Long-term effect of dissolved oxygen on partial nitrification performance and microbial community structure

In this study, the performance of partial nitrification via nitrite and microbial community structure were investigated and compared in two sequencing batch reactors (SBR) with different dissolved oxygen (DO) levels. Both reactors achieved stable partial nitrification with nitrite accumulation ratio of above 95% by using real-time aeration duration control. Compared with high DO (above 3 mg/l on average) SBR, simultaneous nitrification and denitrification (SND) via nitrite was carried out in low DO (0.4–0.8 mg/l) SBR. The average efficiencies of SND in high DO and low DO reactor were 7.7% and 44.9%, and the specific SND rates were 0.20 and 0.83 mg N/(mg MLSS h), respectively. Low DO did not produce sludge with poorer settling properties but attained lower turbidities of the effluent than high DO. Fluorescence in situ hybridization (FISH) analysis in both the reactors showed that ammonia-oxidizing bacteria (AOB) were the dominant nitrifying bacteria and nitrite-oxidizing bacteria (NOB) did not be recovered in spite of exposing nitrifying sludge to high DO. The morphology of the sludge from both two reactors according to scanning electron microscope indicated that small rod-shaped and spherical clusters were dominant, although filamentous bacteria and few long rod-shaped coexisted in the low DO reactor. By selecting properly DO level and adopting process control method is not only of benefit to the achievement of novel biological nitrogen removal technology, but also favorable to sludge population optimization.     

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 the reactor'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.     

焦化废水工业处理装置和实验室装置硝化菌群的分子比较

反应器的群落结构分析有助于对工业装置的故障原因进行诊断。为了解决某焦化废水处理装置硝化功能低下的故障,构建了一套相似的实验室装置作为参照系统,该装置的硝化功能良好。通过工业装置和实验室装置好氧池生物膜16SrDNA克隆文库的比较,分析了它们之间硝化菌群的组成差异。实验室装置克隆文库的构成说明Nitrosomonas europaea-Nitrosoccus mobilis类群和Nitrospira属Ⅰ亚区系分别是该工艺条件下优势的氨氧化菌和亚硝酸氧化菌,但工业装置的克隆文库中却没有找到任何与硝化菌序列相近的克隆,这说明工业装置中硝化菌的多度较低。进一步使用Taqman荧光探针实时定量PCR测定了样品中Nitrospira属的多度,实验室装置中Nitrospira属16S rDNA的拷贝数达到3.4×106个/微克基因组DNA,而工业装置的测定值不到实验室装置的1/300。这些试验结果都表明工业装置好氧池微生物群落中缺少适当的硝化菌群是造成其硝化能力低下的重要原因。提高菌群中Nitrosomonas属和Nitrospira属的多度是解决工业装置硝化能力低下的关键。     

Effect of temperature and free ammonia on nitrification and nitrite accumulation in landfill leachate and analysis of its nitrifying bacterial community by FISH

The cause of seasonal failure of a nitrifying municipal landfill leachate treatment plant utilizing a fixed biofilm was investigated by wastewater analyses and batch respirometric tests at every treatment stage. Nitrification of the leachate treatment plant was severely affected by the seasonal temperature variation. High free ammonia (NH3-N) inhibited not only nitrite oxidizing bacteria (NOB) but also ammonia oxidizing bacteria (AOB). In addition, high pH also increased free ammonia concentration to inhibit nitrifying activity especially when the NH4-N level was high. The effects of temperature and free ammonia of landfill leachate on nitrification and nitrite accumulation were investigated with a semi-pilot scale biofilm airlift reactor. Nitrification rate of landfill leachate increased with temperature when free ammonia in the reactor was below the inhibition level for nitrifiers. Leachate was completely nitrified up to a load of 1.5 kg NH4-N m(-3)d(-1) at 28 degrees C. The activity of NOB was inhibited by NH3-N resulting in accumulation of nitrite. NOB activity decreased more than 50% at 0.7 mg NH3-N L(-1). Fluorescence in situ hybridization (FISH) was carried out to analyze the population of AOB and NOB in the nitrite accumulating nitrifying biofilm. NOB were located close to AOB by forming small clusters. A significant fraction of AOB identified by probe Nso1225 specifically also hybridized with the Nitrosomonas specific probe Nsm156. The main NOB were Nitrobacter and Nitrospira which were present in almost equal amounts in the biofilm as identified by simultaneous hybridization with Nitrobacter specific probe Nit3 and Nitrospira specific probe Ntspa662.     

