Community structure and activity dynamics of nitrifying bacteria in a phosphate-removing biofilm

The microbial community structure and activity dynamics of a phosphate-removing biofilm from a sequencing batch biofilm reactor were investigated with special focus on the nitrifying community. O(2), NO(2)(-), and NO(3)(-) profiles in the biofilm were measured with microsensors at various times during the nonaerated-aerated reactor cycle. In the aeration period, nitrification was oxygen limited and restricted to the first 200 microm at the biofilm surface. Additionally, a delayed onset of nitrification after the start of the aeration was observed. Nitrate accumulating in the biofilm in this period was denitrified during the nonaeration period of the next reactor cycle. Fluorescence in situ hybridization (FISH) revealed three distinct ammonia-oxidizing populations, related to the Nitrosomonas europaea, Nitrosomonas oligotropha, and Nitrosomonas communis lineages. This was confirmed by analysis of the genes coding for 16S rRNA and for ammonia monooxygenase (amoA). Based upon these results, a new 16S rRNA-targeted oligonucleotide probe specific for the Nitrosomonas oligotropha lineage was designed. FISH analysis revealed that the first 100 microm at the biofilm surface was dominated by members of the N. europaea and the N. oligotropha lineages, with a minor fraction related to N. communis. In deeper biofilm layers, exclusively members of the N. oligotropha lineage were found. This separation in space and a potential separation of activities in time are suggested as mechanisms that allow coexistence of the different ammonia-oxidizing populations. Nitrite-oxidizing bacteria belonged exclusively to the genus Nitrospira and could be assigned to a 16S rRNA sequence cluster also found in other sequencing batch systems.     

Community Structure and Activity Dynamics of Nitrifying Bacteria in a Phosphate-Removing Biofilm

The microbial community structure and activity dynamics of a phosphate-removing biofilm from a sequencing batch biofilm reactor were investigated with special focus on the nitrifying community. O₂, NO₂⁻, and NO₃⁻ profiles in the biofilm were measured with microsensors at various times during the nonaerated-aerated reactor cycle. In the aeration period, nitrification was oxygen limited and restricted to the first 200 μm at the biofilm surface. Additionally, a delayed onset of nitrification after the start of the aeration was observed. Nitrate accumulating in the biofilm in this period was denitrified during the nonaeration period of the next reactor cycle. Fluorescence in situ hybridization (FISH) revealed three distinct ammonia-oxidizing populations, related to the Nitrosomonas europaea, Nitrosomonas oligotropha, and Nitrosomonas communis lineages. This was confirmed by analysis of the genes coding for 16S rRNA and for ammonia monooxygenase (amoA). Based upon these results, a new 16S rRNA-targeted oligonucleotide probe specific for the Nitrosomonas oligotropha lineage was designed. FISH analysis revealed that the first 100 μm at the biofilm surface was dominated by members of the N. europaea and the N. oligotropha lineages, with a minor fraction related to N. communis. In deeper biofilm layers, exclusively members of the N. oligotropha lineage were found. This separation in space and a potential separation of activities in time are suggested as mechanisms that allow coexistence of the different ammonia-oxidizing populations. Nitrite-oxidizing bacteria belonged exclusively to the genus Nitrospira and could be assigned to a 16S rRNA sequence cluster also found in other sequencing batch systems.     

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.     

Analysis of size distribution and areal cell density of ammonia-oxidizing bacterial microcolonies in relation to substrate microprofiles in biofilms

