrobacter‐ and Nitrospira‐like NOB (by quantitative PCR) and the community structure of the Nitrobacter‐like NOB (by PCR‐DGGE and cloning‐sequencing targeting the nxrA gene) in soils for four treatments: after establishment of tillage on a previously no‐tillage system, after cessation of tillage on a previously tillage system, and on control tillage and no‐tillage systems. Key soil variables (moisture, organic carbon content and gross mineralization – i.e. ammonification – measured by the ¹⁵N dilution technique) were also surveyed. PNO was always higher for the no‐tillage than tillage treatments. Establishment of tillage led to a strong and rapid decrease in PNO whereas cessation of tillage did not change PNO even after 17 months. PNO was strongly and positively correlated to the abundance of Nitrobacter‐like NOB and was also strongly related to gross mineralization, a proxy of N‐availability; in contrast, PNO was weakly and negatively correlated to the abundance of Nitrospira‐like NOB. Selection of a dominant population was observed under no‐tillage, and PNO was loosely correlated to the community structure of Nitrobacter‐like NOB. Our results demonstrate that Nitrobacter‐like NOB are the key functional players within the NOB community in soils with high N availability and high activity level, and that changes in PNO are due to shifts between Nitrospira‐like and Nitrobacter‐like NOB and to a weaker extent by shifts of populations within Nitrobacter‐like NOB.     

Sequentially aerated membrane biofilm reactors for autotrophic nitrogen removal: microbial community composition and dynamics

Membrane‐aerated biofilm reactors performing autotrophic nitrogen removal can be successfully applied to treat concentrated nitrogen streams. However, their process performance is seriously hampered by the growth of nitrite oxidizing bacteria (NOB). In this work we document how sequential aeration can bring the rapid and long‐term suppression of NOB and the onset of the activity of anaerobic ammonium oxidizing bacteria (AnAOB). Real‐time quantitative polymerase chain reaction analyses confirmed that such shift in performance was mirrored by a change in population densities, with a very drastic reduction of the NOB Nitrospira and Nitrobacter and a 10‐fold increase in AnAOB numbers. The study of biofilm sections with relevant 16S rRNA fluorescent probes revealed strongly stratified biofilm structures fostering aerobic ammonium oxidizing bacteria (AOB) in biofilm areas close to the membrane surface (rich in oxygen) and AnAOB in regions neighbouring the liquid phase. Both communities were separated by a transition region potentially populated by denitrifying heterotrophic bacteria. AOB and AnAOB bacterial groups were more abundant and diverse than NOB, and dominated by the r‐strategists Nitrosomonas europaea and Ca. Brocadia anammoxidans, respectively. Taken together, the present work presents tools to better engineer, monitor and control the microbial communities that support robust, sustainable and efficient nitrogen removal.     

Influence of growth manner on nitrifying bacterial communities and nitrification kinetics in three lab-scale bioreactors

The effects of growth type, including attached growth, suspended growth, and combined growth, on the characteristics of communities of ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) were studied in three lab-scale Anaerobic/Anoxic_m-Oxic_n (AmOn) systems. These systems amplified activated sludge, biofilms, and a mixture of activated sludge and biofilm (AS-BF). Identical inocula were adopted to analyze the selective effects of mixed growth patterns on nitrifying bacteria. Fluctuations in the concentration of nitrifying bacteria over the 120 days of system operation were analyzed, as was the composition of nitrifying bacterial community in the stabilized stage. Analysis was conducted using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) and real-time PCR. According to the DGGE patterns, the primary AOB lineages were Nitrosomonas europaea (six sequences), Nitrosomonas oligotropha (two sequences), and Nitrosospira (one sequence). The primary subclass of NOB community was Nitrospira, in which all identified sequences belonged to Nitrospira moscoviensis (14 sequences). Nitrobacter consisted of two lineages, namely Nitrobacter vulgaris (three sequences) and Nitrobacter alkalicus (two sequences). Under identical operating conditions, the composition of nitrifying bacterial communities in the AS-BF system demonstrated significant differences from those in the activated sludge system and those in the biofilm system. Major varieties included several new, dominant bacterial sequences in the AS-BF system, such as N. europaea and Nitrosospira and a higher concentration of AOB relative to the activated sludge system. However, no similar differences were discovered for the concentration of the NOB population. A kinetic study of nitrification demonstrated a higher maximum specific growth rate of mixed sludge and a lower half-saturation constant of mixed biofilm, indicating that the AS-BF system maintained relatively good nitrifying ability.     

