Nitrite-oxidizing Bacteria Nitrospira Research Articles

Microbial community analysis of membrane bioreactor incorporated with biofilm carriers and activated carbon for nitrification of urine

The integration of powdered activated carbon and biofilm carriers in a membrane bioreactor (MBR) presents a promising approach to address the challenge of long hydraulic retention time (HRT) for nitrification of hydrolysed urine. This study investigated the effect of the incorporation in the MBR on microbial dynamics, focusing on dominant nitrifying bacteria. The results showed that significant shifts in microbial compositions were observed with the feed transition to full-strength urine and across different sludge growth forms. Remarkably, the nitrite-oxidizing bacteria Nitrospira were highly enriched in the suspended sludge. Simultaneously, ammonia-oxidizing bacteria, Nitrosococcaceae thrived in the attached biomass, showing a significant seven-fold increase in relative abundance compared to its suspended counterpart. Consequently, the incorporated MBR displayed 36% higher nitrification rate and 40% HRT reduction compared to the conventional MBR. This study provides valuable insights on the potential development of household or building scale on-site nutrient recovery from urine to fertiliser.

Towards low carbon demand and highly efficient nutrient removal: Establishing denitrifying phosphorus removal in anaerobic/anoxic/oxic + nitrification system

A two-sludge anaerobic/anoxic/oxic + nitrification system with simultaneous nitrogen and phosphorus removal was studied for enhanced low-strength wastewater treatment. After 158 days of operation, excellent NH4+-N, chemical oxygen demand (COD) and PO43−-P removal (99.0 %, 90.0 % and 92.0 %, respectively) were attained under a low carbon/nitrogen ratio of 5, resulting in effluent NH4+-N, COD and PO43−-P concentrations of 0.3, 30.0 and 0.5 mg/L, respectively. The results demonstrate that the anaerobic/anoxic/oxic sequencing batch reactor (A2-SBR) and nitrification sequencing batch reactor (N-SBR) had favorable denitrifying phosphorus removal and nitrification performance, respectively. High-throughput sequencing results indicate that the phosphate-accumulating organisms Dechloromonas (1.1 %) and Tetrasphaera (1.2 %) were enriched in the A2-SBR, while the ammonia-oxidizing bacteria Nitrosomonas (7.8 %) and the nitrite-oxidizing bacteria Nitrospira (18.1 %) showed excellent accumulation in the N-SBR. Further analysis via functional prediction revealed that denitrification is the primary pathway of nitrogen metabolism throughout the system. Overall, the system achieved low carbon and high efficiency nutrient removal.

Global landfill leachate characteristics: Occurrences and abundances of environmental contaminants and the microbiome

Landfill leachates are complex mixtures containing very high concentrations of biodegradable and recalcitrant toxic compounds. Understanding the major contaminant components and microbial community signatures in global landfill leachates is crucial for timely decision-making regarding contaminant management and treatment. Therefore, this study analyzed leachate data from 318 landfill sites primarily used for municipal solid waste disposal, focusing on their chemical and microbiological characteristics. The most prevalent and dominant components in landfill leachates are the chemical oxygen demand (COD, 3.7–75.9 × 103 mg/L) and NH4+ (0.03–0.81 × 104 mg/L), followed by salt species such as SO42- (0.03–5.25 × 103 mg/L), Cl- (3.2–7.8 × 103 mg/L), K+ (0.58–4.20 × 103 mg/L), Na+ (1.3–13.0 × 103 mg/L) and Ca2+ (2.35–230.23 × 103 mg/L), which exhibit significant fluctuations. Heavy metals and metalloids are widely distributed in most landfill leachates but at relatively low concentrations (<182.8 mg/L) compared to conventional parameters. Importantly, there is a distinct global variation in the occurrence of emerging environmental contaminants (ECs). Among these compounds, perfluorooctanoic acid (PFOA, 0.02–7.50 × 103 μg/L) of per- and poly-fluoroalkyl substances (PFAS), bisphenol A (BPA, 0.01–33.46 × 103 μg/L) belonged to endocrine-disrupting compounds (EDCs), together with di-ethyltoluamide (DEET, 1.0–1.0 × 103 μg/L) affiliated to pharmaceuticals and personal care products (PPCPs) are the most frequently detected in landfill leachates. Additionally, the microbial community compositions in most leachates are primarily dominated by Proteobacteria, Bacteroidota, Firmicutes, and Chloroflexi, and some of their abundances are correlated with the concentrations of NH4+, NO3-, Cl-, Na+ and Cr. Notably, the leading microbes driving advanced removal of inorganic nitrogen in the treatment systems are Candidatus Brocadia (anammox), denitrifying Thauera, nitrite-oxidizing bacteria Nitrospira, along with ammonia-oxidizing bacteria Nitrosomonas and Nitrosospira. The findings of this work provide a deeper insight into the leachate characteristics and the sustainable management of landfill leachates, especially presenting a snapshot of the global distribution of pollutants and also the microbiome.