A fine-scale in situ spatial organization of ammonia-oxidizing bacteria (AOB) in biofilms was investigated by combining molecular techniques (i.e., fluorescence in situ hybridization (FISH) and 16S rDNA-cloning analysis) and microelectrode measurements. Important parameters of AOB microcolonies such as size distribution and areal cell density of the microcolonies were determined and correlated with substrate microprofiles in the biofilms. In situ hybridization with a nested 16S rRNA-targeted oligonucleotide probe set revealed two different populations of AOB, Nitrosomonas europaea-lineage and Nitrosospira multiformis-lineage, coexisting in an autotrophic nitrifying biofilm. Nitrosospira formed looser microcolonies, with an areal cell density of 0.51 cells microm(-2), which was half of the cell density of Nitrosomonas (1.12 cells microm(-2)). It is speculated that the formation of looser microcolonies facilitates substrate diffusion into the microcolonies, which might be a survival strategy to low O(2) and NH(4) (+) conditions in the biofilm. A long-term experiment (4-week cultivation at different substrate C/N ratios) revealed that the size distribution of AOB microcolonies was strongly affected by better substrate supply due to shorter distance from the surface and the presence of organic carbon. The microcolony size was relatively constant throughout the autotrophic nitrifying biofilm, while the size increased by approximately 80% toward the depth of the biofilm cultured at the substrate C/N = 1. A short-term ( approximately 3 h) organic carbon addition experiment showed that the addition of organic carbon created interspecies competition for O(2) between AOB and heterotrophic bacteria, which dramatically decreased the in situ NH(4) (+)-uptake activity of AOB in the surface of the biofilms. This result might explain the spatial distribution of AOB microcolony size in the biofilms cultured at the substrate C/N = 1. These experimental results suggest O(2) and organic carbon were the main factors controlling the spatial organization and activity of AOB in biofilms. These findings are significantly important to further improve mathematical models used to describe how the slow-growing AOB develop their niches in biofilms and how that configuration affects nitrification performance in the biofilm.     

Production of NO(2) and N(2)O by Nitrifying Bacteria at Reduced Concentrations of Oxygen

Pure cultures of the marine ammonium-oxidizing bacterium Nitrosomonas sp. were grown in the laboratory at oxygen partial pressures between 0.005 and 0.2 atm (0.18 to 7 mg/liter). Low oxygen conditions induced a marked decrease in the rate for production of NO(2), from 3.6 x 10 to 0.5 x 10 mmol of NO(2) per cell per day. In contrast, evolution of N(2)O increased from 1 x 10 to 4.3 x 10 mmol of N per cell per day. The yield of N(2)O relative to NO(2) increased from 0.3% to nearly 10% (moles of N in N(2)O per mole of NO(2)) as the oxygen level was reduced, although bacterial growth rates changed by less than 30%. Nitrifying bacteria from the genera Nitrosomonas, Nitrosolobus, Nitrosospira, and Nitrosococcus exhibited similar yields of N(2)O at atmospheric oxygen levels. Nitrite-oxidizing bacteria (Nitrobacter sp.) and the dinoflagellate Exuviaella sp. did not produce detectable quantities of N(2)O during growth. The results support the view that nitrification is an important source of N(2)O in the environment.     

Dynamics of specific ammonia-oxidizing bacterial populations and nitrification in response to controlled shifts of ammonium concentrations in wastewater

Ammonia-oxidizing bacteria (AOB) are essential for the nitrification process in wastewater treatment. To retain these slow-growing bacteria in wastewater treatment plants (WWTPs), they are often grown as biofilms, e.g., on nitrifying trickling filters (NTFs) or on carriers in moving bed biofilm reactors (MBBRs). On NTFs, a decreasing ammonium gradient is formed because of the AOB activity, resulting in low ammonium concentrations at the bottom and reduced biomass with depth. To optimize the NTF process, different ammonium feed strategies may be designed. This, however, requires knowledge about AOB population dynamics. Using fluorescence in situ hybridization (FISH) and confocal laser scanning microscopy, we followed biomass changes during 6 months, of three AOB populations on biofilm carriers. These were immersed in aerated MBBR tanks in a pilot plant receiving full-scale wastewater. Tanks were arranged in series, forming a wastewater ammonium gradient mimicking an NTF ammonium gradient. The biomass of one of the dominating Nitrosomonas oligotropha-like populations increased after an ammonium upshift, reaching levels comparable to the high ammonium control in 28 days, whereas a Nitrosomonas europaea-like population increased relatively slowly. The MBBR results, together with competition studies in NTF systems fed with wastewater under controlled ammonium regimes, suggest a differentiation between the two N. oligotropha populations, which may be important for WWTP nitrification.     