宏基因组技术研究泥岩母质发育的三种不同pH紫色土硝化微生物群落演变规律

【目的】揭示典型农田旱地紫色土硝化微生物的群落组成及其对pH的响应规律。【方法】针对同一母质发育但pH差异显著的3种紫色土,利用宏基因组技术深度测序研究土壤中硝化微生物丰度和群落,包括氨氧化古菌(ammonia-oxidizing archaea,AOA),氨氧化细菌(ammonia-oxidizing bacteria,AOB),亚硝酸盐氧化细菌(nitrite-oxidizingbacteria,NOB)和全程氨氧化细菌(completeammoniaoxidizer,Comammox)。【结果】土壤中硝化微生物的丰度占总微生物的2.130%–6.082%。3种紫色土中AOA、AOB和NOB的相对丰度有显著差异:酸性紫色土中AOA的相对丰度显著大于碱性紫色土,而AOB则相反;NOB的相对丰度在中性紫色土中最高。所有土样中均发现了1种全程氨氧化细菌Candidatus Nitrospira inopinata (Ca. N. inopinata),其在中性紫色土中相对丰度最高,占总微生物的0.203%。3种不同pH紫色土中AOA均以Nitrososphaera为主,NOB均以Nitrospira为主;酸性紫色土中AOB以Nitroscoccus为主,而中性和石灰性紫色土中则以Nitrosospira为主。Pearson相关性分析发现,土壤pH和铵态氮是影响硝化微生物丰度最大的两个因子。【结论】Comammox存在于3种不同pH紫色土中,且偏好中性环境;AOA、AOB和NOB群落结构和相对丰度都存在显著差异,结合相关性分析发现土壤pH和铵态氮是导致差异最重要的两个因子。     

Ammonia-oxidizing communities in a highly aerated full-scale activated sludge bioreactor: betaproteobacterial dynamics and low relative abundance of Crenarchaea

Ammonia‐oxidizing bacteria (AOB) have long been considered key to the removal of nitrogen in activated sludge bioreactors. Culture‐independent molecular analyses have established that AOB lineages in bioreactors are dynamic, but the underlying operational or environmental factors are unclear. Furthermore, the contribution of ammonia‐oxidizing archaea (AOA) to nitrogen removal in bioreactors has not been studied. To this end, we investigated the abundance of AOA and AOB as well as correlations between dynamics in AOB lineages and operational parameters at a municipal wastewater treatment plant sampled weekly over a 1 year period. Quantitative PCR measurements of bacterial and archaeal ammonia monooxygenase subunit A (amoA) genes revealed that the bacterial homologue predominated by at least three orders of magnitude in all samples. Archaeal amoA was only detectable in ～15% of these samples. Using terminal restriction fragment length polymorphism analysis, we monitored AOB lineages based on amoA genes. The Nitrosomonas europaea lineage and a novel Nitrosomonas‐like cluster were the dominant AOB signatures, with a Nitrosospira lineage present at lower relative abundance. These lineages exhibited strong temporal oscillations, with one becoming sequentially dominant over the other. Using non‐metric multidimensional scaling and redundancy analyses, we tested correlations between terminal restriction fragment length polymorphism profiles and 20 operational and environmental parameters. The redundancy analyses indicated that the dynamics of AOB lineages correlated most strongly with temperature, dissolved oxygen and influent nitrite and chromium. The Nitrosospira lineage signal had a strong negative correlation to dissolved oxygen and temperature, while the Nitrosomonas‐like (negative correlations) and N. europaea lineages (positive correlations) were inversely linked (relative to one another) to influent nitrite and chromium. Overall, this study suggests that AOA may be minor contributors to ammonia oxidation in highly aerated activated sludge, and provides insight into parameters controlling the diversity and dominance of AOB lineages within bioreactors during periods of stable nitrification.     

