Single-stage autotrophic nitrogen removal process at high loading rate: granular reactor performance, kinetics, and microbial characterization.

For the possible highest performance of single-stage combined partial nitritation/anammox (PNA) process, a continuous complete-mix granular reactor was operated at progressively higher nitrogen loading rate. The variations in bacterial community structure of granules were also characterized using high-throughput pyrosequencing, to give a detail insight to the relationship between reactor performance and functional organism abundance within completely autotrophic nitrogen removal system. In 172 days of operation, a superior total nitrogen (TN) removal rate over 3.9 kg N/(m3/day) was stable implemented at a fixed dissolved oxygen concentration of 1.9 mg/L, corresponding to the maximum specific substrate utilization rate of 0.36/day for TN based on the related kinetics modeling. Pyrosequencing results revealed that the genus Nitrosomonas responsible for aerobic ammonium oxidation was dominated on the granule surface, which was essential to offer the required niche for the selective enrichment of anammox bacteria (genus Candidatus Kuenenia) in the inner layer. And the present of various heterotrophic organisms with general functions, known as fermentation and denitrification, could not be overlooked. In addition, it was believed that an adequate excess of ammonium in the bulk liquid played a key role in maintaining process stability, by suppressing the growth of nitrite-oxidizing bacteria through dual-substrate competitions.

Impact of Heavy Metal Pollution on Ammonia Oxidizers in Soils in the Vicinity of a Tailings Dam, Baotou, China.

Soil heavy metal pollution has received increasing attention due to their toxicity to soil microorganisms. We have analyzed the effects of heavy metal pollution on ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB) in soils in the vicinity of a tailings dam of Baotou region, China. Results showed that AOB were dominated with Nitrosomonas-like clusters, while AOA was dominated by group1.1b (Nitrososphaera cluster). Single Cd and Cr contents, as well as compound heavy metal pollution levels, had a significant negative impact on soil potential nitrification rate and both diversities of AOA and AOB. No clear relationship was found between any single heavy metal and abundance of AOA or AOB. But compound pollution could significantly decrease AOA abundance. The results indicated that heavy metal pollution had an obviously deleterious effect on the abundance, diversity, activity and composition of ammonia oxidizers in natural soils.

Dynamics of the diversity and structure of the overall and nitrifying microbial community in activated sludge along gradient copper exposures.

Diversity and composition of the microbial community, especially the nitrifiers, are essential to the treatment efficiency of wastewater in activated sludge systems. Heavy metals commonly present in the wastewater influent such as Cu can alter the community structure of nitrifiers and lower their activity. However, the dynamics of microbial community along a gradient of metal exposure have largely been unexplored, partially due to the limitations in traditional molecular methods. This study explored the dynamics regarding the diversity and community structures of overall and nitrifying microbial communities in activated sludge under intermittent Cu gradient loadings using Illumina sequencing. We created a new local nitrifying bacterial database for sequence BLAST searches. High Cu loadings (>10.9 mg/L) impoverished microbial diversity and altered the microbial community. Overall, Proteobacteria was the predominant phylum in the activated sludge system, in which Zoogloea, Thauera, and Dechloromonas (genera within the Rhodocyclaceae family of the Beta-proteobacteria class) were the dominant genera in the presence of Cu. The abundance of unclassified bacteria at the phylum level increased substantially with increasing Cu loadings. Nitrosomonas and Nitrospira were the predominant nitrifiers. The nitrifying bacterial community changed through increasing abundance and shifting to Cu-tolerant species to reduce the toxic effects of Cu. Our local nitrifying bacterial database helped to improve the resolution of bacterial identification. Our results provide insights into the dynamics of microbial community in response to various metal concentrations in activated sludge systems and improve our understanding regarding the effect of metals on wastewater treatment efficiency.

A pilot-scale study on PVA gel beads based integrated fixed film activated sludge (IFAS) plant for municipal wastewater treatment.

