Brocadia Anammoxidans Research Articles

Microbial community and population dynamics of single-stage autotrophic nitrogen removal for dilute wastewater at the benchmark oxygen rate supply

Microbial communities and their kinetic performance in a single-stage autotrophic nitrogen-removal filter at an optimal oxygen supply were examined to determine the presence and activity of denitrifiers, anaerobic ammonia-oxidizing (anammox), ammonia-oxidizing, and nitrite-oxidizing bacteria. To this end, different molecular biology techniques such as real-time quantitative polymerase chain reaction (qPCR) and biomarkers such as 16S rRNA revealed a diverse microbial community along the filter. It was important to survey the specific species of anammox bacteria using a newly designed Candidatus Brocadiafulgida (BF) specific primer, as well as Candidatus Brocadia anammoxidans (BA) and Candidatus Kuenenia stuttgartiensis (KS) specific primers. An unexpected finding was that the predominant anammox species switched from KS in concentrated wastewater to BA in dilute wastewaters. The Eckenfelder model of the NH3-N transformation along the filter was Se=S0 exp(-0.192D/L(2.3217)). These results provide a foundational understanding of the microbial structure and reaction kinetics in such systems.

A pilot-scale study on nitrogen removal from dry-spun acrylic fiber wastewater using anammox process

Dry-spun acrylic fiber wastewater (DAFW) is one kind of toxic refractory petrochemical wastewater. The results of GC–MS qualitative analysis demonstrated that the residual organic compounds in DAFW after the biotreatment are non-biodegradable and hardly available for traditional nitrification–denitrification processes. First, the batch test indicated that there was no obvious inhibition or toxicity of DAFW to anaerobic ammonium oxidizing (anammox) bacteria. A pilot test was then carried out to evaluate the feasibility and efficiency of biological nitrogen removal by the anammox process. A rapid start-up was achieved by inoculating enriched anammox sludge to a biofilm system with low inoculated sludge concentration. The pretreatment for anammox process was necessary because DAFW contains high SS which affected the nitrogen removal efficiency. By controlling the reactor temperature and SS content in influent, the removal efficiencies of ammonium and nitrite were 85% and 90%, respectively. The stoichiometric ratios of ammonium conversion, nitrite removal and nitrate production were 1:(1.29):(0.24) for synthetic wastewater and 1:(1.26):(0.22) for DAFW. After 180days of operation, there was a diverse change of the anammox consortium bacteria. Compared with the inoculated sludge (single Brocadia anammoxidans sp.), Kuenenia sp. became the predominant bacteria, with a small amount of Jettenia sp. and B. anammoxidans sp. The results demonstrated that ammonia in effluent of DAFW after two-stage biotreatment could be removed efficiently by the anammox process.

Co-occurrence and distribution of nitrite-dependent anaerobic ammonium and methane-oxidizing bacteria in a paddy soil

The anaerobic ammonium-oxidizing (anammox) and nitrite-dependent anaerobic methane-oxidizing (n-damo) bacteria in a paddy soil core (0-100 cm) were investigated with newly designed primers targeting the hydrazine synthase β-subunit (hzsB) of anammox bacteria and the recently published primers targeting the pmoA and 16S rRNA genes of n-damo bacteria. The hzsB gene was identified as a proper biomarker to explore the anammox bacterial biodiversity and abundance in soil. The anammox bacteria were present throughout the soil core with the highest abundance of 2.7 × 10(6) hzsB copies g(-1) dry soil at 40-50 cm and were not detectable below 70 cm. Sequences related to at least three species of known anammox bacteria, 'Brocadia anammoxidans', 'Brocadia fulgida', and 'Jettenia asiatica' were detected. By combining the analysis of pmoA and 16S rRNA genes, the n-damo bacteria were observed to be present in 30-70 cm with abundance from 6.5 × 10(3) (60-70 cm) to 7.5 × 10(4) (30-40 cm) copies g(-1) dry soil. The pmoA sequences retrieved from different depths closely related to each other and formed a unique clade. Our results showed that anammox and n-damo bacteria co-occurred in the paddy soil. Both of them were abundant in deep layers (30-60 cm) and the community structures changed along depths in the soil core.