Community survey of ammonia-oxidizing bacteria in full-scale activated sludge processes with different solids retention time

AIMS: To study the effects of different solids retention time (SRT) on the nitrification activity and community composition of ammonia-oxidizing bacteria (AOB) in two full-scale activated sludge processes during a 5-month period. METHODS AND RESULTS: The AOB community composition was analysed using fluorescence in situ hybridization (FISH) and denaturing gradient gel electrophoresis (DGGE), and the identified populations were enumerated by quantitative FISH. Potential nitrification rates were determined in batch tests and the in situ rates were calculated from mass balances of nitrogen in the plants. Increased SRT reduced the nitrification activity, but neither the number per mixed liquor suspended solids nor community composition of AOB were affected. Two dominant AOB populations related to Nitrosomonas europaea and Nitrosomonas oligotropha were identified by FISH, whereas only the latter could be detected by DGGE. CONCLUSIONS: The effect of a longer SRT on the activity was probably because of physiological changes in the AOB community rather than a change in community composition. SIGNIFICANCE AND IMPACT OF THE STUDY: Physiological alterations of a stable AOB community are possible and may stabilize activated sludge processes. The commonly used FISH probes designed to target all beta-proteobacterial AOB does not detect certain Nitrosomonas oligotropha populations, leading to an underestimation of AOB if a wider set of probes is not used.     

In situ analysis of nitrifying biofilms as determined by in situ hybridization and the use of microelectrodes.

We investigated the in situ spatial organization of ammonia-oxidizing and nitrite-oxidizing bacteria in domestic wastewater biofilms and autotrophic nitrifying biofilms by using microsensors and fluorescent in situ hybridization (FISH) performed with 16S rRNA-targeted oligonucleotide probes. The combination of these techniques made it possible to relate in situ microbial activity directly to the occurrence of nitrifying bacterial populations. In situ hybridization revealed that bacteria belonging to the genus Nitrosomonas were the numerically dominant ammonia-oxidizing bacteria in both types of biofilms. Bacteria belonging to the genus Nitrobacter were not detected; instead, Nitrospira-like bacteria were the main nitrite-oxidizing bacteria in both types of biofilms. Nitrospira-like cells formed irregularly shaped aggregates consisting of small microcolonies, which clustered around the clusters of ammonia oxidizers. Whereas most of the ammonia-oxidizing bacteria were present throughout the biofilms, the nitrite-oxidizing bacteria were restricted to the active nitrite-oxidizing zones, which were in the inner parts of the biofilms. Microelectrode measurements showed that the active ammonia-oxidizing zone was located in the outer part of a biofilm, whereas the active nitrite-oxidizing zone was located just below the ammonia-oxidizing zone and overlapped the location of nitrite-oxidizing bacteria, as determined by FISH.     

Oxygen-limited autotrophic nitrification/denitrification by ammonia oxidisers enables upward motion towards more favourable conditions

The hypothesis is formulated that in case of oxygen limitation in the sediment, nitrifiers switch from nitrification to oxygen-limited autotrophic nitrification-denitrification (OLAND) in order to survive and maintain activity. During OLAND, ammonium is oxidised using nitrite as e-acceptor to form dinitrogen gas. As an additional advantage they benefit from the gaseous N₂ formed as a means of transport. In this way, the nitrifiers can move out of the sediment and rise through the water column towards more favourable conditions. At the surface, the bacteria could take up oxygen, and recommence nitrification. In order to test this hypothesis, nitrifying sediment with an overlaying water column was simulated in lab-scale columns. Nitrogen transformations and material transport through the water column were followed after addition of different forms of nitrogen under oxygen-limited conditions. ¹⁵N-labelling experiments showed a large contribution of OLAND to the observed nitrogen deficits. Nitrifier enumerations, fluorescent in situ hybridisation and 16S rRNA gene analysis revealed increased populations of ammonia oxidising nitrifiers in the upper water layers. The results presented support the proposed hypothesis of transport using OLAND. Nitrifying activity in the sediment immediately recovered almost completely from prolonged oxygen-limited incubation when oxygen concentrations were increased. Electronic Publication     

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.     