In the present study, a pilot-scale reactor incorporating polyvinyl alcohol gel beads as biomass carrier and operating in biological activated sludge mode (a combination of moving bed biofilm reactor (MBBR) and activated sludge) was investigated for the treatment of actual municipal wastewater. The results, during a monitoring period of 4 months, showed effective removal of chemical oxygen demand (COD), biological oxygen demand (BOD) and NH3-N at optimum conditions with 91%, ∼92% and ∼90% removal efficiencies, respectively. Sludge volume index (SVI) values of activated sludge varied in the range of 25-72 mL/g, indicating appreciable settling characteristics. Furthermore, soluble COD and BOD in the effluent of the pilot plant were reduced to levels well below discharge limits of the Punjab Pollution Control Board, India. A culture dependent method was used to enrich and isolate abundant heterotrophic bacteria in activated sludge. In addition to this, 16S rRNA genes analysis was performed to identify diverse dominant bacterial species in suspended and attached biomass. Results revealed that Escherichia coli, Pseudomonas sp. and Nitrosomonas communis played a significant role in biomass carrier, while Acinetobactor sp. were dominant in activated sludge of the pilot plant. Identification of ciliated protozoa populations rendered six species of ciliates in the plant, among which Vorticella was the most dominant.

Analysis of microbial population dynamics in a partial nitrifying SBR at ambient temperature.

In this study, a lab-scale partial nitrifying sequencing batch reactor (SBR) was developed to investigate partial nitrification at ambient temperature (16-22 °C). Techniques of denaturing gradient gel electrophoresis (DGGE), cloning, and fluorescence in situ hybridization (FISH) were utilized simultaneously to study microbial population dynamics. Partial nitrification was effectively achieved in response to shifts of influent ammonium concentrations. DGGE results showed that higher ammonia concentration referred to lower ammonia-oxidizing bacteria (AOB) diversity in the SBR. Phylogenetic analysis revealed that all the predominant AOB was affiliated with Nitrosomonas genus. FISH analysis illustrated AOB was the predominant nitrifying bacteria of microbial compositions when SBR achieved partial nitrification (PN) at ambient temperature.

Diversity and dynamics of ammonia-oxidizing bacterial communities in a sponge-based trickling filter treating effluent from a UASB reactor.

Changes in ammonia-oxidizing bacterial (AOB) population dynamics were examined in a new sponge-based trickling filter (TF) post-upflow anaerobic sludge blanket (UASB) reactor by denaturating gradient gel electrophoresis (DGGE), and these changes were linked to relevant components influencing nitrification (chemical oxygen demand (COD), nitrogen (N)). The sponge-based packing media caused strong concentration gradients along the TF, providing an ecological selection of AOB within the system. The organic loading rate (OLR) affected the population dynamics, and under higher OLR or low ammonium-nitrogen (NH4(+)-N) concentrations some AOB bands disappeared, but maintaining the overall community function for NH4(+)-N removal. The dominant bands present in the upper portions of the TF were closely related to Nitrosomonas europaea and distantly affiliated to Nitrosomonas eutropha, and thus were adapted to higher NH4(+)-N and organic matter concentrations. In the lower portions of the TF, the dominant bands were related to Nitrosomonas oligotropha, commonly found in environments with low levels of NH4(+)-N. From a technology point of view, changes in AOB structure at OLR around 0.40-0.60 kgCOD m(-3) d(-1) did not affect TF performance for NH4(+)-N removal, but AOB diversity may have been correlated with the noticeable stability of the sponge-based TF for NH4(+)-N removal at low OLR. This study is relevant because molecular biology was used to observe important features of a bioreactor, considering realistic operational conditions applied to UASB/sponge-based TF systems.

Characteristics of aerobic and anaerobic ammonium-oxidizing bacteria in the hyporheic zone of a contaminated river.

Both ß-proteobacterial aerobic ammonium-oxidizing bacteria (AOB) and anaerobic ammonium-oxidizing (ANAMMOX) bacteria were investigated in the hyporheic zone of a contaminated river in China containing high ammonium levels and low chemical oxygen demand. Fluorescence in-situ hybridization (FISH), denaturing gradient gel electrophoresis (DGGE) and cloning-sequencing were employed in this study. FISH analysis illustrated that AOB (average population of 3.5 %) coexisted with ANAMMOX bacteria (0.7 %). The DGGE profile revealed a high abundance and diversity of bacteria at the water-air-soil interface rather than at the water-soil interface. The redundancy analysis correlated analysis showed that the diversity of ANAMMOX bacteria was positively related to the redox potential. The newly detected sequences of ANAMMOX organisms principally belonged to the genus Candidatus "Brocadia", while most ammonia monooxygenase subunit-A gene amoA sequences were affiliated with Nitrosospira and Nitrosomonas. These results suggest that the water-air-soil interface performs an important function in the nitrogen removal process and that the bioresources of AOB and ANAMMOX bacteria can potentially be utilized for the eutrophication of rivers.