Unravelling the reasons for disproportion in the ratio of AOB and NOB in aerobic granular sludge

In this study, we analysed the nitrifying microbial community (ammonium-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB)) within three different aerobic granular sludge treatment systems as well as within one flocculent sludge system. Granular samples were taken from one pilot plant run on municipal wastewater as well as from two lab-scale reactors. Fluorescent in situ hybridization (FISH) and quantitative PCR (qPCR) showed that Nitrobacter was the dominant NOB in acetate-fed aerobic granules. In the conventional system, both Nitrospira and Nitrobacter were present in similar amounts. Remarkably, the NOB/AOB ratio in aerobic granular sludge was elevated but not in the conventional treatment plant suggesting that the growth of Nitrobacter within aerobic granular sludge, in particular, was partly uncoupled from the lithotrophic nitrite supply from AOB. This was supported by activity measurements which showed an approximately threefold higher nitrite oxidizing capacity than ammonium oxidizing capacity. Based on these findings, two hypotheses were considered: either Nitrobacter grew mixotrophically by acetate-dependent dissimilatory nitrate reduction (ping-pong effect) or a nitrite oxidation/nitrate reduction loop (nitrite loop) occurred in which denitrifiers reduced nitrate to nitrite supplying additional nitrite for the NOB apart from the AOB.     

Structure and activity of multiple nitrifying bacterial populations co-existing in a biofilm

A biofilm from a nitrifying pilot-scale sequencing batch reactor was investigated for effects of varying process conditions on its microscale activity and structure. Microsensor measurements of oxygen, substrates and products of nitrification were applied under incubation at different ammonium and oxygen concentrations which reflected various situations during a treatment cycle. A high net N loss was observed under high ammonium (HA) concentrations in contrast to low ones. Additionally, results indicated inhibition of nitrite-oxidizing bacteria (NOB), but not of ammonia-oxidizing bacteria (AOB) by free ammonia under HA conditions. Diversity, spatial distribution, and abundance of nitrifying bacteria as analysed by fluorescence in situ hybridization (FISH) revealed six different nitrifying populations with heterogeneous distributions. Nitrosococcus mobilis formed conspicuous microcolonies locally surrounded by cells of the dominating N. europaea/eutropha-related AOB population. A third less abundant population was affiliated to N. oligotropha. Nitrite-oxidizing bacteria of the genera Nitrobacter and Nitrospira (with at least two distinct populations) showed a large scale heterogeneity in their distribution. Nitrospira spp. were also found in deeper inactive layers where they might persist rather than thrive, and act as seed population when detached. Results of functional and structural analyses are discussed with respect to specific niches of individual populations in this system.     

Anammox enrichment from reject water on blank biofilm carriers and carriers containing nitrifying biomass: operation of two moving bed biofilm reactors (MBBR)