Community structures and activities of nitrifying and denitrifying bacteria in industrial wastewater-treating biofilms

The bacterial community structure, in situ spatial distributions and activities of nitrifying and denitrifying bacteria in biofilms treating industrial wastewater were investigated by combination of the 16S rRNA gene clone analysis, fluorescence in situ hybridization (FISH) and microelectrodes. These results were compared with the nitrogen removal capacity of the industrial wastewater treatment plant (IWTP). Both nitrification and denitrification occurred in the primary denitrification (PD) tank and denitrification occurred in the secondary denitrification (SD) tank. In contrast, nitrification and denitrification rates were very low in the nitrification (N) tank. 16S rRNA gene clone sequence analysis revealed that the bacteria affiliated with Alphaproteobacteria, followed by Betaproteobacteria, were numerically important microbial groups in three tanks. The many clones affiliated with Alphaproteobacteria were closely related to the denitrifying bacteria (e.g., Hyphomicrobium spp., Rhodopseudomonas palustris, and Rhodobacter spp.). In addition, Methylophilus leisingeri affiliated with Betaproteobacteria, which favorably utilized methanol, was detected only in the SD-tank to which methanol was added. Nitrosomonas europaea and Nitrosomonas marina were detected as the ammonia-oxidizing bacteria affiliated with Betaproteobacteria throughout this plant, although the dominant species of them was different among three tanks. Nitrifying bacteria were mainly detected in the upper parts of the PD-biofilm whereas their populations were low in the upper parts of the N-biofilm. The presence of denitrifying bacteria affiliated with Hyphomicrobium spp. in SD- and N-biofilms was verified by FISH analysis. Microelectrode measurements showed that the nitrifying bacteria present in the N- and PD-biofilms were active and the bacteria present in the SD-biofilm could denitrify.     

Influence of different cultivars on populations of ammonia-oxidizing bacteria in the root environment of rice

Comparisons of the activities and diversities of ammonia-oxidizing bacteria (AOB) in the root environment of different cultivars of rice (Oryza sativa L.) indicated marked differences despite identical environmental conditions during growth. Gross nitrification rates obtained by the 15N dilution technique were significantly higher in a modern variety, IR63087-1-17, than in two traditional varieties. Phylogenetic analysis based on the ammonium monooxygenase gene (amoA) identified strains related to Nitrosospira multiformis and Nitrosomonas europaea as the predominant AOB in our experimental rice system. A method was developed to determine the abundance of AOB on root biofilm samples using fluorescently tagged oligonucleotide probes targeting 16S rRNA. The levels of abundance detected suggested an enrichment of AOB on rice roots. We identified 40 to 69% of AOB on roots of IR63087-1-17 as Nitrosomonas spp., while this subpopulation constituted 7 to 23% of AOB on roots of the other cultivars. These results were generally supported by denaturing gradient gel electrophoresis of the amoA gene and analysis of libraries of cloned amoA. In hydroponic culture, oxygen concentration profiles around secondary roots differed significantly among the tested rice varieties, of which IR63087-1-17 showed maximum leakage of oxygen. The results suggest that varietal differences in the composition and activity of root-associated AOB populations may result from microscale differences in O2 availability.     

Nitrifying bacterial community structure of a full-scale integrated fixed-film activated sludge process as investigated by pyrosequencing

Nitrifying bacterial community structures of suspended and attached biomasses in a full-scale integrated fixed-film activated sludge process were investigated by analyzing 16S rRNA gene sequences obtained from pyrosequencing. The suspended biomass had a higher number of ammoniaoxidizing bacterial sequences (0.8% of total sequences) than the attached biomass (0.07%), although most of the sequences were within the Nitrosomonas oligotropha lineage in both biomasses. Nitrospira-like nitrite-oxidizing bacterial sequences were retrieved in the suspended biomass (0.06%), not in the attached biomass, whereas the existence of Nitrobacter-like sequences was not evident. The suspended biomass had higher nitrification activity (1.13 mg N/TSS/h) than the attached biomass (0.07 mg N/TSS/h). Overall, the results made it possible to conclude the importance of the suspended biomass, rather than the attached biomass, in nitrification in the wastewater treatment process studied.     

Effect of bulk liquid BOD concentration on activity and microbial community structure of a nitrifying,membrane-aerated biofilm