重庆紫色水稻土中“全程”和“半程”氨氧化微生物的垂直分异

【目的】系统评估全程氨氧化细菌(complete ammonia oxidizing bacteria, Comammox bacteria)、半程氨氧化细菌(AOB)和古菌(AOA)在典型水稻土剖面的垂直分异规律。2015年发现的"全程"氨氧化细菌(Comammox Nitrospira)可将氨分子一步氧化为硝酸盐,实现硝化作用。而经典的"半程"氨氧化细菌(AOB)或古菌(AOA)将氨分子氧化为亚硝酸盐后,再由系统发育完全不同的硝化细菌将其氧化为硝酸盐。全程氨氧化细菌实现了一步硝化全过程,根本改变了学术界对2类微生物分步硝化的经典认知,但相关研究仍处于初步阶段。【方法】选择重庆北碚地区2017年典型水稻土并采集5、10、20和40 cm不同深度土壤(剖面采样点的上下误差不超过1cm),提取水稻土总DNA后,利用标靶功能基因amoA,通过实时荧光定量PCR技术分析全程氨氧化细菌(Comammox)、半程氨氧化细菌(AOB)和古菌(AOA)在水稻土不同深度的数量变异规律。【结果】半程氨氧化细菌AOB和古菌AOA均随土壤深度增加呈显著下降趋势。然而,全程氨氧化细菌的两大类微生物则表现出相反的规律,Comammox Clade A的丰度随着土壤剖面的加深而显著增加(P0.05),但Clade B并未有类似规律。Clade A在水稻土不同层次的土层中均比Clade B高出1个数量级,在5 cm和40 cm处的最低和最高值分别为3.42×10~7、8.46×10~7 copies/g。AOA与AOB的丰度大致相当,5cm剖面处数量最高分别为1.23×10~7、1.83×10~5copies/g,但其平均丰度远低于全程氨氧化细菌,Comammox与AOA、AOB amoA功能基因拷贝数之比为10–2000。【结论】全程氨氧化细菌(Comammox bacteria)广泛分布于水稻土不同土层中,且数量远高于"半程"氨氧化细菌和古菌,意味着Comammox可能在水稻土硝化作用中起重要作用。     

Autotrophic growth of nitrifying community in an agricultural soil

The two-step nitrification process is an integral part of the global nitrogen cycle, and it is accomplished by distinctly different nitrifiers. By combining DNA-based stable isotope probing (SIP) and high-throughput pyrosequencing, we present the molecular evidence for autotrophic growth of ammonia-oxidizing bacteria (AOB), ammonia-oxidizing archaea (AOA) and nitrite-oxidizing bacteria (NOB) in agricultural soil upon ammonium fertilization. Time-course incubation of SIP microcosms indicated that the amoA genes of AOB was increasingly labeled by ¹³CO₂ after incubation for 3, 7 and 28 days during active nitrification, whereas labeling of the AOA amoA gene was detected to a much lesser extent only after a 28-day incubation. Phylogenetic analysis of the ¹³C-labeled amoA and 16S rRNA genes revealed that the Nitrosospira cluster 3-like sequences dominate the active AOB community and that active AOA is affiliated with the moderately thermophilic Nitrososphaera gargensis from a hot spring. The higher relative frequency of Nitrospira-like NOB in the ¹³C-labeled DNA suggests that it may be more actively involved in nitrite oxidation than Nitrobacter-like NOB. Furthermore, the acetylene inhibition technique showed that ¹³CO₂ assimilation by AOB, AOA and NOB occurs only when ammonia oxidation is not blocked, which provides strong hints for the chemolithoautotrophy of nitrifying community in complex soil environments. These results show that the microbial community of AOB and NOB dominates the nitrification process in the agricultural soil tested.     