The anammox bacteria were enriched from reject water of anaerobic digestion of municipal wastewater sludge onto moving bed biofilm reactor (MBBR) system carriers-the ones initially containing no biomass (MBBR1) as well as the ones containing nitrifying biomass (MBBR2). Duration of start-up periods of the both reactors was similar (about 100?days), but stable total nitrogen (TN) removal efficiency occurred earlier in the system containing nitrifying biomass. Anammox TN removal efficiency of 70% was achieved by 180?days in both 20?l volume reactors at moderate temperature of 26.0°C. During the steady state phase of operation of MBBRs the average TN removal efficiencies and maximum TN removal rates in MBBR1 were 80% (1,000?g-N/m(3)/day, achieved by 308?days) and in MBBR2 85% (1,100?g-N/m(3)/day, achieved by 266?days). In both reactors mixed bacterial cultures were detected. Uncultured Planctomycetales bacterium clone P4, Candidatus Nitrospira defluvii and uncultured Nitrospira sp. clone 53 were identified by PCR-DGGE from the system initially containing blank biofilm carriers as well as from the nitrifying biofilm system; from the latter in addition to these also uncultured ammonium oxidizing bacterium clone W1 and Nitrospira sp. clone S1-62 were detected. FISH analysis revealed that anammox microorganisms were located in clusters in the biofilm. Using previously grown nitrifying biofilm matrix for anammox enrichment has some benefits over starting up the process from zero, such as less time for enrichment and protection against severe inhibitions in case of high substrate loading rates.     

Achieving nitrite accumulation in a continuous system treating low-strength domestic wastewater: switchover from batch start-up to continuous operation with process control

Although biological nitrogen removal via nitrite is recognized as one of the cost-effective and sustainable biological nitrogen removal processes, nitrite accumulation has proven difficult to achieve in continuous processes treating low-strength nitrogenous wastewater. Partial nitrification to nitrite was achieved and maintained in a lab-scale completely stirred tank reactor (CSTR) treating real domestic wastewater. During the start-up period, sludge with ammonia-oxidizing bacteria (AOB) but no nitrite-oxidizing bacteria (NOB) was obtained by batch operation with aeration time control. The nitrifying sludge with the dominance of AOB was then directly switched into continuous operation. It was demonstrated that partial nitrification to nitrite in the continuous system could be repeatedly and reliably achieved using this start-up strategy. The ratio of dissolved oxygen to ammonium loading rate (DO/ALR) was critical to maintain high ammonium removal efficiency and nitrite accumulation ratio. Over 85% of nitrite accumulation ratio and more than 95% of ammonium removal efficiency were achieved at DO/ALR ratios in an optimal range of 4.0–6.0 mg O₂/g N d, even under the disturbances of ammonium loading rate. Microbial population shift was investigated, and fluorescence in situ hybridization analysis indicated that AOB were the dominant nitrifying bacteria over NOB when stable partial nitrification was established.     

Identification of novel small molecule enhancers of protein production by cultured mammalian cells

Shortcut nitrogen removal, that is, removal via formation and reduction of nitrite rather than nitrate, has been observed in membrane-aerated biofilms (MABs), but the extent, the controlling factors, and the kinetics of nitrite formation in MABs are poorly understood. We used a special MAB reactor to systematically study the effects of the dissolved oxygen (DO) concentration at the membrane surface, which is the biofilm base, on nitrification rates, extent of shortcut nitrification, and microbial community structure. The focus was on anoxic bulk liquids, which is typical in MAB used for total nitrogen (TN) removal, although aerobic bulk liquids were also studied. Nitrifying MABs were grown on a hollow-fiber membrane exposed to 3 mg N/L ammonium. The MAB intra-membrane air pressure was varied to achieve different DO concentrations at the biofilm base, and the bulk liquid was anoxic or with 2 g m(-3) DO. With 2.2 and 3.5 g m(-3) DO at the biofilm base, and with an anoxic bulk-liquid, the ammonium fluxes were 0.75 and 1.0 g N m(-2) day(-1), respectively, and nitrite was the main oxidized nitrogen product. However, with membrane DO of 5.5 g m(-3), and either zero or 2 g m(-3) DO in the bulk, the ammonium flux was around 1.3 g N m(-2) day(-1), and nitrate flux increased significantly. For all experiments, the cell density of ammonium oxidizing bacteria (AOB) was relatively uniform throughout the biofilm, but the density of nitrite oxidizing bacteria (NOB) decreased with decreasing biofilm DO. Among NOB, Nitrobacter spp. were dominant in biofilm regions with 2 g m(-3) DO or greater, while Nitrospira spp. were dominant in regions with less than 2 g m(-3) DO. A biofilm model, including AOB, Nitrobacter spp., and Nitrospira spp., was developed and calibrated with the experimental results. The model predicted the greatest extent of nitrite formation (95%) and the lowest ammonium oxidation flux (0.91 g N m(-2) day(-1)) when the membrane DO was 2 g m(-3) and the bulk liquid was anoxic. Conversely, the model predicted the lowest extent of nitrite formation (40%) and the highest ammonium oxidation flux (1.5 g N m(-2) day(-1)) when the membrane-DO and bulk-DO were 8 g m(-3) and 2 g m(-3), respectively. The estimated kinetic parameters for Nitrospira spp., revealed a high affinity for nitrite and oxygen. This explains the dominance of Nitrospira spp. over Nitrobacter spp. in regions with low nitrite and oxygen concentrations. Our results suggest that shortcut nitrification can effectively be controlled by manipulating the DO at the membrane surface. A tradeoff is made between increased nitrite accumulation at lower DO, and higher nitrification rates at higher DO.     

