Effects of environmental conditions on the nitrifying population dynamics in a pilot wastewater treatment plant

The effect of environmental conditions, especially ammonium concentration, on community composition and nitrification activity of nitrifying bacterial biofilms in a pilot wastewater treatment plant was examined. A decreasing ammonium gradient was created when four aerated tanks with suspended carrier material were serially fed with wastewater. Community composition was analysed using fluorescence in situ hybridization (FISH) probes as well as partial 16S rRNA and amoA gene analysis using polymerase chain reaction-denaturating gradient gel electrophoresis (PCR-DGGE) and sequencing. Fluorescence in situ hybridization probes identified at least five ammonia-oxidizing bacterial (AOB) and two nitrite-oxidizing bacterial (NOB) populations. A change in nitrifying community was detected in the tanks, indicating that ammonium was an important structuring factor. Further, we found support for different autoecology within the Nitrosomonas oligotropha lineage, as at least one population within this lineage increased in relative abundance with ammonium concentration while another population decreased. Absolute numbers of AOB and NOB growing in biofilms on the carriers were determined and the cell specific nitrification rates calculated seemed strongly correlated to ammonium concentration. Oxygen could also be limiting in the biofilms of the first tank with high ammonium concentrations. The response of the nitrifying community to increased ammonium concentrations differed between the tanks, indicating that activity correlates with community structure.     

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.     

Investigation of total bacterial and ammonia-oxidizing bacterial community composition in a full-scale aerated submerged biofilm reactor for drinking water pretreatment in China

The community composition of total bacteria and ammonia-oxidizing bacteria in a full-scale aerated submerged biofilm reactor for drinking water pretreatment was characterized by analysis of 16S rRNA gene and the functional gene amoA, respectively. Sampling was performed in February and in July. 16S rRNA gene clone libraries revealed 13 bacterial divisions. At both sampling dates, the majority of clone sequences were related to the Alpha- and Betaproteobacteria. A minor proportion belonged to the following groups: Gammaproteobacteria, Deltaproteobacteria, Nitrospira, Firmicutes, Acidobacteria, Verrucomicrobia, Actinobacteria, Planctomycetes, Chloroflexi, Gemmatimonadetes and the Cytophaga-Flavobacterium-Bacteroides group. Some sequences related to bacteria owning high potential metabolic capacities were detected in both samples, such as Rhodobacter-like rRNA gene sequences. Surveys of cloned amoA genes from the two biofilm samples revealed ammonia-oxidizing bacterial sequences affiliated with the Nitrosomonas oligotropha lineage, Nitrosomonas communis lineage. An unknown Nitrosomonas group of amoA gene sequences was also detected.     

Use of stable-isotope probing, full-cycle rRNA analysis, and fluorescence in situ hybridization-microautoradiography to study a methanol-fed denitrifying microbial community

A denitrifying microbial consortium was enriched in an anoxically operated, methanol-fed sequencing batch reactor (SBR) fed with a mineral salts medium containing methanol as the sole carbon source and nitrate as the electron acceptor. The SBR was inoculated with sludge from a biological nutrient removal activated sludge plant exhibiting good denitrification. The SBR denitrification rate improved from less than 0.02 mg of NO(3)(-)-N mg of mixed-liquor volatile suspended solids (MLVSS)(-1) h(-1) to a steady-state value of 0.06 mg of NO(3)(-)-N mg of MLVSS(-1) h(-1) over a 7-month operational period. At this time, the enriched microbial community was subjected to stable-isotope probing (SIP) with [(13)C]methanol to biomark the DNA of the denitrifiers. The extracted [(13)C]DNA and [(12)C]DNA from the SIP experiment were separately subjected to full-cycle rRNA analysis. The dominant 16S rRNA gene phylotype (group A clones) in the [(13)C]DNA clone library was closely related to those of the obligate methylotrophs Methylobacillus and Methylophilus in the order Methylophilales of the Betaproteobacteria (96 to 97% sequence identities), while the most abundant clone groups in the [(12)C]DNA clone library mostly belonged to the family Saprospiraceae in the Bacteroidetes phylum. Oligonucleotide probes for use in fluorescence in situ hybridization (FISH) were designed to specifically target the group A clones and Methylophilales (probes DEN67 and MET1216, respectively) and the Saprospiraceae clones (probe SAP553). Application of these probes to the SBR biomass over the enrichment period demonstrated a strong correlation between the level of SBR denitrification and relative abundance of DEN67-targeted bacteria in the SBR community. By contrast, there was no correlation between the denitrification rate and the relative abundances of the well-known denitrifying genera Hyphomicrobium and Paracoccus or the Saprospiraceae clones visualized by FISH in the SBR biomass. FISH combined with microautoradiography independently confirmed that the DEN67-targeted cells were the dominant bacterial group capable of anoxic [(14)C]methanol uptake in the enriched biomass. The well-known denitrification lag period in the methanol-fed SBR was shown to coincide with a lag phase in growth of the DEN67-targeted denitrifying population. We conclude that Methylophilales bacteria are the dominant denitrifiers in our SBR system and likely are important denitrifiers in full-scale methanol-fed denitrifying sludges.     