Nitrification inhibitors effectively target N2O-producing Nitrosospira spp. in tropical soil

The nitrification inhibitors (NIs) 3,4-dimethylpyrazole (DMPP) and dicyandiamide (DCD) can effectively reduce N₂O emissions; however, which species are targeted and the effect of these NIs on the microbial nitrifier community is still unclear. Here, we identified the ammonia oxidizing bacteria (AOB) species linked to N₂O emissions and evaluated the effects of urea and urea with DCD and DMPP on the nitrifying community in a 258 day field experiment under sugarcane. Using an amoA AOB amplicon sequencing approach and mining a previous dataset of 16S rRNA sequences, we characterized the most likely N₂O-producing AOB as a Nitrosospira spp. and identified Nitrosospira (AOB), Nitrososphaera (archaeal ammonia oxidizer) and Nitrospira (nitrite-oxidizer) as the most abundant, present nitrifiers. The fertilizer treatments had no effect on the alpha and beta diversities of the AOB communities. Interestingly, we found three clusters of co-varying variables with nitrifier operational taxonomic units (OTUs): the N₂O-producing AOB Nitrosospira with N₂O, NO₃⁻, NH₄⁺, water-filled pore space (WFPS) and pH; AOA Nitrososphaera with NO₃⁻, NH₄⁺ and pH; and AOA Nitrososphaera and NOB Nitrospira with NH₄⁺, which suggests different drivers. These results support the co-occurrence of non-N₂O-producing Nitrososphaera and Nitrospira in the unfertilized soils and the promotion of N₂O-producing Nitrosospira under urea fertilization. Further, we suggest that DMPP is a more effective NI than DCD in tropical soil under sugarcane.     

Abundance,community structure and diversity of nitrifying bacterial enrichments from low and high saline brackishwater environments

The study reports diversity in nitrifying microbial enrichments from low (0·5–5‰) and high (18–35‰) saline ecosystems. Microbial community profiling of ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) enrichments was analysed by sequencing 16S rRNA and was processed using Mothur pipeline. The α-diversity indices showed the richness of nitrifying bacterial consortia from the high saline environment and were clustering based on the source of the sample. AOB and NOB enrichments from both the environments showed diverse lineages of phyla distributed in both groups with 38 and 34 phyla from low saline and 53 and 40 phyla in high saline sources, respectively. At class level, α- and γ-proteobacteria were found to be more dominant in both the enrichments. AOBs and NOBs in enrichments from low saline environments were dominated by Nitrosomonadaceae, Gallionellaceae (Nitrotoga sp.) and Ectothiorhodospiraceae and Nitrospira, respectively. Though Chromatiaceae were present in both AOB and NOB enrichments, Nitrosoglobus and Nitrosococcus dominated the AOBs while NOBs were dominated by uncultured genera, whereas Rhizobiales were found in both the enrichments. AOBs and NOBs in enrichments from high saline environments were dominated by Nitrospira-like AOBs, Nitrosomonas and Nitrosococcus genera, whereas ammonia-oxidizing archaea (AOA) group included Nitrosopumilus and Nitrososphaera genera comprising and Nitrospirae, respectively. The majority of the genera obtained in both the salinities were found to be either uncultured or unclassified groups. Results of the study suggest that the AOB and NOB consortia have unique and diverse microbes in each of the enrichments, capable of functioning in aquaculture systems practised at different salinities (0–60 ppt).     

Differential contributions of ammonia oxidizers and nitrite oxidizers to nitrification in four paddy soils

