Anaerobic Ammonium-Oxidizing (Anammox) Bacteria and Associated Activity in Fixed-Film Biofilters of a Marine Recirculating Aquaculture System

Microbial communities in the biological filter and waste sludge compartments of a marine recirculating aquaculture system were examined to determine the presence and activity of anaerobic ammonium-oxidizing (anammox) bacteria. Community DNA was extracted from aerobic and anaerobic fixed-film biofilters and the anaerobic sludge waste collection tank and was analyzed by amplifying 16S rRNA genes by PCR using anammox-selective and universal GC-clamped primers. Separation of amplified PCR products by denaturing gradient gel electrophoresis and sequencing of the different phylotypes revealed a diverse biofilter microbial community. While Planctomycetales were found in all three communities, the anaerobic denitrifying biofilters contained one clone that exhibited high levels of sequence similarity to known anammox bacteria. Fluorescence in situ hybridization studies using an anammox-specific probe confirmed the presence of anammox Planctomycetales in the microbial biofilm from the denitrifying biofilters, and anammox activity was observed in these biofilters, as detected by the ability to simultaneously consume ammonia and nitrite. To our knowledge, this is the first identification of anammox-related sequences in a marine recirculating aquaculture filtration system, and our findings provide a foundation for incorporating this important pathway for complete nitrogen removal in such systems.     

Anammox bacteria in different compartments of recirculating aquaculture systems

Strict environmental restrictions force the aquaculture industry to guarantee optimal water quality for fish production in a sustainable manner. The implementation of anammox (anaerobic ammonium oxidation) in biofilters would result in the conversion of both ammonium and nitrite (both toxic to aquatic animals) into harmless dinitrogen gas. Both marine and freshwater aquaculture systems contain populations of anammox bacteria. These bacteria are also present in the faeces of freshwater and marine fish. Interestingly, a new planctomycete species appears to be present in these recirculation systems too. Further exploitation of anammox bacteria in different compartments of aquaculture systems can lead to a more environmentally friendly aquaculture practice.     

Potential roles of anaerobic ammonium and methane oxidation in the nitrogen cycle of wetland ecosystems

Anaerobic ammonium oxidation (anammox) and anaerobic methane oxidation (ANME coupled to denitrification) with nitrite as electron acceptor are two of the most recent discoveries in the microbial nitrogen cycle. Currently the anammox process has been relatively well investigated in a number of natural and man-made ecosystems, while ANME coupled to denitrification has only been observed in a limited number of freshwater ecosystems. The ubiquitous presence of anammox bacteria in marine ecosystems has changed our knowledge of the global nitrogen cycle. Up to 50% of N₂ production in marine sediments and oxygen-depleted zones may be attributed to anammox bacteria. However, there are only few indications of anammox in natural and constructed freshwater wetlands. In this paper, the potential role of anammox and denitrifying methanotrophic bacteria in natural and artificial wetlands is discussed in relation to global warming. The focus of the review is to explore and analyze if suitable environmental conditions exist for anammox and denitrifying methanotrophic bacteria in nitrogen-rich freshwater wetlands.     

Microbiology and application of the anaerobic ammonium oxidation ('anammox') process

Ten years ago, an anaerobic ammonium oxidation ('anammox') process was discovered in a denitrifying pilot plant reactor. From this system, a highly enriched microbial community was obtained, dominated by a single deep-branching planctomycete, Candidatus Brocadia anammoxidans. Phylogenetic inventories of different wastewater treatment plants with anammox activity have suggested that at least two genera in Planctomycetales can catalyse the anammox process. Electron microscopy of the ultrastructure of B. anammoxidans has shown that several membrane-bounded compartments are present inside the cytoplasm. Hydroxylamine oxidoreductase, a key anammox enzyme, is found exclusively inside one of these compartments, tentatively named the 'anammoxosome'.     

厌氧氨氧化菌的物种多样性与生态分布

厌氧氨氧化是微生物和环境领域的重大发现,厌氧氨氧化可同时去除氨氮和亚硝氮,在环境工程上具有重大开发价值.由于厌氧氨氧化菌生长极慢,倍增时间长达11 d以上,严重制约了该反应的开发进程,因此,对厌氧氨氧化菌的研究具有重要意义.厌氧氨氧化菌种类丰富,除了人们最早认识的浮霉状菌外,还有硝化细菌和反硝化细菌,这些菌群生态分布广泛,为开辟新的厌氧氨氧化菌种资源创造了条件.硝化细菌和反硝化细菌兼有厌氧氨氧化能力,其代谢多样性为加速厌氧氨氧化反应器的启动提供了依据.厌氧消化污泥可呈现硫酸盐型厌氧氨氧化活性,可为新型生物脱氮工艺的研发奠定基础.探明厌氧氨氧化菌种资源及其生态分布,将有利于厌氧氨氧化的开发应用.     