Effects of oxytetracycline on anammox activity

Batch experiments were conducted to investigate the effects of oxytetracycline on anaerobic ammonium oxidation (anammox) process. The short-and long-term effects on anammox activity were studied by measuring ammonium (NH4 +), nitrite (NO2 −), and nitrate (NO3 −) concentrations over time. Experiments were conducted at NH4 +and NO2 − concentrations of 60–90 mg N/L and 60–190 mg N/L, respectively (NH4 +:NO2 − ratio from 1:1–1:2.25), oxytetracycline concentrations of 10–100 mg/L, and biomass concentrations of 300–800 mg/L. In the short-term study, anammox activity was inhibited by all oxytetracycline concentrations studied. However, daily addition of oxytetracycline to a concentration of 5 ± 3.5 mg/L in the anammox sequencing batch reactor completely inhibited anammox activity in the fifth week. Fluorescent in situ hybridization was used to identify autotrophic ammonium oxidizing bacteria (Nitrosomonas spp., Nitrobacter spp., Nitrospira spp., Candidatus Brocadia anammoxidans, and Candidatus Kuenenia stuttgartiensis). The population of anammox culture was significantly decreased while Nitrosomonas spp. and Nitrospira spp. increased in the fifth week compared with the first week of experiment. A not-competitive model fit the anammox inhibition data at oxytetracycline concentrations of 0–100 mg/L quite well with Vmax of 0.0435 mg N/mg VSS-hr and Ki of 54.66 mg/L.

Inhibitory effects of free ammonia on Anammox bacteria

Anammox bacteria can effectively treat high ammonia and nitrite concentrations under anoxic environments. However, the presence of high ammonia and nitrite concentrations may cause free ammonia and nitrous acid inhibition at high pH and temperature environments. In this study, the inhibitory effect of free ammonia on Anammox bacteria was investigated in a lab-scale upflow fixed-bed reactor with Kaldnes biofilm carriers. Results of continuous operation showed that inhibition was not observed in the Anammox reactor when the free ammonia concentration gradually increased up to 150mg/L. However, Anammox activity suddenly dropped to 10 % when the free ammonia concentration reached to 190mg/L. Nevertheless, high influent ammonia and nitrite concentrations up to 1,500 and 500mg/L, respectively, did not noticeably inhibit the Anammox activity. Gradually decreasing Anammox activity was also supported by fluorescent in situ hybridization (FISH) analysis. FISH and 16S rRNA gene analysis results revealed that main Anammox organisms were phylogenetically related to Candidatus Kuenenia stuttgartiensis, Candidatus Jettenia asiatica and Candidatus Brocadia anammoxidans.

Is the CANON reactor an alternative for nitrogen removal from pre-treated swine slurry?

The use of the completely autotrophic nitrogen removal over nitrite (CANON) process as a post-treatment for nitrogen removal from anaerobically pre-treated swine slurry is studied in the present work. The ammonium removal, under oxygen-limited conditions, in a system with anammox bacteria mainly in the form of granules and aerobic ammonium oxidizing bacteria mainly as dispersed biomass was researched in an air pulsing sequencing batch reactor operated at room temperature. The achieved nitrogen removal rate was of 0.46kgN/(m3d) treating 300mg NH4+-N/L with values of nitrogen removal efficiencies around 75%.The presence of slowly or non-biodegradable organic matter (from 260 to 45mgCOD/L) did not affect the operation of the process. By means of the FISH technique, Nitrosomonas were detected as the majority of ammonia oxidizing bacteria in the sample, and Candidatus “Brocadia fulgida” and Candidatus “Brocadia anammoxidans” as the anammox bacteria.The comparison of this aerobic process with other post-treatments for effluents from anaerobic digesters showed that the CANON process is a promising alternative to remove nitrogen from effluents generated in pig farms.