Crude oil-degradation and plasmid profile of nitrifying bacteria isolated from oil-impacted mangrove sediment in the Niger Delta of Nigeria.

The crude oil degradability and plasmid profile of autotrophic nitrifying bacteria, Nitrosomonas and Nitrobacter species, isolated from mangrove sediment in the Niger Delta of Nigeria were studied. The effects of temperature, pH and optical density on the utilization of different carbon sources by the bacteria were also investigated. Results showed that nitrifying bacteria could utilize kerosene, diesel oil, jet fuel and engine oil as carbon sources. None utilized hexane and xylene but moderate growth was observed in benzene, phenol and toluene. However, their ability to utilized crude oil varied both in rates of utilization and in growth profiles. Mixed culture of the isolates degrades 52 % of crude oil introduced into the medium followed by Nitrosomonas sp. with 40 % degradation. The least was Nitrobacter sp. with 20 % degradation. The ability of the autotrophs to degrade crude oil was found to be plasmid-mediated through curing experiment and electrophoresis. The size of the plasmid involved was estimated to be 23 kb. The high crude oil utilization of the mixed culture implies that nitrifying bacteria isolated from contaminated ecosystem are excellent crude oil degraders and can be harnessed for bioremediation purposes.

Genome sequence of Nitrosomonas sp. strain AL212, an ammonia-oxidizing bacterium sensitive to high levels of ammonia.

Nitrosomonas sp. strain AL212 is an obligate chemolithotrophic ammonia-oxidizing bacterium (AOB) that was originally isolated in 1997 by Yuichi Suwa and colleagues. This organism belongs to Nitrosomonas cluster 6A, which is characterized by sensitivity to high ammonia concentrations, higher substrate affinity (lower K(m)), and lower maximum growth rates than strains in Nitrosomonas cluster 7, which includes Nitrosomonas europaea and Nitrosomonas eutropha. Genome-informed studies of this ammonia-sensitive cohort of AOB are needed, as these bacteria are found in freshwater environments, drinking water supplies, wastewater treatment systems, and soils worldwide.

Ammonia-oxidizing bacteria in biofilters removing trihalomethanes are related to Nitrosomonas oligotropha.

Ammonia-oxidizing bacteria (AOB) in nitrifying biofilters degrading four regulated trihalomethanes-trichloromethane, bromodichloromethane, dibromochloromethane, and tribromomethane-were related to Nitrosomonas oligotropha. N. oligotropha is associated with chloraminated drinking water systems, and its presence in the biofilters might indicate that trihalomethane tolerance is another reason that this bacterium is dominant in chloraminated systems.

Low-dissolved-oxygen nitrifying systems exploit ammonia-oxidizing bacteria with unusually high yields.

In wastewater treatment plants, nitrifying systems are usually operated with elevated levels of aeration to avoid nitrification failures. This approach contributes significantly to operational costs and the carbon footprint of nitrifying wastewater treatment processes. In this study, we tested the effect of aeration rate on nitrification by correlating ammonia oxidation rates with the structure of the ammonia-oxidizing bacterial (AOB) community and AOB abundance in four parallel continuous-flow reactors operated for 43 days. Two of the reactors were supplied with a constant airflow rate of 0.1 liter/min, while in the other two units the airflow rate was fixed at 4 liters/min. Complete nitrification was achieved in all configurations, though the dissolved oxygen (DO) concentration was only 0.5 ± 0.3 mg/liter in the low-aeration units. The data suggest that efficient performance in the low-DO units resulted from elevated AOB levels in the reactors and/or putative development of a mixotrophic AOB community. Denaturing gel electrophoresis and cloning of AOB 16S rRNA gene fragments followed by sequencing revealed that the AOB community in the low-DO systems was a subset of the community in the high-DO systems. However, in both configurations the dominant species belonged to the Nitrosomonas oligotropha lineage. Overall, the results demonstrated that complete nitrification can be achieved at low aeration in lab-scale reactors. If these findings could be extended to full-scale plants, it would be possible to minimize the operational costs and greenhouse gas emissions without risk of nitrification failure.