Presence and activity of anammox and denitrification process in low ammonium-fed bioreactors

A combination of anammox and denitrification process was studied for 300 days in low ammonium-fed bioreactors under the support of organic carbon. Nutrient profiles, (15)N-labelling techniques and qualitative fluorescence in situ hybridization (FISH) probes were used to confirm the nitrogen removal pathways and intercompetition among different bacteria populations. About 80% of nitrogen removal was achieved throughout the study period. The results confirmed that anammox bacteria were absent in the bioreactor inoculated with anaerobic granules only but they were present and active in the central anoxic parts of biopellets in the bioreactor inoculated with mixed microbial consortium from activated sludge and anaerobic granules. It also showed that the anammox bacteria were successfully enriched in the low ammonium-fed bioreactors. Results of this study clearly demonstrated that anammox and denitrification processes could coexist in same environment and anammox bacteria were less competitive than denitrifying bacteria.     

Spatial Variation in the Bacterial and Denitrifying Bacterial Community in a Biofilter Treating Subsurface Agricultural Drainage

Denitrifying biofilters can remove agricultural nitrates from subsurface drainage, reducing nitrate pollution that contributes to coastal hypoxic zones. The performance and reliability of natural and engineered systems dependent upon microbially mediated processes, such as the denitrifying biofilters, can be affected by the spatial structure of their microbial communities. Furthermore, our understanding of the relationship between microbial community composition and function is influenced by the spatial distribution of samples. In this study we characterized the spatial structure of bacterial communities in a denitrifying biofilter in central Illinois. Bacterial communities were assessed using automated ribosomal intergenic spacer analysis for bacteria and terminal restriction fragment length polymorphism of nosZ for denitrifying bacteria. Non-metric multidimensional scaling and analysis of similarity (ANOSIM) analyses indicated that bacteria showed statistically significant spatial structure by depth and transect, while denitrifying bacteria did not exhibit significant spatial structure. For determination of spatial patterns, we developed a package of automated functions for the R statistical environment that allows directional analysis of microbial community composition data using either ANOSIM or Mantel statistics. Applying this package to the biofilter data, the flow path correlation range for the bacterial community was 6.4 m at the shallower, periodically inundated depth and 10.7 m at the deeper, continually submerged depth. These spatial structures suggest a strong influence of hydrology on the microbial community composition in these denitrifying biofilters. Understanding such spatial structure can also guide optimal sample collection strategies for microbial community analyses.     

Bacterial community responses to increasing ammonia concentrations in model recirculating vertical flow saline biofilters

This study represents the first analysis of ammonia removal and bacterial communities in gravel biofilters treating saline wastewater and is of relevance to the growing inland marine aquaculture industry. As part of a study to gain greater understanding of the microbial processes occurring in a newly constructed limestone gravel wetland at a commercial marine fish farm, this study was designed to establish the ammonia removal capacity of model biofilters treating saline aquaculture wastewater and to investigate changes to total bacterial communities and ammonia-oxidizing bacterial communities as the biofilters are exposed to increasing ammonia concentrations. Three replicate laboratory-scale gravel biofilters were constructed and the limits of nitrification capacity were tested by dosing with aquaculture wastewater supplemented with increasing amounts of ammonium chloride. The experiment was run over a 12-week period with the water temperature between 24.5 and 28 °C and salinity between 28 and 38 ppt. Greater than 97% ammonia removal in each weekly treatment period was observed with ammonia concentrations of up to 600 ppm. At higher concentrations of ammonia, a lower percentage of ammonia was removed, and on occasion nitrite accumulation was observed. A drop in the number of operational taxonomic units (OTUs) detected in the bacterial community as measured by 16s rRNA T-RFLP was observed concurrent with the decrease in percentage ammonia removal. T-RFLP of the amoA gene showed the experimental biofilters to be dominated by three different OTUs of ammonia-oxidizing bacteria. A synchronous successional pattern among these three ammonia oxidizers was observed. The three OTUs were identified as belonging to three different nitrosomonad clusters. This study demonstrates that the vertical flow gravel biofilters have the ability to treat saline aquaculture wastewater that has a high ammonia concentration and that the microbial community within saline biofilters has the capacity to adapt to changing ammonia levels while maintaining nitrification activity.     