Rice paddy fields are characterized by regular flooding and nitrogen fertilization, but the functional importance of aerobic ammonia oxidizers and nitrite oxidizers under unique agricultural management is poorly understood. In this study, we report the differential contributions of ammonia-oxidizing archaea (AOA), bacteria (AOB) and nitrite-oxidizing bacteria (NOB) to nitrification in four paddy soils from different geographic regions (Zi-Yang (ZY), Jiang-Du (JD), Lei-Zhou (LZ) and Jia-Xing (JX)) that are representative of the rice ecosystems in China. In urea-amended microcosms, nitrification activity varied greatly with 11.9, 9.46, 3.03 and 1.43 μg NO₃⁻-N g⁻¹ dry weight of soil per day in the ZY, JD, LZ and JX soils, respectively, over the course of a 56-day incubation period. Real-time quantitative PCR of amoA genes and pyrosequencing of 16S rRNA genes revealed significant increases in the AOA population to various extents, suggesting that their relative contributions to ammonia oxidation activity decreased from ZY to JD to LZ. The opposite trend was observed for AOB, and the JX soil stimulated only the AOB populations. DNA-based stable-isotope probing further demonstrated that active AOA numerically outcompeted their bacterial counterparts by 37.0-, 10.5- and 1.91-fold in ¹³C-DNA from ZY, JD and LZ soils, respectively, whereas AOB, but not AOA, were labeled in the JX soil during active nitrification. NOB were labeled to a much greater extent than AOA and AOB, and the addition of acetylene completely abolished the assimilation of ¹³CO₂ by nitrifying populations. Phylogenetic analysis suggested that archaeal ammonia oxidation was predominantly catalyzed by soil fosmid 29i4-related AOA within the soil group 1.1b lineage. Nitrosospira cluster 3-like AOB performed most bacterial ammonia oxidation in the ZY, LZ and JX soils, whereas the majority of the ¹³C-AOB in the JD soil was affiliated with the Nitrosomona communis lineage. The ¹³C-NOB was overwhelmingly dominated by Nitrospira rather than Nitrobacter. A significant correlation was observed between the active AOA/AOB ratio and the soil oxidation capacity, implying a greater advantage of AOA over AOB under microaerophilic conditions. These results suggest the important roles of soil physiochemical properties in determining the activities of ammonia oxidizers and nitrite oxidizers.     

Biological inhibition of soil nitrification by forest tree species affects Nitrobacter populations

Some temperate tree species are associated with very low soil nitrification rates, with important implications for forest N dynamics, presumably due to their potential for biological nitrification inhibition (BNI). However, evidence for BNI in forest ecosystems is scarce so far and the nitrifier groups controlled by BNI-tree species have not been identified. Here, we evaluated how some tree species can control soil nitrification by providing direct evidence of BNI and identifying the nitrifier group(s) affected. First, by comparing 28 year-old monocultures of several tree species, we showed that nitrification rates correlated strongly with the abundance of the nitrite oxidizers Nitrobacter (50- to 1000-fold changes between tree monocultures) and only weakly with the abundance of ammonia oxidizing archaea (AOA). Second, using reciprocal transplantation of soil cores between low and high nitrification stands, we demonstrated that nitrification changed 16 months after transplantation and was correlated with changes in the abundance of Nitrobacter, not AOA. Third, extracts of litter or soil collected from the low nitrification stands of Picea abies and Abies nordmanniana inhibited the growth of Nitrobacter hamburgensis X14. Our results provide for the first time direct evidence of BNI by tree species directly affecting the abundance of Nitrobacter.     

Mimicking the oxygen minimum zones: stimulating interaction of aerobic archaeal and anaerobic bacterial ammonia oxidizers in a laboratory‐scale model system

In marine oxygen minimum zones (OMZs), ammonia‐oxidizing archaea (AOA) rather than marine ammonia‐oxidizing bacteria (AOB) may provide nitrite to anaerobic ammonium‐oxidizing (anammox) bacteria. Here we demonstrate the cooperation between marine anammox bacteria and nitrifiers in a laboratory‐scale model system under oxygen limitation. A bioreactor containing ‘Candidatus Scalindua profunda’ marine anammox bacteria was supplemented with AOA (Nitrosopumilus maritimus strain SCM1) cells and limited amounts of oxygen. In this way a stable mixed culture of AOA, and anammox bacteria was established within 200 days while also a substantial amount of endogenous AOB were enriched. ‘Ca. Scalindua profunda’ and putative AOB and AOA morphologies were visualized by transmission electron microscopy and a C₁₈ anammox [3]‐ladderane fatty acid was highly abundant in the oxygen‐limited culture. The rapid oxygen consumption by AOA and AOB ensured that anammox activity was not affected. High expression of AOA, AOB and anammox genes encoding for ammonium transport proteins was observed, likely caused by the increased competition for ammonium. The competition between AOA and AOB was found to be strongly related to the residual ammonium concentration based on amoA gene copy numbers. The abundance of archaeal amoA copy numbers increased markedly when the ammonium concentration was below 30 μM finally resulting in almost equal abundance of AOA and AOB amoA copy numbers. Massive parallel sequencing of mRNA and activity analyses further corroborated equal abundance of AOA and AOB. PTIO addition, inhibiting AOA activity, was employed to determine the relative contribution of AOB versus AOA to ammonium oxidation. The present study provides the first direct evidence for cooperation of archaeal ammonia oxidation with anammox bacteria by provision of nitrite and consumption of oxygen.     