Structure and dynamics of nitrifier populations in a full-scale submerged membrane bioreactor during start-up.

Changes of microbial characteristics in a full-scale submerged membrane bioreactor system (capacity, 60,000 m(3) day(-1)) treating sewage were monitored over the start-up period (96 days). Fluorescence in situ hybridization analysis showed that the percentages of ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (nitrobacter-related population) in total bacteria counted with DAPI staining increased significantly from 1.9% and 0.9% to 4.5% and 2.8%, corresponding to an increase of the specific ammonium oxidizing rate (from 0.06 to 0.12 kg N kg(-1) mixed liquor suspended solids (MLSS) per day) and the specific nitrate forming rate (from 0.05 to 0.10 kg N kg(-1) MLSS day(-1)). Both the denaturing gradient gel electrophoresis of polymerase chain reaction and clone library results showed that the AOB was dominated by the genus Nitrosomonas, the diversity of which increased markedly with operational time. Most of the day 2 clones were closely related with the uncultured Nitrosomonas sp. clone Ninesprings-49S amoA gene (AY356450.1) originated from activated sludge, while the day 96 clone library showed a more diverse distribution characterized by the appearance of the oligotrophic nitrifiers like the Nitrosomonas oligotropha- and Nitrosomonas ureae-like bacteria, perhaps due to the interception by membrane and the low food-to-microorganisms ratio environment. The above results show that the membrane bioreactor system was characterized by the increased diversity and percentage of nitrifiers, which made it possible to achieve a stable and high efficient nitrification. Ammonia-oxidizing archaea with the changing population structures were also detected, but their roles for ammonia oxidation in the system need further studies.

The community analysis of ammonia-oxidizing bacteria in wastewater treatment plants revealed by the combination of double labeled T-RFLP and sequencing.

The functional gene of amoA, which produces the α-subunit of ammonia monooxygenase (AMO), has been analyzed to reveal the microbial community structure of ammonia-oxidizing bacteria (AOB) by culture-independent methods. In this study, the distribution of the amoA gene in 10 wastewater treatment plants (WWTPs) was revealed by the fingerprinting method of terminal restriction fragment length polymorphism (T-RFLP) and comparative sequencing. T-RFLP showed diverse communities of AOB in the modified Ludzack-Ettinger process, in the anaerobic-anoxic-oxic processes, in the hanging biological contactor, and in the sequencing batch reactor. In all of these environments, long solid retention time (SRT) was expected to be the critical factor for maintaining the diverse AOB community structure. Because T-RFLP does not offer sufficient information to confirm the phylogenetic information of AOB, the microbial community structures were analyzed by comparative sequencing for seven samples that were selected by the statistical categorization using principal component analysis (PCA) among 14 samples. The phylogenetic tree of 21 operational taxonomic units (OTUs) among 88 clones obtained in this study revealed that AOB of Nitrosomonas oligotropha and europaea lineages were predominant in WWTPs. Double labeled T-RFLP produced group-specific terminal restriction fragments (T-RFs) representing several groups of AOB and offered advanced resolution comparing with the single labeled T-RFLP.

Enrichment of marine anammox bacteria from seawater-related samples and bacterial community study.

Anaerobic ammonium oxidation (anammox) is a novel nitrogen pathway catalyzed by anammox bacteria which are obligate anaerobic chemoautotrophs. In this study, enrichment culture of marine anammox bacteria (MAAOB) from the samples related to seawater was conducted. Simultaneous removal of ammonium and nitrite was confirmed in continuous culture inoculated with sediment of a sea-based waste disposal site within 50 days. However, no simultaneous nitrogen removal was observed in cultures inoculated with seawater-acclimated denitrifying sludge or with muddy sediment of tideland even during 200 days. Nitrogen removal rate of 0.13 kg/m(3)/day was achieved at nitrogen loading rate of 0.16 kg/m(3)/day after 320th days in the culture inoculated with the sediment of waste disposal site. The nitrogen removal ratio between ammonium nitrogen and nitrite nitrogen was 1:1.07. Denaturing gradient gel electrophoresis (DGGE) analysis indicated that an abundance of the bacteria close to MAAOB and coexistence of ammonium oxidizing bacteria and denitrifying bacteria in the culture.