Revealing the behavior of perfluorooctane sulfonic acid in an aerobic granular sludge system: Fate and impact

The biogeochemistry (i.e., migration and transformation) and impacts of perfluorooctane sulfonic acid (PFOS) in an aerobic granular sludge (AGS) system have been studied. The results suggested that PFOS removal rates under 0.1, 0.5 and 5.0 mg/L PFOS treatment were 89.23 ± 0.92 %, 71.92 ± 1.45 % and 48.15 ± 1.90 %, respectively, which was mainly attributed to the adsorption mechanism involving electrostatic, hydrophobic and ionic bridging interactions. Meanwhile, the adsorbed PFOS deteriorated sludge flocculation. Compared with the control, 0.1 mg/L PFOS treatment increased the concentration of effluent suspended solids (ESS) from 70 ± 5 to 238 ± 15 mg/L. Interestingly, the denitrification performance of the AGS improved (i.e., total nitrogen (TN) average removal rate increased from 87.05 ± 0.21 % to 96.56 ± 0.05 %), which was contrary to the outcomes for activated sludge. Further analysis showed that PFOS reduced the percentage of norank_f_norank_o_Ardenticatenales, norank_f_Sphingomonadaceae and Candidatus_Competibacter maintaining the particle stability, which led to the decrease in AGS flocculation. In addition, the relative abundance of nitrite-oxidizing bacteria Nitrospira decreased under PFOS exposure, while the total abundance of denitrifying bacteria (mainly Dechloromonas) increased. This might enhance the short-cut nitrification and denitrification capacity of the AGS system and promote the removal of TN. However, PFOS had no obvious impact on the settleability of AGS and the transformation of chemical oxygen demand (COD) and soluble phosphorus (SOP). Our findings will provide theoretical insights and practical support for risk control in the process of treating fluoride-containing wastewater.

Metabolic versatility of the nitrite-oxidizing bacterium Nitrospira marina and its proteomic response to oxygen-limited conditions

The genus Nitrospira is the most widespread group of nitrite-oxidizing bacteria and thrives in diverse natural and engineered ecosystems. Nitrospira marina Nb-295T was isolated from the ocean over 30 years ago; however, its genome has not yet been analyzed. Here, we investigated the metabolic potential of N. marina based on its complete genome sequence and performed physiological experiments to test genome-derived hypotheses. Our data confirm that N. marina benefits from additions of undefined organic carbon substrates, has adaptations to resist oxidative, osmotic, and UV light-induced stress and low dissolved pCO2, and requires exogenous vitamin B12. In addition, N. marina is able to grow chemoorganotrophically on formate, and is thus not an obligate chemolithoautotroph. We further investigated the proteomic response of N. marina to low (∼5.6 µM) O2 concentrations. The abundance of a potentially more efficient CO2-fixing pyruvate:ferredoxin oxidoreductase (POR) complex and a high-affinity cbb3-type terminal oxidase increased under O2 limitation, suggesting a role in sustaining nitrite oxidation-driven autotrophy. This putatively more O2-sensitive POR complex might be protected from oxidative damage by Cu/Zn-binding superoxide dismutase, which also increased in abundance under low O2 conditions. Furthermore, the upregulation of proteins involved in alternative energy metabolisms, including Group 3b [NiFe] hydrogenase and formate dehydrogenase, indicate a high metabolic versatility to survive conditions unfavorable for aerobic nitrite oxidation. In summary, the genome and proteome of the first marine Nitrospira isolate identifies adaptations to life in the oxic ocean and provides insights into the metabolic diversity and niche differentiation of NOB in marine environments.