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。     

Structure and function of denitrifying and nitrifying bacterial communities in relation to the plant species in a constructed wetland

The community structure and potential activities of nitrifying and denitrifying bacteria were studied in the rhizosphere of Typha latifolia and Phragmites australis present in a free water system constructed wetland (CW). Potential nitrate reduction and nitrification activities were shown to be significantly higher in the rhizosphere when compared with the nonvegetated sediment. Higher rates were generally obtained for P. australis . The community structure of denitrifying bacteria in the rhizosphere differed from that found at the bulk sediment, as revealed by PCR-denaturing gradient gel electrophoresis (DGGE) of the nitrous oxide reductase encoding gene nosZ . Results also show a greater nosZ genotype diversification and suggest a plant species effect in rhizosphere samples obtained during events of low hydraulic retention times. Ammonia-oxidizing communities were less complex on the basis of PCR-DGGE analysis of the 16S rRNA gene. Retrieved sequences were all related to Nitrosomonas marina and Nitrosomonas ureae , being both present in rhizosphere and bulk sediment regardless of environmental changes. The results demonstrate the effect of vegetation on the functioning and structure of bacterial communities involved in the removal of nitrogen in the treatment cells of a CW and point to the use of vegetation coverage to promote nitrification or denitrification in particular areas.     

Functional robustness and gene pools of a wastewater nitrification reactor: comparison of dispersed and intact biofilms when stressed by low oxygen and low pH

The functional robustness of biofilms in a wastewater nitrification reactor, and the gene pools therein, were investigated. Nitrosomonas and Nitrosospira spp. were present in similar amounts (cloning-sequencing of ammonia-oxidizing bacteria 16S rRNA gene), and their estimated abundance (1.1 x 10(9) cells g(-1) carrier material, based on amoA gene real-time PCR) was sufficient to explain the observed nitrification rates. The biofilm also had a diverse community of heterotrophic denitrifying bacteria (cloning-sequencing of nirK). Anammox 16S rRNA genes were detected, but not archaeal amoA. Dispersed biofilms (DB) and intact biofilms (IB) were incubated in gas-tight reactors at different pH levels (4.5 and 5.5 vs. 6.5) while monitoring O(2) depletion and concentrations of NO, N(2)O and N(2) in the headspace. Nitrification was severely reduced by suboptimal O(2) concentrations (10-100 microM) and low pH (IB was more acid tolerant than DB), but the N(2)O/NO(3)(-) product ratio of nitrification remained low (<10(-3)). The NO(2)(-) concentrations during nitrification were generally 10 times higher in DB than in IB. Transient NO and N(2)O accumulation at the onset of denitrification was 10-10(3) times higher in DB than in IB (depending on the pH). The contrasting performance of DB and IB suggests that the biofilm structure, with anoxic/micro-oxic zones, helps to stabilize functions during anoxic spells and low pH.     

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.     