Nitrifying bacterial communities in an aquaculture wastewater treatment system using fluorescence in situ hybridization (FISH), 16S rRNA gene cloning, and phylogenetic analysis

Aquaculture, especially shrimp farming, has played a major role in the growth of Thailand's economy in recent years, as well as in many South East Asian countries. However, the nutrient discharges from these activities have caused adverse impacts on the quality of the receiving waterways. In particular nitrogenous compounds, which may accumulate in aquaculture ponds, can be toxic to aquatic animals and cause environmental problems such as eutrophication. The mineralization process is well known, but certain aspects of the microbial ecology of nitrifiers, the microorganisms that convert ammonia to nitrate, are poorly understood. A previously reported enrichment of nitrifying bacteria (ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB)) from a shrimp farm inoculated in a sequencing batch reactor (SBR) was studied by molecular methods. The initial identification and partial quantification of the nitrifying bacteria (AOB and NOB) were carried out by fluorescence in situ hybridization (FISH) using previously published 16S rRNA-targeting oligonucleotide probes. The two dominant bacterial groups detected by FISH were from the Cytophaga-Flavobacterium-Bacteroides and Proteobacteria (beta subdivision) phyla. Published FISH probes for Nitrobacter and Nitrospira did not hybridize to any of the bacterial cells. Therefore it is likely that new communities of NOBs, differing from previously reported ones, exist in the enrichments. Molecular genetic techniques (cloning, sequencing, and phylogenetic analysis) targeting the 16S rRNA genes from the nitrifying enrichments were performed to identify putative AOBs and NOBs.     

Estimation of nitrifier abundances in a partial nitrification reactor treating ammonium-rich saline wastewater using DGGE, T-RFLP and mathematical modeling

The bacterial community in a partial nitrification reactor was analyzed on the basis of 16S rRNA gene by cloning–sequencing method, and the percentages of ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) in the activated sludge were quantified by three independent methods, namely, denaturing gradient gel electrophoresis (DGGE), terminal restriction fragment length polymorphism (T-RFLP) and Double Monod modeling. The clone library results suggested that there were only a dominant AOB and a dominant NOB species in the reactor, belonging to Nitrosomonas genus and Nitrospira genus, respectively. The percentages of NOB in total bacterial community increased from almost 0% to 30% when dissolved oxygen (DO) levels were changed from 0.15 mg/L to 0.5 mg/L, coinciding with the accumulation and conversion of nitrite, while the percentages of AOB changed little in the two phases. The results confirmed the importance of low DO level for inhibiting NOB to achieve partial nitrification. Furthermore, the percentages of AOB and NOB in the total bacteria community were estimated based on the results of batch experiments using Double Monod model, and the results were comparable with those determined according to profiles of DGGE and T-RFLP.     