Treatment of coking wastewater in biofilm-based bioaugmentation process: Biofilm formation and microbial community analysis

Coking wastewater (CWW) containing complicated organic compositions and strong toxicity cause potential hazards to natural water bodies as well as human health. The aim of this study was integrating newly isolated Comamonas sp. ZF-3, biofilm-based bioaugmentation and fluidized bed reactor into an anoxic filter-fluidized bed reactor (AF-FBR) system to treat actual CWW. The results showed that 93 % of chemical oxygen demand (COD) and 97 % of ammonia nitrogen (NH4+-N) removal efficiency were achieved with hydraulic retention time of 70 h. The main pollutants including phenolic compounds, heterocyclic compounds and polycyclic aromatic hydrocarbons could be removed via biofilm-based process in AF-FBR. The formation of carrier biofilm was consistent with the system performance as well as the biofilm community evolution, during which the microbial community was gradually dominated by some functional genus (e.g., Comamonas, Thiobacillus, Pseudomonas and Thauera), meanwhile, ammonium-oxidizing bacteria Nitrosomonas, nitrite-oxidizing bacteria Nitrospira and denitrifiers (e.g., Pseudomonas, Thiobacillus and Bacillus) coexisted in biofilm to form a microbial community for biological nitrogen removal. Such microbial community structure explained the observed simultaneous removal of COD and NH4+-N in the AF-FBR.

Free ammonia shock treatment eliminates nitrite-oxidizing bacterial activity for mainstream biofilm nitritation process

Nitritation (NH4+ → NO2−) is a critical step to provide nitrite for the followed anammox in a two-stage nitrogen removal system. In the mainstream line of wastewater treatment plants, the nitritation has not been applied to date, because of the significant difficulties in the suppression of nitrite-oxidizing bacteria (NOB), especially in biofilm systems. This study aims to systematically assess the effect of free ammonia (FA) shock treatment on the mainstream biofilm nitritation process through an integration of laboratory reactor operation, microbial community analysis, incubation tests, and kinetic model evaluation. In a laboratory nitrifying moving bed biofilm reactor (MBBR) fed with domestic-strength synthetic wastewater, it was shown that with the exposure of carrier-biofilms containing ammonia-oxidizing bacteria (AOB) Nitrosomonas and NOB Nitrospira to a high-level of FA (1068 mg NH3-N/L) over two days, a much higher residual AOB level was retained in the biofilm in comparison to NOB. The higher residual AOB on biofilm led to much faster recovery of AOB over NOB after the shock treatment, when normal operation resumed with the dissolved oxygen (DO) controlled at around 0.2 mg/L. The faster recovery of AOB than NOB subsequently gave rise to a stable, high nitrite accumulation ratio (nearly 100%) over a long period (two months). Collectively, these results suggest that FA shock treatment in conjunction with limited DO control is effective in eliminating NOB for mainstream biofilm nitritation process. The chemical cost would be marginal given the intermittent nature of the FA shock strategy and the readily available ammonium in the anaerobic sludge digestion liquor.

Niche differentiation of ammonia and nitrite oxidizers along a salinity gradient from the Pearl River estuary to the South China Sea

Abstract. The niche differentiation of ammonia and nitrite oxidizers is controversial because they display disparate patterns in estuarine, coastal, and oceanic regimes. We analyzed diversity and abundance of ammonia-oxidizing archaea (AOA) and β-proteobacteria (AOB), nitrite-oxidizing bacteria (NOB), and nitrification rates to identify their niche differentiation along a salinity gradient from the Pearl River estuary to the South China Sea. AOA were generally more abundant than β-AOB; however, AOB more clearly attached to particles compared with AOA in the upper reaches of the Pearl River estuary. The NOB Nitrospira had higher abundances in the upper and middle reaches of the Pearl River estuary, while Nitrospina was dominant in the lower estuary. In addition, AOB and Nitrospira could be more active than AOA and Nitrospina since significantly positive correlations were observed between their gene abundance and the nitrification rate in the Pearl River estuary. There is a significant positive correlation between ammonia and nitrite oxidizer abundances in the hypoxic waters of the estuary, suggesting a possible coupling through metabolic interactions between them. Phylogenetic analysis further revealed that the AOA and NOB Nitrospina subgroups can be separated into different niches based on their adaptations to substrate levels. Water mass mixing is apparently crucial in regulating the distribution of nitrifiers from the estuary to open ocean. However, when eliminating water mass effect, the substrate availability and the nitrifiers' adaptations to substrate availability via their ecological strategies essentially determine their niche differentiation.