Nitrification in hybrid bioreactors treating simulated domestic wastewater

Aim

To provide deeper insights into nitrification process within aerobic bioreactors containing supplemental physical support media (hybrid bioreactors).

Methods and Results

Three bench‐scale hybrid bioreactors with different media size and one control bioreactor were operated to assess how biofilm integrity influences microbial community conditions and bioreactor performance. The systems were operated initially at a 5‐day hydraulic retention time (HRT), and all reactors displayed efficient nitrification and chemical oxygen demand (COD) removal (>95%). However, when HRT was reduced to 2·5 days, COD removal rates remained high, but nitrification efficiencies declined in all reactors after 19 days. To explain reduced performance, nitrifying bacterial communities (ammonia‐oxidizing bacteria, AOB; nitrite‐oxidizing bacteria, NOB) were examined in the liquid phase and also on the beads using qPCR, FISH and DGGE. Overall, the presence of the beads in a reactor promoted bacterial abundances and diversity, but as bead size was increased, biofilms with active coupled AOB–NOB activity were less apparent, resulting in incomplete nitrification.

Conclusions

Hybrid bioreactors have potential to sustain effective nitrification at low HRTs, but support media size and configuration type must be optimized to ensure coupled AOB and NOB activity in nitrification.

Significance and Impact of the Study

This study shows that AOB and NOB coupling must be accomplished to minimize nitrification failure.     

Effective and robust partial nitrification to nitrite by real-time aeration duration control in an SBR treating domestic wastewater

Achieving sustainable partial nitrification to nitrite has been proven difficult in treating low strength nitrogenous wastewater. Real-time aeration duration control was used to achieve efficient partial nitrification to nitrite in a sequencing batch reactor (SBR) to treat low strength domestic wastewater. Above 90% nitrite accumulation ratio was maintained for long-term operation at normal condition, or even lower water temperature in winter. Partial nitrification established by controlling aeration duration showed good performance and robustness even though encountering long-term extended aeration and starvation period. Process control enhanced the successful accumulation of ammonia oxidizing bacteria (AOB) and washout of nitrite oxidizing bacteria (NOB). Scanning electron microscope observations indicated that the microbial morphology showed a shift towards small rod-shaped clusters. Fluorescence in situ hybridization (FISH) results demonstrated AOB were the dominant nitrifying bacteria, up to 8.3 ± 1.1% of the total bacteria; on the contrary, the density of NOB decreased to be negligible after 135 days operation since adopting process control.     

Distinctive microbial ecology and biokinetics of autotrophic ammonia and nitrite oxidation in a partial nitrification bioreactor