Characterization of the Microbial Community in a Partial Nitrifying Sequencing Batch Biofilm Reactor

A lab-scale partial nitrifying sequencing batch biofilm reactor was a successful start-up. Denaturing gradient gel electrophoresis (DGGE) was used to investigate the bacterial community dynamics in three periods together with inocula sludge at ambient temperature. The DGGE profiles of bacteria and Shannon–Wiener index (H′) results showed that high free ammonia (FA) concentration referred to lower diversity in the bioreactor system. Cluster analysis indicated that microorganism in period III was similar with inocula sludge and was different from that in periods I and II. Similar results also appeared in ammonia-oxidizing bacteria (AOB) community structure and nitrite-oxidizing bacteria (NOB) community structure, and at least four AOB species and two NOB species were present in period III, respectively. Phylogenetic analysis of amoA gene sequences showed that Nitrosomonas eutropha cluster was predominant in all the three periods. With lower ammonium loads, three new operational taxonomic units formed and consisted Nitrosomonas sp. Cluster. This article demonstrated that microbial community, AOB, and NOB diversity were related with FA concentration closely at ambient temperature.     

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.     

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.     

Multi‐species nitrifying biofilm model (MSNBM) including free ammonia and free nitrous acid inhibition and oxygen limitation

A multi‐species nitrifying biofilm model (MSNBM) is developed to describe nitrite accumulation by simultaneous free ammonia (FA) and free nitrous acid (FNA) inhibition, direct pH inhibition, and oxygen limitation in a biofilm. The MSNBM addresses the spatial gradient of pH with biofilm depth and how it induces changes of FA and FNA speciation and inhibition. Simulations using the MSNBM in a completely mixed biofilm reactor show that influent total ammonia nitrogen (TAN) concentration, bulk dissolved oxygen (DO) concentration, and buffer concentration exert significant control on the suppression of nitrite‐oxidizing bacteria (NOB) and shortcut biological nitrogen removal (SBNR), but the pH in the bulk liquid has a weaker influence. Ammonium oxidation increases the nitrite concentration and decreases the pH, which together can increase FNA inhibition of NOB in the biofilm. Thus, a low buffer concentration can accentuate SBNR. DO and influent TAN concentrations are efficient means to enhance DO limitation, which affects NOB more than ammonia‐oxidizing bacteria (AOB) inside the biofilm. With high influent TAN concentration, FA inhibition is dominant at an early phase, but finally DO limitation becomes more important as TAN degradation and biofilm growth proceed. MSNBM results indicate that oxygen depletion and FNA inhibition throughout the biofilm continuously suppress the growth of NOB, which helps achieve SBNR with a lower TAN concentration than in systems without concentration gradients. Biotechnol. Bioeng. 2010;105: 1115–1130. © 2009 Wiley Periodicals, Inc.     

Nitrite-oxidizing bacteria guild ecology associated with nitrification failure in a continuous-flow reactor

Nitrification is an important process for nitrogen removal in many wastewater treatment plants, which requires the mutualistic oxidation of ammonia to nitrate by ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB). However, this process can be quite unpredictable because both guilds are conditionally sensitive to small changes in operating conditions. Here, dynamics are examined within the NOB guild in two parallel chemostats operated at low and high dilution rates (0.10 and 0.83 day(-1), respectively) during periods of varying nitrification performance. NOB and AOB guild abundances and nitrogen-oxidation efficiency were relatively constant over time in the 0.10 day(-1) reactor; however, the 0.83 day(-1) reactor had two major disturbance episodes that caused destabilization of the NOB guild, which ultimately led to nitrification failure. The first episode caused the extinction of Nitrospira spp. from the system, resulting in chronic incomplete ammonia oxidation and nitrite accumulation. The second episode caused complete loss of nitrification activity, likely resulting from metal toxicity and the previous extinction of Nitrospira spp. from the system. These results exemplify the types of changes that can occur within the NOB guild that result in process impairment or failure, and provide one possible explanation for why nitrification is often unstable at higher dilution rates.     