The relative contribution of nitrifiers to autotrophic nitrification across a pH-gradient in a vegetable cropped soil

Microbial nitrification plays an important role in nitrogen cycling in ecosystems. Nitrification is performed by ammonia-oxidizing archaea (AOA), ammonia-oxidizing bacteria (AOB), and nitrite-oxidizing bacteria (NOB) including complete ammonia oxidizers. However, the relative importance of nitrifiers in autotrophic nitrification in relation to soil pH is still unclear. Combining DNA-based stable isotope probing (SIP) and molecular biological techniques, we investigated the abundance, structure, and activity of AOA, AOB, and NOB along a pH-gradient (3.97–7.04) in a vegetable cropped soil. We found that AOA abundance outnumbered AOB abundance and had a significantly negative relationship with soil pH. The abundances of NOB Nitrospira 16S rRNA, nxrB gene, and Nitrobacter nxrA gene were affected by soil pH. Incubation of soil with 13CO2 and DNA-SIP analysis demonstrated that significant 13CO2 assimilation by AOA rather than by AOB occurred in the acidic soils, whereas the labeled 13C level of AOA was much less in the neutral soil than in the acidic soils. There was no evidence of 13CO2 assimilation by NOB except for Nitrobacter with NxrB gene at pH 3.97. Phylogenetic analysis of AOA amoA gene in the 13C- and 12C-labeled treatments showed that the active AOA mainly belonged to Nitrososphaera in the acidic soils. These results suggested that the main performer of nitrification was AOA in the acidic soils, but both AOA and AOB participated in nitrification in the neutral soil with low nitrification activity. NOB Nitrospira and Nitrobacter did not grow in the soils with pH 4.82–7.04 and other populations of NOB were probably involved in nitrite oxidation in the vegetable cropped soil.

Performance and microbial characteristics of biomass in a full-scale aerobic granular sludge wastewater treatment plant

By modification of the operational conditions of batch reactors, a municipal wastewater treatment plant was upgraded from activated sludge to aerobic granular sludge (AGS) technology. After upgrading, the volume of the biological reactors was reduced by 30%, but the quality of the effluent substantially improved. The concentration of biomass in the reactors increased twofold; the average biomass yield was 0.6 g MLVSS/g COD, and excess granular sludge was efficiently stabilized in aerobic conditions. Canonical correspondence analysis based on the results of next-generation sequencing showed that the time of adaptation significantly influenced the microbial composition of the granules. In mature granules, the abundance of ammonium-oxidizing bacteria was very low, while the abundance of the nitrite-oxidizing bacteria Nitrospira sp. was 0.5 ± 0.1%. The core genera were Tetrasphaera, Sphingopyxis, Dechloromonas, Flavobacterium, and Ohtaekwangia. Bacteria belonging to these genera produce extracellular polymeric substances, which stabilize granule structure and accumulate phosphorus. The results of this study will be useful for designers of AGS wastewater treatment plants, and molecular data given here provide insight into the ecology of mature aerobic granules from a full-scale facility.

Functional diversity of microbial communities in pristine aquifers inferred by PLFA- and sequencing-based approaches