Evaluation of oxygen adaptation and identification of functional bacteria composition for anammox consortium in non-woven biological rotating contactor

In this study, the anammox consortium was found to adapt to the wastewater containing dissolved oxygen (DO), as the DO was gradually increased. Batch tests indicated the maximum aerobic ammonium oxidizing activity of the consortium was 1.38mmolNH(4)(+)-N(gVSS)(-1)day(-1), which played key roles in the oxygen consumption process; the maximum anaerobic ammonium oxidizing activity was slightly decreased after long-term oxygen exposure, but only from 21.23mmolNH(4)(+)-N(gVSS)(-1)day(-1) to 20.23mmolNH(4)(+)-N(gVSS)(-1)day(-1). Microbiological community analysis identified two strains similar to Nitrosomonas eutropha were responsible for oxygen consumption, which were able to exist in the autotrophic anaerobic condition for long periods and protect anammox bacteria Planctomycetales from the influence of oxygen. Microbiological composition analysis showed Nitrosomonas and Planctomycetales approximately accounted for 10% and 70% of the bacteria, respectively. The possibility of cultivation anammox consortium in presence of DO will lead to substantial savings of energy and resource in the industrial application.     

Anaerobic ammonium-oxidizing bacteria in marine environments: widespread occurrence but low diversity

Laboratory and field studies have indicated that anaerobic ammonium oxidation (anammox) is an important process in the marine nitrogen cycle. In this study 11 additional anoxic marine sediment and water column samples were studied to substantiate this claim. In a combined approach using the molecular methods, polymerase chain reaction (PCR), qualitative and quantitative fluorescence in situ hybridization (FISH), as well as (15)N stable isotope activity measurements, it was shown that anammox bacteria were present and active in all samples investigated. The anammox activity measured in the sediment samples ranged from 0.08 fmol cell(-1) day(-1) N(2) in the Golfo Dulce (Pacific Ocean, Costa Rica) sediment to 0.98 fmol cell(-1) day(-1) N(2) in the Gullmarsfjorden (North Sea, Sweden) sediment. The percentage of anammox cell of the total population (stained with DAPI) as assessed by quantitative FISH was highest in the Barents Sea (9% +/- 4%) and in most of the samples well over 2%. Fluorescence in situ hybridization and phylogenetic analysis of the PCR products derived from the marine samples indicated the exclusive presence of members of the Candidatus'Scalindua' genus. This study showed the ubiquitous presence of anammox bacteria in anoxic marine ecosystems, supporting previous observations on the importance of anammox for N cycling in marine environments.     

Diversity of ammonium-oxidizing bacteria in a granular sludge anaerobic ammonium-oxidizing (anammox) reactor

The ammonium-oxidizing microbial community was investigated in a granular sludge anaerobic ammonium-oxidizing (anammox) reactor that was operated for about 1 year with high anaerobic ammonium oxidation activity (up to 0.8 kg NH(4)(+)-N m(-3) day(-1)). A Planctomycetales-specific 16S rRNA gene library was constructed to analyse the diversity of the anaerobic ammonium-oxidizing bacteria (AnAOB). Most of the specifically amplified sequences (15/16) were similar to each other (> 99%) but were distantly related to all of the previously recognized sequences (< 94%), with the exception of an unclassified anammox-related clone, KSU-1 (98%). An ammonia monooxygenase (amoA) gene library was also analysed to investigate the diversity of 'aerobic' ammonium-oxidizing bacteria (AAOB) from the beta-Proteobacteria. Most of the amoA gene fragments (53/55) clustered in the Nitrosomonas europaea-Nitrosococcus mobilis group which has been reported to prevail under oxygen-limiting conditions. The quantitative results from real-time polymerase chain reaction (PCR) amplification showed that the dominant AnAOB comprised approximately 50% of the total bacterial 16S rRNA genes in the reactor, whereas the AAOB of beta-Proteobacteria represented only about 3%. A large fragment (4008 bp) of the rRNA gene cluster of the dominant AnAOB (AS-1) in this reactor sludge was sequenced and compared with sequences of other Planctomycetales including four anammox-related candidate genera. The partial sequence of hydrazine-oxidizing enzyme (hzo) of dominant AnAOB was also identified using new designed primers. Based on this analysis, we propose to tentatively name this new AnAOB Candidatus'Jettenia asiatica'.     