Biological nitrogen removal (BNR) based on partial nitrification and denitrification via nitrite is a cost-effective alternate to conventional nitrification and denitrification (via nitrate). The goal of this study was to investigate the microbial ecology, biokinetics, and stability of partial nitrification. Stable long-term partial nitrification resulting in 82.1 +/- 17.2% ammonia oxidation, primarily to nitrite (77.3 +/- 19.5% of the ammonia oxidized) was achieved in a lab-scale bioreactor by operation at a pH, dissolved oxygen and solids retention time of 7.5 +/- 0.1, 1.54 +/- 0.87 mg O(2)/L, and 3.0 days, respectively. Bioreactor ammonia oxidizing bacteria (AOB) and nitrite oxidizing bacteria (NOB) populations were most closely related to Nitrosomonas europaea and Nitrobacter spp., respectively. The AOB population fraction varied in the range 61 +/- 45% and was much higher than the NOB fraction, 0.71 +/- 1.1%. Using direct measures of bacterial concentrations in conjunction with independent activity measures and mass balances, the maximum specific growth rate (micro(max)), specific decay (b) and observed biomass yield coefficients (Y(obs)) for AOB were 1.08 +/- 1.03 day(-1), 0.32 +/- 0.34 day(-1), and 0.15 +/- 0.06 mg biomass COD/mg N oxidized, respectively. Corresponding micro(max), b, and Y(obs) values for NOB were 2.6 +/- 2.05 day(-1), 1.7 +/- 1.9 day(-1), and 0.04 +/- 0.02 mg biomass COD/mg N oxidized, respectively. The results of this study demonstrate that the highly selective partial nitrification operating conditions enriched for a narrow diversity of rapidly growing AOB and NOB populations unlike conventional BNR reactors, which host a broader diversity of nitrifying bacteria. Further, direct measures of microbial abundance enabled not only elucidation of mixed community microbial ecology but also estimation of key engineering parameters describing bioreactor systems supporting these communities.     

Response of nitrifying bacterial communities to the increased thiocyanate concentration in pre-denitrification process

Changes in process performance and the nitrifying bacterial community associated with an increase of thiocyanate (SCN⁻) loading were investigated in a pre-denitrification process treating industrial wastewater. The increased SCN⁻ loading led to the concentration of total nitrogen (TN) in the final effluent, but increasing the internal recycling ratio as an operation parameter from 2 to 5 resulted in a 21% increase in TN removal efficiency. In the aerobic reactor, we found that the Nitrosomonas europaea lineage was the predominant ammonia oxidizing bacteria (AOB) and the percentages of the AOB population within the total bacteria increased from about 4.0% to 17% with increased SCN⁻ concentration. The increase of nitrite loading seemed to change the balance between Nitrospira and Nitrobacter, resulting in the high dominance of Nitrospira over Nitrobacter. Meanwhile, a Thiobacillus thioparus was suggested to be the main microorganism responsible for the SCN⁻ biodegradation observed in the system.     

Effects of Aeration Cycles on Nitrifying Bacterial Populations and Nitrogen Removal in Intermittently Aerated Reactors

The effects of the lengths of aeration and nonaeration periods on nitrogen removal and the nitrifying bacterial community structure were assessed in intermittently aerated (IA) reactors treating digested swine wastewater. Five IA reactors were operated in parallel with different aeration-to-nonaeration time ratios (ANA). Populations of ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) were monitored using 16S rRNA slot blot hybridizations. AOB species diversity was assessed using amoA gene denaturant gradient gel electrophoresis. Nitrosomonas and Nitrosococcus mobilis were the dominant AOB and Nitrospira spp. were the dominant NOB in all reactors, although Nitrosospira and Nitrobacter were also detected at lower levels. Reactors operated with the shortest aeration time (30 min) showed the highest Nitrosospira rRNA levels, and reactors operated with the longest anoxic periods (3 and 4 h) showed the lowest levels of Nitrobacter, compared to the other reactors. Nitrosomonas sp. strain Nm107 was detected in all reactors, regardless of thereactor's performance. Close relatives of Nitrosomonas europaea, Nitrosomonas sp. strain ENI-11, and Nitrosospira multiformis were occasionally detected in all reactors. Biomass fractions of AOB and effluent ammonia concentrations were not significantly different among the reactors. NOB were more sensitive than AOB to long nonaeration periods, as nitrite accumulation and lower total NOB rRNA levels were observed for an ANA of 1 h:4 h. The reactor with the longest nonaeration time of 4 h performed partial nitrification, followed by denitrification via nitrite, whereas the other reactors removed nitrogen through traditional nitrification and denitrification via nitrate. Superior ammonia removal efficiencies were not associated with levels of specific AOB species or with higher AOB species diversity.