Microbial composition of the activated sludge of Moscow wastewater treatment plants

The contribution of the major technologically important microbial groups (ammonium- and nitrite-oxidizing, phosphate-accumulating, foam-inducing, and anammox bacteria, as well as planctomycetes and methanogenic archaea) was characterized for the aeration tanks of the Moscow wastewater treatment facilities. FISH investigation revealed that aerobic sludge were eubacterial communities; the metabolically active archaea contributed insignificantly. Stage II nitrifying microorganisms and planctomycetes were significant constituents of the bacterial component of activated sludges, with Nitrobacter spp. being the dominant nitrifiers. No metabolically active anammox bacteria were revealed in the sludge from aeration tanks. The sludge from the aeration tanks using different wastewater treatment technologies were found to have differing characteristics. Abundance of the nitrifying and phosphate-accumulating bacteria in the sludge generally correlated with microbial activity in microcosms and with efficiency of nitrogen and phosphorus removal from wastewater. The highest microbial numbers and activity were found in the sludge of the tanks operating according to the technologies developed in the universities of Hannover and Cape Town. The activated sludge from the Novokur’yanovo facilities, where abundant growth of filamentous bacteria resulted in foam formation, exhibited the lowest activity. The group of foaming bacteria included Gordonia spp. and Acinetobacter spp utilizing petroleum and motor oils, Sphaerotilus spp. utilizing unsaturated fatty acids, and Candidatus ‘Microthrix parvicella’. Thus, the data on abundance and composition of metabolically active microorganisms obtained by FISH may be used for the technological control of wastewater treatment.     

Nitrification, denitrification, and dechlorination in bleached kraft pulp mill wastewater

This study deals with combining the biologi cal removal of organic halogens with the removal of nitrogen from bleached kraft pulp mill wastewater in fluidized-bed reactors under nitrifying and denitrifying conditions. Untreated and biotreated bleached kraft pulp mill wastewaters had no detrimental effect on nitrification or denitrification. The nitrifying biofilm reactor, pregrown on synthetic inorganic feed with ammonia, removed without a lag phase adsorbable organic halogens [7.2 mg Cl (g biomass volatile solids)⁻¹day⁻¹] from bleached kraft pulp mill wastewater and selected chlorophenols from synthetic wastewater. Electron microscopical examination of the biofilm showed that bacteria, morphologically similar to the nitrifying species Nitrosomonas or Nitrobacter, and Nitrosospira were dominant. The denitrifying fluidized-bed reactor, pregrown on nitrate and methanol, denitrified without a lag phase bleached kraft pulp mill wastewater. Under denitrifying conditions, 35% of the total organic carbon content of untreated bleached kraft pulp mill waste water was removed. The reducing power delivered by untreated bleached kraft pulp mill wastewater for denitrification was 2 mmol electrons/mmol carbon mineralized. Dechlorination under denitrifying conditions was negligible. Received: 21 November 1996 / Received revision: 27 January 1997 / Accepted: 1 February 1997     

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.     

Molecular and ecological analyses of microbial community structures in biofilms of a full-scale Aerated Up-Flow Biobead process

Molecular and cultivation techniques were used to characterize the bacterial communities of biobead reactor biofilms in a sewage treatment plant to which an Aerated Up-Flow Biobead process was applied. With this biobead process, the monthly average values of various chemical parameters in the effluent were generally kept under the regulation limits of the effluent quality of the sewage treatment plant during the operation period. Most probable number (MPN) analysis revealed that the population of denitrifying bacteria was abundant in the biobead #1 reactor, denitrifying and nitrifying bacteria coexisted in the biobead #2 reactor, and nitrifying bacteria prevailed over denitrifying bacteria in the biobead #3 reactor. The results of the MPN test suggested that the biobead #2 reactor was a transition zone leading to acclimated nitrifying biofilms in the biobead #3 reactor. Phylogenetic analysis of 16S rDNA sequences cloned from biofilms showed that the biobead #1 reactor, which received a high organic loading rate, had much diverse microorganisms, whereas the biobead #2 and #3 reactors were dominated by the members of Proteobacteria. DGGE analysis with the ammonia monooxygenase (amoA) gene supported the observation from the MPN test that the biofilms of September were fully developed and specialized for nitrification in the biobead reactor #3. All of the DNA sequences of the amoA DGGE bands were very similar to the sequence of the amoA gene of Nitrosomonas species, the presence of which is typical in the biological aerated filters. The results of this study showed that organic and inorganic nutrients were efficiently removed by both denitrifying microbial populations in the anaerobic tank and heterotrophic and nitrifying bacterial biofilms well-formed in the three functional biobead reactors in the Aerated Up-Flow Biobead process.     