Abstract. Microorganisms in groundwater play an important role in aquifer biogeochemical cycles and water quality. However, the mechanisms linking the functional diversity of microbial populations and the groundwater physico-chemistry are still not well understood due to the complexity of interactions between surface and subsurface. Within the framework of Hainich (north-western Thuringia, central Germany) Critical Zone Exploratory of the Collaborative Research Centre AquaDiva, we used the relative abundances of phospholipid-derived fatty acids (PLFAs) to link specific biochemical markers within the microbial communities to the spatio-temporal changes of the groundwater physico-chemistry. The functional diversities of the microbial communities were mainly correlated with groundwater chemistry, including dissolved O2, Fet and NH4+ concentrations. Abundances of PLFAs derived from eukaryotes and potential nitrite-oxidizing bacteria (11Me16:0 as biomarker for Nitrospira moscoviensis) were high at sites with elevated O2 concentration where groundwater recharge supplies bioavailable substrates. In anoxic groundwaters more rich in Fet, PLFAs abundant in sulfate-reducing bacteria (SRB), iron-reducing bacteria and fungi increased with Fet and HCO3− concentrations, suggesting the occurrence of active iron reduction and the possible role of fungi in meditating iron solubilization and transport in those aquifer domains. In more NH4+-rich anoxic groundwaters, anammox bacteria and SRB-derived PLFAs increased with NH4+ concentration, further evidencing the dependence of the anammox process on ammonium concentration and potential links between SRB and anammox bacteria. Additional support of the PLFA-based bacterial communities was found in DNA- and RNA-based Illumina MiSeq amplicon sequencing of bacterial 16S rRNA genes, which showed high predominance of nitrite-oxidizing bacteria Nitrospira, e.g. Nitrospira moscoviensis, in oxic aquifer zones and of anammox bacteria in more NH4+-rich anoxic groundwater. Higher relative abundances of sequence reads in the RNA-based datasets affiliated with iron-reducing bacteria in more Fet-rich groundwater supported the occurrence of active dissimilatory iron reduction. The functional diversity of the microbial communities in the biogeochemically distinct groundwater assemblages can be largely attributed to the redox conditions linked to changes in bioavailable substrates and input of substrates with the seepage. Our results demonstrate the power of complementary information derived from PLFA-based and sequencing-based approaches.

Evaluating the feasibility of ratio control strategy for achieving partial nitritation in a continuous floccular sludge reactor: Experimental demonstration

To investigate the applicability of ratio control strategy to other systems, a continuous floccular sludge reactor was used in this study. It was found that nitrite accumulation was barely detected throughout 70days’ investigation, being the average concentration in the effluent of 0.7±0.4mg/L. Batch experiments indicated that low dissolved oxygen (DO<0.3mg·L−1) greatly repressed the ammonium oxidizing bacteria (AOB) but only slightly inhibited the nitrite oxidizing bacteria (NOB). However, high-throughput sequencing revealed that the ratio of abundance between Nitrospira and Nitrosomonas, being the dominant NOB and AOB respectively, was considerably low (1.2%/18.7%). The weak oxygen gradients in floccular sludge and the selectively enriched K-strategist NOB Nitrospira under oxygen-limited conditions were both contributed to the failure of achieving partial nitritation; therefore, the rapid start-up of partial nitritation process based on proposed ratio control strategy is not feasible for continuous floccular sludge systems treating low-strength wastewater.

Biological treatment of actual petrochemical wastewater using anaerobic/anoxic/oxic process and the microbial diversity analysis.

A novel process integrating anaerobic hydrolysis-acidification (HA) and anoxic/oxic (A/O) reactors was developed to treat the actual petrochemical wastewater, which was operated for more than 8months, the removal efficiency of COD and NH4+-N was monitored, and the microbial community was analyzed. The results showed that the effluent concentrations were maintained at around 99 and 1.3mg/L, with the removal efficiency of 70.6 and 95.4%, respectively at a total hydraulic retention time (HRT) of 20h. The major pollutants in the influent were identified as hydrocarbons, aldehydes, heterocyclic matters, amines, alcohols, phenols, ketones, etc. by GC-MS analysis, while only heterocyclic compounds, ketones, and esters were detected in the effluent after HA-A/O treatment. Bacteria belonging to phyla Chloroflexi, Proteobacteria, and Bacteroidetes were highly enriched in the system. The predominant genera in HA, anoxic, and oxic tanks were Anaerolineaceae uncultured and Desulfobacter, Blastocatella and Anaerolineaceae uncultured, Saprospiraceae uncultured and Nitrosomonadaceae uncultured, respectively. The sulfate-reducing bacteria Desulfobacter, Desulfofustis and Desulfomicrobium were detected only in HA reactor. The ammonium-oxidizing bacteria Nitrosomonadaceae and Nitrosomonas and nitrite-oxidizing bacteria Nitrospira were highly enriched in A/O reactor, which is consistent with the good nitrification performance.

Microbial community in anoxic-oxic-settling-anaerobic sludge reduction process revealed by 454 pyrosequencing analysis.