Vertical distribution of nitrifying populations in bacterial biofilms from a full-scale nitrifying trickling filter

Cryosectioned biofilm from three depths (0.5, 3.0 and 6.0 m) in a full-scale nitrifying trickling filter (NTF) were studied using fluorescence in situ hybridization (FISH). A large number of sections were used to determine how the biofilm thickness, structure and community composition varied with depth along the ammonium concentration gradient in the NTF, and how the ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) were distributed vertically within the biofilm. Both the biofilm thickness and relative biomass content of the biofilm decreased with depth, along with structural differences such as void size and surface roughness. Four AOB populations were found, with two Nitrosomonas oligotropha populations dominating at all depths. A smaller population of Nitrosomonas europaea was present only at 0.5 m, while a population of Nitrosomonas communis increased with depth. The two N. oligotropha populations showed different vertical distribution patterns within the biofilm, indicating different ecophysiologies even though they belong to the same AOB lineage. All NOB were identified as Nitrospira sp., and were generally more associated with the biofilm base than the surface-associated dominating AOB population. Additionally, a small population of anaerobic ammonia-oxidizers was found at 6.0 m, even though the biofilm was well aerated.     

Development and application of a PCR-denaturing gradient gel electrophoresis tool to study the diversity of Nitrobacter-like nxrA sequences in soil

A new PCR-denaturing gel gradient electrophoresis (DGGE) tool based on the functional gene nxrA encoding the catalytic subunit of the nitrite oxidoreductase in nitrite-oxidizing bacteria (NOB) has been developed. The first aim was to determine if the primers could target representatives of NOB genera: Nitrococcus and Nitrospira. The primers successfully amplified nxrA gene sequences from Nitrococcus mobilis, but not from Nitrospira marina. The second aim was to develop a PCR-DGGE tool to characterize NOB community structure on the basis of Nitrobacter-like partial nrxA gene sequences (Nb-nxrA). We tested (1) the ability of this tool to discriminate between Nitrobacter strains, and (2) its ability to reveal changes in the community structure of NOB harbouring Nb-nrxA sequences induced by light grazing or intensive grazing in grassland soils. The DGGE profiles clearly differed between the four Nitrobacter strains tested. Differences in the structure of NOB community were revealed between grazing regimes. Phylogenetic analysis of the sequences corresponding to different DGGE bands showed that Nb-nxrA sequences did not group in management-specific clusters. Most of the nxrA sequences obtained from soils differed from nxrA sequences of NOB strains. Along with existing tools for characterizing the community structure of nitrifiers, this new approach is a significant step forward to performing comprehensive studies on nitrification.     

Bioaugmentation as a tool for improving the start-up and stability of a pilot-scale partial nitrification biofilm airlift reactor

The effectiveness of bioaugmentation in the improvement of the start-up of a biofilm airlift reactor to perform partial nitrification was investigated. Two identical biofilm airlift reactors were inoculated. The non-bioaugmented reactor (NB-reactor) was inoculated with conventional activated sludge, whereas the bioaugmented reactor (B-reactor) was seeded with the same conventional activated sludge but bioaugmented with nitrifying activated sludge from a pilot plant performing full nitritation under stable conditions (100% oxidation of influent ammonium to nitrite). The fraction of specialized nitrifying activated sludge in the inoculum of the B-reactor was only 6% (measured as dry matter). To simplify comparison of the results, operational parameters were equivalent for both reactors. Partial nitrification was achieved significantly faster in the B-reactor, showing a very stable operation. The results obtained by fluorescence in situ hybridization assays showed that the specialized nitrifying biomass added to the B-reactor remained in the biofilm throughout the start-up period.     

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.