Membrane-aerated biofilms (MABs) are an effective means to achieve nitrification and denitrification of wastewater. In this research, microsensors, fluorescence in situ hybridization (FISH), and modeling were used to assess the impact of bulk liquid biological oxygen demand (BOD) concentrations on the activity and microbial community structure of nitrifying MABs. With 1 g m⁻³ BOD in the bulk liquid, the nitrification rate was 1.3 g N m⁻² day⁻¹, slightly lower than the 1.5 g N m⁻² day⁻¹ reported for no bulk liquid BOD. With bulk liquid BOD concentrations of 3 and 10 g m⁻³, the rates decreased to 1 and 0.4 g N m⁻² day⁻¹, respectively. The percent denitrification increased from 20% to 100% when the BOD increased from 1 to 10 g m⁻³ BOD. FISH results indicated increasing abundance of heterotrophs with increasing bulk liquid BOD, consistent with the increased denitrification rates. Modeling was used to assess the effect of BOD on nitrification rates and to compare an MAB to a conventional biofilm. The model-predicted nitrification rates were consistent with the experimental results. Also, nitrification in the MAB was much less sensitive to BOD inhibition than the conventional biofilm. The MAB achieved concurrent nitrification and denitrification, whereas little denitrification occurred in the conventional biofilm.     

Nitrification in a Biofilm at Low pH Values: Role of In Situ Microenvironments and Acid Tolerance

The sensitivity of nitrifying bacteria to acidic conditions is a well-known phenomenon and generally attributed to the lack and/or toxicity of substrates (NH₃ and HNO₂) with decreasing pHs. In contrast, we observed strong nitrification at a pH around 4 in biofilms grown on chalk particles and investigated the following hypotheses: the presence of less acidic microenvironments and/or the existence of acid-tolerant nitrifiers. Microelectrode measurements (in situ and under various experimental conditions) showed no evidence of a neutral microenvironment, either within the highly active biofilm colonizing the chalk surface or within a control biofilm grown on a nonbuffering (i.e., sintered glass) surface under acidic pH. A 16S rRNA approach (clone libraries and fluorescence in situ hybridizations) did not reveal uncommon nitrifying (potentially acid-tolerant) strains. Instead, we found a strongly acidic microenvironment, evidence for a clear adaptation to the low pH in situ, and the presence of nitrifying populations related to subgroups with low K_ms for ammonia (Nitrosopira spp., Nitrosomonas oligotropha, and Nitrospira spp.). Acid-consuming (chalk dissolution) and acid-producing (ammonia oxidation) processes are equilibrated on a low-pH steady state that is controlled by mass transfer limitation through the biofilm. Strong affinity to ammonia and possibly the expression of additional functions, e.g., ammonium transporters, are adaptations that allow nitrifiers to cope with acidic conditions in biofilms and other habitats.     

Nitrosomonas Nm143-like ammonia oxidizers and Nitrospira marina-like nitrite oxidizers dominate the nitrifier community in a marine aquaculture biofilm

Zero-discharge marine aquaculture systems are an environmentally friendly alternative to conventional aquaculture. In these systems, water is purified and recycled via microbial biofilters. Here, quantitative data on nitrifier community structure of a trickling filter biofilm associated with a recirculating marine aquaculture system are presented. Repeated rounds of the full-cycle rRNA approach were necessary to optimize DNA extraction and the probe set for FISH to obtain a reliable and comprehensive picture of the ammonia-oxidizing community. Analysis of the ammonia monooxygenase gene (amoA) confirmed the results. The most abundant ammonia-oxidizing bacteria (AOB) were members of the Nitrosomonas sp. Nm143-lineage (6.7% of the bacterial biovolume), followed by Nitrosomonas marina-like AOB (2.2% of the bacterial biovolume). Both were outnumbered by nitrite-oxidizing bacteria of the Nitrospira marina-lineage (15.7% of the bacterial biovolume). Although more than eight other nitrifying populations were detected, including Crenarchaeota closely related to the ammonia-oxidizer 'Nitrosopumilus maritimus', their collective abundance was below 1% of the total biofilm volume; their contribution to nitrification in the biofilter is therefore likely to be negligible.     

Isotope labeling and microautoradiography of active heterotrophic bacteria on the basis of assimilation of 14CO(2)