Investigation of an acetate-fed denitrifying microbial community by stable isotope probing, full-cycle rRNA analysis, and fluorescent in situ hybridization-microautoradiography

The acetate-utilizing microbial consortium in a full-scale activated sludge process was investigated without prior enrichment using stable isotope probing (SIP). [13C]acetate was used in SIP to label the DNA of the denitrifiers. The [13C]DNA fraction that was extracted was subjected to a full-cycle rRNA analysis. The dominant 16S rRNA gene phylotypes in the 13C library were closely related to the bacterial families Comamonadaceae and Rhodocyclaceae in the class Betaproteobacteria. Seven oligonucleotide probes for use in fluorescent in situ hybridization (FISH) were designed to specifically target these clones. Application of these probes to the sludge of a continuously fed denitrifying sequencing batch reactor (CFDSBR) operated for 16 days revealed that there was a significant positive correlation between the CFDSBR denitrification rate and the relative abundance of all probe-targeted bacteria in the CFDSBR community. FISH-microautoradiography demonstrated that the DEN581 and DEN124 probe-targeted cells that dominated the CFDSBR were capable of taking up [14C]acetate under anoxic conditions. Initially, DEN444 and DEN1454 probe-targeted bacteria also dominated the CFDSBR biomass, but eventually DEN581 and DEN124 probe-targeted bacteria were the dominant bacterial groups. All probe-targeted bacteria assessed in this study were denitrifiers capable of utilizing acetate as a source of carbon. The rapid increase in the number of organisms positively correlated with the immediate increase in denitrification rates observed by plant operators when acetate is used as an external source of carbon to enhance denitrification. We suggest that the impact of bacteria on activated sludge subjected to intermittent acetate supplementation should be assessed prior to the widespread use of acetate in the wastewater industry to enhance denitrification.     

喹啉与吲哚驯化的反硝化反应器的微生物群落结构分析比较

[目的]本研究旨在比较分析分别以喹啉和吲哚为底物,在相同条件下驯化的两个反硝化生物反应器的微生物群落结构.[方法]采用相同的种子污泥和相同的驯化条件,经过大约6周的驯化后,两个反应器均达到稳定而高效的污染物去除能力,通过16S rDNA克隆文库技术对两个反应器的微生物群落结构进行研究.[结果]研究发现,微生物群落结构表现出很大的差异.喹啉驯化的群落中所有的OTU都属于Betaproteobacteria,而吲哚驯化的群落中Betaproteobacteria占56.3%,吲哚驯化的群落具有更高的多样性.两个群落的优势OTU也不同,喹啉驯化群落中Thauera及其它Rhodocyclaceae科的微生物占整个群落的73%,而吲哚驯化群落中优势OTU为Comamonadaceae科、Alcaligenaceae科和Rhodocyclaceae科等类型的微生物,其中Comamonadaceae科的一个OTU占整个群落的28.7%.[结论]不同的驯化底物对微生物群落的组成具有较强的选择作用.首次报道并比较了可高效降解喹啉和吲哚的反硝化生物反应器的微生物群落结构.     

Analysis of the phylogenetic diversity of estrone-degrading bacteria in activated sewage sludge using microautoradiography-fluorescence in situ hybridization

In situ uptake of [2,4,6,7-3H(N)]estrone ([3H]E1) by the major phylogenetic groups present in activated sludge samples from two different municipal wastewater treatment plants was investigated using microautoradiography-fluorescence in situ hybridization (MAR-FISH). Approximately 1-2% of the total cells confined in the samples by an EUB probe mix contributed to E1 assimilation. Almost all the detected E1-assimilating cells involved in the early phase of E1 degradation were affiliated with the Beta- and Gammaproteobacteria. In the early phase of E1 degradation, no E1-assimilating cells affiliated with the Alphaproteobacteria, Actinobacteria, the Cytophaga-Flavobacterium cluster of phylum Bacteroidetes, or the phyla Chloroflexi, Nitrospira and Planctomycetes were detected. Bacteria affiliated with the Betaproteobacteria in the shape of long rods or chains of rods were found to contribute most to in situ E1 degradation. They contributed 61% and 82% of total E1-assimilating cells in cultures from two sources of activated sludge spiked with [3H]E1. The E1-degrading bacteria related to the Betaproteobacteria differed phylogenetically from the aerobic E1-degrading bacterial isolates reported in previous studies. In addition, MAR-FISH revealed the significant contribution of E1-degrading bacteria affiliated with the Gammaproteobacteria in the degradation of E1 in activated sludge.     