Modification of the anoxic-oxic (AO) process by inserting a sludge holding tank (SHT) into the sludge return line forms an anoxic-oxic-settling-anaerobic (A+OSA) process that can achieve a 48.98% sludge reduction rate. The 454 pyrosequencing method was used to obtain the microbial communities of the AO and A+OSA processes. Results showed that the microbial community structures of the 2 processes were different as a result of the SHT insertion. Bacteria assigned to the phyla Proteobacteria and Bacteroidetes commonly existed and dominated the microbial populations of the 2 processes. However, the relative abundance of these populations shifted in the presence of SHT. The relative abundance of Proteobacteria decreased during the A+OSA process. A specific comparison at the class level showed that Sphingobacteria was enriched in the A+OSA process. The result suggested that the fermentative bacteria Sphingobacteria may have key functions in reducing the sludge from the A+OSA process. Uncultured Nitrosomonadaceae gradually became the dominant ammonia-oxidizing bacteria, and the nitrite-oxidizing bacterium Nitrospira was enriched in the A+OSA process. Both occurrences were favorable for stabilized nitrogen removal. The known denitrifying species in the A+OSA process were similar to those in the AO process; however, their relative abundance also decreased.

Impact of ozonation and residual ozone-produced oxidants on the nitrification performance of moving-bed biofilters from marine recirculating aquaculture systems

In marine recirculating aquaculture systems (RAS) ozone is often used in combination with biofiltration for the improvement of process water quality. Especially for disinfection purposes ozone residuals are required, that lead to a fast formation of secondary oxidants in seawater, summed up as ozone-produced oxidants (OPO). We studied the impact of OPO on nitrifying biofilter bacteria in a series of laboratory batch experiments by exposing (i) cell suspensions of the ammonia-oxidizing bacteria (AOB) Nitrosomonas marina strain 22 and the nitrite-oxidizing bacteria (NOB) Nitrospira strain Ecomares 2.1, (ii) a pure culture of the NOB Nitrospira strain immobilized on biocarriers, as well as (iii) a heterogeneous biofilm culture settled on biocarriers from a marine RAS for 1h to different OPO concentrations up to 0.6mg/l chlorine equivalent. Subsequent activity tests detected a negative linear correlation between OPO concentration and nitrifying activity of suspended pure cultures. Immobilization on biocarriers increased the tolerance of AOB and NOB dramatically, suggesting the biofilm matrix to be highly protective against OPO. Furthermore, we investigated the chronic effect of moderate ozonation at OPO concentrations of 0, 0.05, 0.10 and 0.15mg/l chlorine equivalent on biofilter performance in a 21 d exposure experiment using 12 experimental RAS, stocked with tilapia (Oreochromis niloticus). Chronic exposure experiments could not reveal any harmful impact on biofilter performance for OPO concentrations up to 0.15mg/l, even at continuous exposure. Surprisingly, nitrifying activity was enhanced at all OPO concentrations compared to the control without ozonation, suggesting moderate ozonation to promote biological nitrification. It can be concluded that rather health, welfare and performance of most cultivated fish species are the limiting factors for ozone dosage than nitrification performance of biofilters. The results may further have practical implications in relation to design and operational strategy of water treatment processes in RAS and might thus contribute to the optimization of an effective and safe treatment combination of biofiltration and ozonation.

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

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

Comparison of Conventional and Integrated Fixed‐Film Activated Sludge Systems: Attached‐ and Suspended‐Growth Functions and Quantitative Polymerase Chain Reaction Measurements

Pilot-scale integrated fixed-film activated sludge (IFAS) and non-IFAS control systems were compared, with respect to overall performance and functional behaviors and microbial population composition in the attached and suspended phases. The suspended phases of the control and IFAS systems exhibited similar rates of ammonia consumption; the attached phase in the second aerobic IFAS reactor had significantly higher rates of ammonia consumption and nitrate production than any other biomass source, and the attached biomass from the first aerobic reactor had the lowest ammonia consumption rates. Quantitative polymerase chain reaction (qPCR) indicated the presence of the ammonia-oxidizing bacteria Nitrosomonas oligotropha and the nitrite-oxidizing bacteria Nitrospira spp. and Nitrobacter spp. Mathematical modeling and qPCR both indicated greater concentrations of nitrifiers in the attached phases of a downstream aerobic reactor relative to the upstream reactor, possibly because of increased competition from heterotrophs for space in the attached phase of the upstream aerobic reactor.