Most heterotrophic bacteria assimilate CO(2) in various carboxylation reactions during biosynthesis. In this study, assimilation of (14)CO(2) by heterotrophic bacteria was used for isotope labeling of active microorganisms in pure cultures and environmental samples. Labeled cells were visualized by microautoradiography (MAR) combined with fluorescence in situ hybridization (FISH) to obtain simultaneous information about activity and identity. Cultures of Escherichia coli and Pseudomonas putida assimilated sufficient (14)CO(2) during growth on various organic substrates to obtain positive MAR signals. The MAR signals were comparable with the traditional MAR approach based on uptake of (14)C-labeled organic substrates. Experiments with E. coli showed that (14)CO(2) was assimilated during both fermentation and aerobic and anaerobic respiration. The new MAR approach, HetCO(2)-MAR, was evaluated by targeting metabolic active filamentous bacteria, including "Candidatus Microthrix parvicella" in activated sludge. "Ca. Microthrix parvicella" was able to take up oleic acid under anaerobic conditions, as shown by the traditional MAR approach with [(14)C]oleic acid. However, the new HetCO(2)-MAR approach indicated that "Ca. Microthrix parvicella," did not significantly grow on oleic acid under anaerobic conditions with or without addition of NO(2)(-), whereas the addition of O(2) or NO(3)(-) initiated growth, as indicated by detectable (14)CO(2) assimilation. This is a metabolic feature that has not been described previously for filamentous bacteria. Such information could not have been derived by using the traditional MAR procedure, whereas the new HetCO(2)-MAR approach differentiates better between substrate uptake and substrate metabolism that result in growth. The HetCO(2)-MAR results were supported by stable isotope analysis of (13)C-labeled phospholipid fatty acids from activated sludge incubated under aerobic and anaerobic conditions in the presence of (13)CO(2). In conclusion, the novel HetCO(2)-MAR approach expands the possibility for studies of the ecophysiology of uncultivated microorganisms.     

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。     

Sequencing batch membrane biofilm reactor for simultaneous nitrogen and phosphorus removal: novel application of membrane-aerated biofilm

A sequencing batch membrane biofilm reactor (SBMBfR) was developed for simultaneous carbon, nitrogen, and phosphorus removal from wastewater. This reactor was composed of two functional parts: (1) a gas-permeable membrane on which a nitrifying biofilm formed and (2) a bulk solution in which bacteria, mainly denitrifying polyphosphate-accumulating organisms (DNPAOs), were suspended. The reactor was operated sequentially under anaerobic condition and then under membrane aeration condition in one cycle. During the anaerobic period, organic carbon was consumed by DNPAOs; this was accompanied by phosphate release. During the subsequent membrane aeration period, nitrifying bacteria utilized oxygen supplied directly to them from the inside of the membrane. Consequently, the nitrite and nitrate products diffused into the bulk solution, where they were used by DNPAOs as electron acceptors for phosphate uptake. In a long-term sequencing batch operation, the mean removal efficiencies of total organic carbon (TOC), total nitrogen (T-N), and total phosphorus (T-P) under steady-state condition were 99%, 96%, and 90%, respectively. In addition, fluorescence in situ hybridization (FISH) clearly demonstrated the difference in bacterial community structure between the membrane biofilm and the suspended sludge: ammonia-oxidizing bacteria belonging to the Nitrosomonas group were dominant in the region adjacent to the membrane throughout the operation, and the occupation ratio of the well-known polyphosphate-accumulating organism (PAO) Candidatus "Accumulibacter phosphates" in the suspended sludge gradually increased to a maximum of 37%.     

Structural and Functional Dynamics of Sulfate-Reducing Populations in Bacterial Biofilms

We describe the combined application of microsensors and molecular techniques to investigate the development of sulfate reduction and of sulfate-reducing bacterial populations in an aerobic bacterial biofilm. Microsensor measurements for oxygen showed that anaerobic zones developed in the biofilm within 1 week and that oxygen was depleted in the top 200 to 400 μm during all stages of biofilm development. Sulfate reduction was first detected after 6 weeks of growth, although favorable conditions for growth of sulfate-reducing bacteria (SRB) were present from the first week. In situ hybridization with a 16S rRNA probe for SRB revealed that sulfate reducers were present in high numbers (approximately 10⁸ SRB/ml) in all stages of development, both in the oxic and anoxic zones of the biofilm. Denaturing gradient gel electrophoresis (DGGE) showed that the genetic diversity of the microbial community increased during the development of the biofilm. Hybridization analysis of the DGGE profiles with taxon-specific oligonucleotide probes showed that Desulfobulbus and Desulfovibrio were the main sulfate-reducing bacteria in all biofilm samples as well as in the bulk activated sludge. However, different Desulfobulbus and Desulfovibrio species were found in the 6th and 8th weeks of incubation, respectively, coinciding with the development of sulfate reduction. Our data indicate that not all SRB detected by molecular analysis were sulfidogenically active in the biofilm.