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 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.     

Simultaneous nitrification and denitrification by controlling vertical and horizontal microenvironment in a membrane-aerated biofilm reactor

Nitrogen and carbon components in domestic modified wastewater were completely removed by simultaneous nitrification and denitrification using a membrane-aerated biofilm reactor where biofilm was fixed on a hollow-fiber membrane. To measure the spatial distribution of pH, ammonium and nitrate ions and to observe microbes inside the biofilm fixed on the membrane, microelectrodes and the fluorescence in situ hybridization (FISH) method were applied. Due to plug flow in the vertical direction (from the bottom to the top of the reactor), ammonium nitrogen was gradually removed and negligible nitrate nitrogen was detected throughout the reactor. FISH revealed that ammonia-oxidizing bacteria were mainly distributed inside the biofilm and other bacteria, which included denitrifying bacteria, were mainly distributed outside the biofilm and over the suspended sludge. In order to characterize bacterial activity in the vertical direction of the reactor, nitrification rates at lower, central and upper points were calculated using microelectrode data. The nitrification rate at the lower point was 7 and 125 times higher than those at the central and upper points, respectively. These results show that the removal of carbon and nitrogen compounds was accomplished efficiently by using various kinds of bacteria distributed vertically and horizontally in a single reactor.     

Effect of the Earthworms Lumbricus terrestris and Aporrectodea caliginosa on Bacterial Diversity in Soil

Earthworms ingest large amounts of soil and have the potential to radically alter the biomass, activity, and structure of the soil microbial community. In this study, the diversity of eight bacterial groups from fresh soil, gut, and casts of the earthworms Lumbricus terrestris and Aporrectodea caliginosa were studied by single-strand conformation polymorphism (SSCP) analysis using both newly designed 16S rRNA gene-specific primer sets targeting Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Deltaproteobacteria, Bacteroidetes, Verrucomicrobia, Planctomycetes, and Firmicutes and a conventional universal primer set for SSCP, with RNA and DNA as templates. In parallel, the study of the relative abundance of these taxonomic groups in the same samples was performed using fluorescence in situ hybridization. Bacteroidetes, Alphaproteobacteria, and Betaproteobacteria were predominant in communities from the soil and worm cast samples. Representatives of classes Flavobacteria and Sphingobacteria (Bacteroidetes) and Pseudomonas spp. (low-abundant Gammaproteobacteria) were detected in soil and worm cast samples with conventional and taxon-targeting SSCP and through the sequence analysis of 16S rRNA clone libraries. Physiologically active unclassified Sphingomonadaceae (Alphaproteobacteria) and Alcaligenes spp. (Betaproteobacteria) also maintained their diversities during transit through the earthworm intestine and were found on taxon-targeting SSCP profiles from the soil and worm cast samples. In conclusion, our results suggest that some specific bacterial taxonomic groups maintain their diversity and even increase their relative numbers during transit through the gastrointestinal tract of earthworms.     

Nitrification and occurrence of salt-tolerant nitrifying bacteria in the Negev desert soils

Ammonia oxidation potential, major ammonia oxidizers and occurrence of salt-tolerant nitrifying bacteria were studied in soil samples collected from diverse ecosystems along the northern Negev desert. Great diversity in ammonia oxidation potential was observed among the soil samples, and ammonia oxidizers were the rate-limiting step of nitrification. Denaturing gradient gel electrophoresis and partial 16S rRNA gene sequences indicate that members of the genus Nitrosospira are the major ammonia oxidizers in the natural desert soil samples. Upon enrichment with different salt concentrations, salt-tolerant nitrifying enrichments were established from several soil samples. In two enrichments, nitrification was not inhibited by 400 mM NaCl. Electrophoretic analysis and partial 16S rRNA gene sequences indicate that Nitrosomonas species were dominant in the 400 mM salt enrichment. The results point towards the potential of the desert ecosystem as a source of stress-tolerant nitrifying bacteria or other microorganisms with important properties.