Community composition of ammonia-oxidizing bacteria and archaea in rice field soil as affected by nitrogen fertilization.

Little information is available on the ecology of ammonia-oxidizing bacteria (AOB) and archaea (AOA) in flooded rice soils. Consequently, a microcosm experiment was conducted to determine the effect of nitrogen fertilizer on the composition of AOB and AOA communities in rice soil by using molecular analyses of ammonia monooxygenase gene (amoA) fragments. Experimental treatments included three levels of N (urea) fertilizer, i.e. 50, 100 and 150 mgNkg(-1) soil. Soil samples were operationally divided into four fractions: surface soil, bulk soil deep layer, rhizosphere and washed root material. NH(4)(+)-N was the dominant form of N in soil porewater and increased with N fertilization. Cloning and sequencing of amoA gene fragments showed that the AOB community in the rice soil consisted of three major groups, i.e. Nitrosomonas communis cluster, Nitrosospira cluster 3a and cluster 3b. The sequences related to Nitrosomonas were predominant. There was a clear effect of N fertilizer and soil depth on AOB community composition based on terminal restriction fragment length polymorphism fingerprinting. Nitrosomonas appeared to be more abundant in the potentially oxic or micro-oxic fractions, including surface soil, rhizosphere and washed root material, than the deep layer of anoxic bulk soil. Furthermore, Nitrosomonas increased relatively in the partially oxic fractions and that of Nitrosospira decreased with the increasing application of N fertilizer. However, AOA community composition remained unchanged according to the denaturing gradient gel electrophoresis analyses.

Greenhouse gas microbiology in wet and dry straw crust covering pig slurry.

Liquid manure (slurry) storages are sources of gases such as ammonia (NH(3)) and methane (CH(4)). Danish slurry storages are required to be covered to reduce NH(3) emissions and often a floating crust of straw is applied. This study investigated whether physical properties of the crust or crust microbiology had an effect on the emission of the potent greenhouse gases CH(4) and nitrous oxide (N(2)O) when crust moisture was manipulated ("dry", "moderate", and "wet"). The dry crust had the deepest oxygen penetration (45 mm as compared to 20 mm in the wet treatment) as measured with microsensors, the highest amounts of nitrogen oxides (NO(2)(-) and NO(3)(-)) (up to 36 mumol g(-1) wet weight) and the highest emissions of N(2)O and CH(4). Fluorescent in situ hybridization and gene-specific polymerase chain reaction (PCR) were used to detect occurrence of bacterial groups. Ammonia-oxidizing bacteria (AOB) were abundant in all three crust types, whereas nitrite-oxidizing bacteria (NOB) were undetectable and methane-oxidizing bacteria (MOB) were only sparsely present in the wet treatment. A change to anoxia did not affect the CH(4) emission indicating the virtual absence of aerobic methane oxidation in the investigated 2-mo old crusts. However, an increase in N(2)O emission was observed in all crusted treatments exposed to anoxia, and this was probably a result of denitrification based on NO(x)(-) that had accumulated in the crust during oxic conditions. To reduce overall greenhouse gas emissions, floating crust should be managed to optimize conditions for methanotrophs.

Bacterial community structure in rotating biological contactor treating coke wastewater in relation to medium composition.

Biological wastewater treatment using biofilm systems is an effective way to treat difficult wastewater, such as coke wastewater. The information about the structure and the dynamics of this microbial community in biofilm, which are responsible for wastewater treatment, is relevant in the context of treatment efficacy and the biochemical potential to remove various pollutants. However, physico-chemical factors can influence the biofilm community significantly, causing performance disturbances. Therefore, we decided to examine the structure of microbial community in rotating biological contactor (RBC) biofilm during coke wastewater treatment and to investigate the possible shift in the community structure caused by the feeding medium change from synthetic to real coke wastewater. The experiment performed with high-throughput next-generation sequencing (NGS) revealed that bacteria commonly present in wastewater treatment plant (WWTP) systems, responsible for nitrite oxidizing, such as Nitrospira or Nitrobacter, were absent or below detection threshold, while Nitrosomonas, responsible for ammonia oxidizing, was detected in a relatively small number especially after shift to real coke wastewater. This research indicates that medium change could cause the change from autotrophic into heterotrophic nitrification led by Acinetobacter. Moreover, biofilm systems can be also a potential source of bacteria possessing high biochemical potential for pollutants removal but less known in WWTP systems, as well as potentially pathogenic microorganisms.