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.     

Candidatus "Scalindua brodae", sp. nov., Candidatus "Scalindua wagneri", sp. nov., two new species of anaerobic ammonium oxidizing bacteria

Anaerobic ammonium oxidation (anammox) is both a promising process in wastewater treatment and a long overlooked microbial physiology that can contribute significantly to biological nitrogen cycling in the world's oceans. Anammox is mediated by a monophyletic group of bacteria that branches deeply in the Planctomycetales. Here we describe a new genus and species of anaerobic ammonium oxidizing planctomycetes, discovered in a wastewater treatment plant (wwtp) treating landfill leachate in Pitsea, UK. The biomass from this wwtp showed high anammox activity (5.0 +/- 0.5 nmol/mg protein/min) and produced hydrazine from hydroxylamine, one of the unique features of anammox bacteria. Eight new planctomycete 16S rRNA gene sequences were present in the 16S rRNA gene clone library generated from the biomass. Four of these were affiliated to known anammox 16S rRNA gene sequences, but branched much closer to the root of the planctomycete line of descent. Fluorescence in situ hybridization (FISH) with oligonucleotide probes specific for these new sequences showed that two species (belonging to the same genus) together made up > 99% of the planctomycete population which constituted 20% of the total microbial community. The identification of these organisms as typical anammox bacteria was confirmed with electron microscopy and lipid analysis. The new species, provisionally named Candidatus "Scalindua brodae" and "Scalindua wagneri" considerably extend the biodiversity of the anammox lineage on the 16S rRNA gene level, but otherwise resemble known anammox bacteria. Simultaneously, another new species of the same genus, Candidatus "Scalindua sorokinii", was detected in the water column of the Black Sea, making this genus the most widespread of all anammox bacteria described so far.     

Aerobic and Anaerobic Ammonia-Oxidizing Microorganisms in Low-Temperature Hydrothermal Fe-Si-rich Precipitates of the Southwestern Pacific Ocean

Fe-Si-rich hydrothermal precipitates are distributed widely in low-temperature diffusing hydrothermal fields. Due to the significant contribution of Fe-oxidizing bacteria (FeOB) to the formation of this type of hydrothermal precipitates, previous studies focus mostly on investigating FeOB-related microbial populations, albeit these precipitates actually accommodate abundant other microbial communities, particularly those involved in marine nitrogen cycle. In this study, we investigated the composition, diversity, and abundance of aerobic and anaerobic ammonia-oxidizing microorganisms dwelling in low-temperature Fe-Si-rich hydrothermal precipitates of the Lau Integrated Study Site based on ammonia monooxygenase (amoA) gene and 16S rRNA gene. Phylogenetic analysis revealed the common presence of ammonia-oxidizing archaea (AOA), Nitrosospira-like ammonia-oxidizing bacteria (AOB) and anaerobic ammonium-oxidizing anammox (bacteria) in the Fe-Si-rich hydrothermal precipitates. Quantitative PCR analysis showed that AOA dominated the whole microbial community and the abundance of archaeal amoA gene was 2–3 orders of magnitude higher than that of AOB and anammox bacteria. Result of glycerol dialkyl glycerol tetraether analysis confirmed the presence and abundance of AOA. Our results suggest that microbial ammonia oxidations, especially archaeal aerobic ammonia oxidation, are prevalent and pivotal processes in low-temperature diffusing hydrothermal fields.

Supplemental materials are available for this article. Go to the publisher's online edition of Geomicrobiology Journal to view the supplemental file.     

The microbial nitrogen cycle

This special issue highlights several recent discoveries in the microbial nitrogen cycle including the diversity of nitrogen-fixing bacteria in special habitats, distribution and contribution of aerobic ammonium oxidation by bacteria and crenarchaea in various aquatic and terrestrial ecosystems, regulation of metabolism in nitrifying bacteria, the molecular diversity of denitrifying microorganisms and their enzymes, the functional diversity of freshwater and marine anammox bacteria, the physiology of nitrite-dependent anaerobic methane oxidation and the degradation of recalcitrant organic nitrogen compounds. Simultaneously the articles in this issue show that many questions still need to be addressed, and that the microbes involved in catalyzing the nitrogen conversions still harbour many secrets that need to be disclosed to fully understand the biogeochemical nitrogen cycle, and make future predictions and global modelling possible.     