Life without light: microbial diversity and evidence of sulfur- and ammonium-based chemolithotrophy in Movile Cave

Microbial diversity in Movile Cave (Romania) was studied using bacterial and archaeal 16S rRNA gene sequence and functional gene analyses, including ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), soxB (sulfate thioesterase/thiohydrolase) and amoA (ammonia monooxygenase). Sulfur oxidizers from both Gammaproteobacteria and Betaproteobacteria were detected in 16S rRNA, soxB and RuBisCO gene libraries. DNA-based stable-isotope probing analyses using 13C-bicarbonate showed that Thiobacillus spp. were most active in assimilating CO2 and also implied that ammonia and nitrite oxidizers were active during incubations. Nitrosomonas spp. were detected in both 16S rRNA and amoA gene libraries from the 'heavy' DNA and sequences related to nitrite-oxidizing bacteria Nitrospira and Candidatus 'Nitrotoga' were also detected in the 'heavy' DNA, which suggests that ammonia/nitrite oxidation may be another major primary production process in this unique ecosystem. A significant number of sequences associated with known methylotrophs from the Betaproteobacteria were obtained, including Methylotenera, Methylophilus and Methylovorus, supporting the view that cycling of one-carbon compounds may be an important process within Movile Cave. Other sequences detected in the bacterial 16S rRNA clone library included Verrucomicrobia, Firmicutes, Bacteroidetes, alphaproteobacterial Rhodobacterales and gammaproteobacterial Xanthomonadales. Archaeal 16S rRNA sequences retrieved were restricted within two groups, namely the Deep-sea Hydrothermal Vent Euryarchaeota group and the Miscellaneous Crenarchaeotic group. No sequences related to known sulfur-oxidizing archaea, ammonia-oxidizing archaea, methanogens or anaerobic methane-oxidizing archaea were detected in this clone library. The results provided molecular biological evidence to support the hypothesis that Movile Cave is driven by chemolithoautotrophy, mainly through sulfur oxidation by sulfur-oxidizing bacteria and reveal that ammonia- and nitrite-oxidizing bacteria may also be major primary producers in Movile Cave.

Real-time PCR quantification of nitrifying bacteria in a municipal wastewater treatment plant.

Real-time PCR assays using TaqMan or Molecular Beacon probes were developed and optimized for the quantification of total bacteria, the nitrite-oxidizing bacteria Nitrospira, and Nitrosomonas oligotropha-like ammonia oxidizing bacteria (AOB) in mixed liquor suspended solids (MLSS) from a municipal wastewater treatment plant (WWTP) using a single-sludge nitrification process. The targets for the real-time PCR assays were the 16S rRNA genes (16S rDNA) for bacteria and Nitrospira spp. and the amoA gene for N. oligotropha. A previously reported assay for AOB 16S rDNA was also tested for its application to activated sludge. The Nitrospira 16S rDNA, AOB 16S rDNA, and N. oligotropha-like amoA assays were log-linear over 6 orders of magnitude and the bacterial 16S rDNA real-time PCR assay was log-linear over 4 orders of magnitude with DNA standards. When these real-time PCR assays were applied to DNA extracted from MLSS, dilution of the DNA extracts was necessary to prevent PCR inhibition. The optimal DNA dilution range was broad for the bacterial 16S rDNA (1000-fold) and Nitrospira 16S rDNA assays (2500-fold) but narrow for the AOB 16S rDNA assay (10-fold) and N. oligotropha-like amoA real-time PCR assay (5-fold). In twelve MLSS samples collected over one year, mean cell per L values were 4.3 +/- 2.0 x 10(11) for bacteria, 3.7 +/- 3.2 x 10(10) for Nitrospira, 1.2 +/- 0.9 x 10(10) for all AOB, and 7.5 +/- 6.0 x 10(9) for N. oligotropha-like AOB. The percent of the nitrifying population was 1.7% N. oligotropha-like AOB based on the N. oligotropha amoA assay, 2.9% total AOB based on the AOB 16S rDNA assay, and 8.6% nitrite-oxidizing bacteria based on the Nitrospira 16S rDNA assay. Ammonia-oxidizing bacteria in the wastewater treatment plant were estimated to oxidize 7.7 +/- 6.8 fmol/hr/cell based on the AOB 16S rDNA assay and 12.4 +/- 7.3 fmol/hr/cell based on the N. oligotropha amoA assay.