Effects of the Wastewater Flow Rate on Interactions between the Genus Nitrosomonas and Diverse Populations in an Activated Sludge Microbiome.

The present study characterized the interactions of microbial populations in activated sludge systems during the operational period after an increase in the wastewater flow rate and consequential ammonia accumulation using a 16S rRNA gene sequencing-based network analysis. Two hundred microbial populations accounting for 81.8% of the total microbiome were identified. Based on a co-occurrence analysis, Nitrosomonas-type ammonia oxidizers had one of the largest number of interactions with diverse bacteria, including a bulking-associated Thiothrix organism. These results suggest that an increased flow rate has an impact on constituents by changing ammonia concentrations and also that Nitrosomonas- and Thiothrix-centric responses are critical for ammonia removal and microbial community recovery.

Primer and probe sets for group-specific quantification of the genera Nitrosomonas and Nitrosospira using real-time PCR.

Use of quantitative real-time PCR (QPCR) with TaqMan probes is increasingly popular in various environmental works to detect and quantify a specific microorganism or a group of target microorganism. Although many aspects of conducting a QPCR assay have become very easy to perform, a proper design of oligonucleotide sequences comprising primers and a probe is still considered as one of the most important aspects of a QPCR application. This work was conducted to design group specific primer and probe sets for the detection of ammonia oxidizing bacteria (AOB) using a real-time PCR with a TaqMan system. The genera Nitrosomonas and Nitrosospira were grouped into five clusters based on similarity of their 16S rRNA gene sequences. Five group-specific AOB primer and probe sets were designed. These sets separately detect four subgroups of Nitrosomonas (Nitrosomonas europaea-, Nitrosococcus mobilis-, Nitrosomonas nitrosa-, and Nitrosomonas cryotolerans-clusters) along with the genus Nitrosospira. Target-group specificity of each primer and probe set was initially investigated by analyzing potential false results in silico, followed by a series of experimental tests for QPCR efficiency and detection limit. In general, each primer and probe set was very specific to the target group and sensitive to detect target DNA as low as two 16S rRNA gene copies per reaction mixture. QPCR efficiency, higher than 93.5%, could be achieved for all primer and probe sets. The primer and probe sets designed in this study can be used to detect and quantify the beta-proteobacterial AOB in biological nitrification processes and various environments.

Comparative analysis of ammonia monooxygenase (amoA) genes in the water column and sediment-water interface of two lakes and the Baltic Sea.

The functional gene amoA was used to compare the diversity of ammonia-oxidizing bacteria (AOB) in the water column and sediment-water interface of the two freshwater lakes Plusssee and Schöhsee and the Baltic Sea. Nested amplifications were used to increase the sensitivity of amoA detection, and to amplify a 789-bp fragment from which clone libraries were prepared. The larger part of the sequences was only distantly related to any of the cultured AOB and is considered to represent new clusters of AOB within the Nitrosomonas/Nitrosospira group. Almost all sequences from the water column of the Baltic Sea and from 1-m depth of Schöhsee were related to different Nitrosospira clusters 0 and 2, respectively. The majority of sequences from Plusssee and Schöhsee were associated with sequences from Chesapeake Bay, from a previous study of Plusssee and from rice roots in Nitrosospira-like cluster A, which lacks sequences from Baltic Sea. Two groups of sequences from Baltic Sea sediment were related to clonal sequences from other brackish/marine habitats in the purely environmental Nitrosospira-like cluster B and the Nitrosomonas-like cluster. This confirms previous results from 16S rRNA gene libraries that indicated the existence of hitherto uncultivated AOB in lake and Baltic Sea samples, and showed a differential distribution of AOB along the water column and sediment of these environments.