'FloraArray' for screening of specific DNA probes representing the characteristics of a certain microbial community

To investigate uncharacterized microbial communities, a custom DNA microarray named 'FloraArray' was developed for screening specific probes that would represent the characteristics of a microbial community. The array was prepared by spotting 2000 plasmid DNAs from a genomic shotgun library of a sludge sample on a DNA microarray. By comparative hybridization of the array with two different samples of genomic DNA, one from the activated sludge and the other from a nonactivated sludge sample of an anaerobic ammonium oxidation (anammox) bacterial community, specific spots were visualized as a definite fluctuating profile in an MA (differential intensity ratio vs. spot intensity) plot. About 300 spots of the array accounted for the candidate probes to represent anammox reaction of the activated sludge. After sequence analysis of the probes and examination of the results of blastn searches against the reported anammox reference sequence, complete matches were found for 161 probes (58.3%) and >90% matches were found for 242 probes (87.1%). These results demonstrate that 'FloraArray' could be a useful tool for screening specific DNA molecules of unknown microbial communities.     

Adaptation of a freshwater anammox population to high salinity wastewater

For the successful application of anaerobic ammonium oxidation (anammox) in wastewater practice it is important to know how to seed new anammox reactors with biomass from existing reactors. In this study, a new high salinity anammox reactor was inoculated with biomass from a freshwater system. The changes in activity and population shifts were monitored. It was shown that freshwater anammox bacteria could adapt to salt concentrations as high as 30 gl(-1) provided the salt concentration was gradually increased. Higher salt concentrations reversibly inhibited anammox bacteria. The nitrogen removal efficiency and maximum anammox activity of the salt adapted sludge was very similar to the reference freshwater sludge. Fluorescence in situ hybridization analysis revealed that the freshwater anammox species Candidatus "Kuenenia stuttgartiensis" was the dominant in both salt adapted sludge and freshwater sludge. These results show that gradual adaptation may be the key to successful seeding of anammox bioreactors.     

Distribution and activity of microorganisms in the deep repository for liquid radioactive waste at the Siberian Chemical Combine

The physicochemical conditions, composition of microbial communities, and the rates of anaerobic processes in the deep sandy horizons used as a repository for liquid radioactive wastes (LRW) at the Siberian Chemical Combine (Seversk, Tomsk oblast), were studied. Formation waters from the observation wells drilled into the production horizons of the radioactive waste disposal site were found to be inhabited by microorganisms of different physiological groups, including aerobic organotrophs, anaerobic fermentative, denitrifying, sulfate-reducing, and methanogenic bacteria. The density of microbial population, as determined by cultural methods, was low and usually did not exceed 10(4) cells/ml. Enrichment cultures of microorganisms producing gases (hydrogen, methane, carbon dioxide, and hydrogen sulfide) and capable of participation in the precipitation of metal sulfides were obtained from the waters of production horizons. The contemporary processes of sulfate reduction and methanogenesis were assayed; the rates of these terminal processes of organic matter destruction were found to be low. The denitrifying bacteria from the underground repository were capable of reducing the nitrates contained in the wastes, provided sources of energy and biogenic elements were available. Biosorption of radionuclides by the biomass of aerobic bacteria isolated from groundwater was demonstrated. The results obtained give us insight into the functional structure of the microbial community inhabiting the waters of repository production horizons. This study indicates that the numbers and activity of microbial cells are low both inside and outside the zone of radioactive waste dispersion, in spite of the long period of waste discharge.     

Anaerobic ammonium oxidation (Anammox) in immobilized activated sludge biofilms during the treatment of weak wastewater

This work studied the formation of molecular nitrogen by the microbial population of immobilized activated sludge of the domestic wastewater treatment plants (WWTP) that employ the technology developed by ZAO ECOS Company. The technology includes physicochemical water pretreatment and treated water recycling. A hard flexible fibrous brush carrier is used for the immobilization of microorganisms. The presence of both aerobic and anaerobic microorganisms and functioning of the methanogenic microbial community was shown in the biofilms developing on the carrier fibers and in suspended sludge. The high efficiency of nitrogen removal at a low C/N ratio was established to be due to the conjugated nitrification, denitrification, and anammox processes, whose functioning was demonstrated by laboratory cultivation methods and by studying the processes in batch and continuous reactors. Fluorescence in situ hybridization with 16S rRNA-targeted oligonucleotide probes (FISH) revealed bacteria belonging to the order Planctomycetales, particularly their anammox group. This work is the first evidence of the important role of the anammox process in the combined system of physicochemical and biological treatment of weak wastewater (BCDEAMOX).