白洋淀富营养化湖泊湿地厌氧氨氧化菌的分布及对氮循环的影响

PDF HTML阅读 XML下载 导出引用 引用提醒 白洋淀富营养化湖泊湿地厌氧氨氧化菌的分布及对氮循环的影响 DOI: 10.5846/stxb201109151354 作者: 作者单位: 中国科学院生态环境研究中心 环境水质学国家重点实验室,中国科学院生态环境研究中心 环境水质学国家重点实验室,中国科学院青藏高原所 环境变化与地表过程国家重点实验室;中国科学院青藏高原所 环境变化与地表过程国家重点实验室,中国科学院生态环境研究中心 环境水质学国家重点实验室 作者简介: 通讯作者: 中图分类号: 基金项目: 国家自然科学基金项目(20877086); 国家973项目(2009CB421103); 国家科技重大专项(2008ZX07421-001); 北京市科技新星计划项目(2011095) Widespread of anaerobic ammonia oxidation bacteria in an eutrophic freshwater lake wetland and its impact on nitrogen cycle Author: Affiliation: State Key Laboratory of Environmental Aquatic Quality,Research Center for Eco-Environmental Sciences,Chinese Academy of Sciences,State Key Laboratory of Environmental Aquatic Quality,Research Center for Eco-Environmental Sciences,Chinese Academy of Sciences,Key Laboratory of Tibetan Environment Changes and Land Surface Processes,Chinese Academy of Sciences,State Key Laboratory of Environmental Aquatic Quality,Research Center for Eco-Environmental Sciences,Chinese Academy of Sciences Fund Project: 摘要 | 图/表 | 访问统计 | 参考文献 | 相似文献 | 引证文献 | 资源附件 | 文章评论 摘要:随着生物反应器和海洋生态系统中厌氧氨氧化反应的发现,自然生态系统的氮循环过程被重新认识,但是目前厌氧氨氧化过程是否也存在于富营养化湖泊湿地并发挥着重要作用,还未见报道。结合15N同位素示踪与分子生物学技术研究了白洋淀湖泊湿地沉积物中厌氧氨氧化菌的分布、菌群结构特性、生物多样性及其活性。结果表明,在藻类影响导致的高氨氮沉积物中,厌氧氨氧化菌具有广泛存在性。通过构建16S rRNA克隆文库发现,沉积物中厌氧氨氧化菌的生物多样性相对较低,在2%差异度的条件下,30个克隆序列只分为5个操作分类单元 (OTUs),代表序列与Genebank数据库中已探明的厌氧氨氧化菌Candidatus Brocadia fulgida和 Candidatus Brocadia anammoxidans同源性最高,分别可达97%和96%。同位素示踪结果表明,白洋淀湖泊湿地沉积物中厌氧氨氧化活性为0.19-7.78 nmol N g-1 h-1,空间差异较大,产生的氮气占此沉积物总氮气生成量的0.64%-20.65%,体现了湿地的异质性。通过得出的厌氧氨氧化反应速率推算每年由厌氧氨氧化反应损失的氮量为1.8-78 gN m-2 a-1,对白洋淀氮循环起到非常重要的作用。富营养化湖泊湿地沉积物中厌氧氨氧化反应的发现为研究厌氧氨氧化对氮循环的重要影响提供了新证据。 Abstract:Anaerobic ammonia oxidation (anammox) is one of the most innovative discoveries in field of microbial nitrogen cycle with its direct oxidation of ammonia to nitrogen gas with no N2O production. At present the anammox researches are limited to two areas:biological wastewater treatment and marine ecosystems. Evidence for anammox was obtained from Baiyangdian, the largest freshwater lake in North China, using an isotope pairing technique and molecular analysis. This lake is characterized by hyper-eutrophic conditions with a great amount of accumulated ammonia nitrogen (95-336 mg/kg) in surface sediments (0-5cm). The anammox processes were detected on an extensive spatial scale (n=10) in the lake sediments by PCR and 16S rRNA gene clone libraries, using primers combination pla46f-630r and Amx368f-Amx820r. The 30 sequences sharing 98% similarity were grouped into only five operational taxonomic unit (OTU) using software DOTUR by the furthest neighbor approach. The biodiversity indicator "Shannon (1.4) and Chao 1 estimator(5)" were also calculated with DOTUR software. The result of biodiversity analysis showed that anammox bacteria biodiversity in the lake sediments was low. Phylogenetic trees were constructed by neighbor-joining (NJ) with the Jukes-Cantor correction in MEGA 4 package. The 16S rRNA gene sequences of anammox bacteria in the surface sediments were closely related to the known anammox bacteria Candidatus Brocadia fulgida (97%) and Candidatus Brocadia anammoxidans (96%), and it revealed most of anammox bacteria from Baiyangdian Lake may belong to "Brocadia". Isotope analyses were based on 15N labeled ammonium and nitrate. Three treatments (1) 15NH4+ (negative control) (2) 15NH4+ and 14NO3- (positive control) (3) 14NH4+ and 15NO3- (test group) was prepared and experiments were operated in hermetic anaerobic containers at 19℃. Outcome gaseous N2 was delivered to Gas chromatography-isotope ratio mass spectrometry for separation of 28N2, 29N2 and 30N2. Then the rates of anammox and denitrification activities were calculated. The anammox rates ranged between 0.19 to 7.78 nmol N g-1 h-1 with significant spatial heterogeneity, which contributed 0.64 to 20.65 precent to sediments N2 production with the remainder being due to denitrification. It was estimated that the total removed N (1.8-78 gN m-2 a-1) was linked to anammox in this eutrophic freshwater lake. About 56 to 9426 t/a of N was removed by way of anammox process with the water level changing from 5.5 to 9.5 m of Baiyangdian Lake. This research provided some new knowledge on anammox in freshwater ecosystems. 参考文献 相似文献 引证文献

Impact of reactor configuration on anammox process start-up: MBR versus SBR

Anaerobic ammonium oxidation (anammox) is an energy saving biological nitrogen removal process which was limited to slow growth rate of anammox bacteria during start-up period. This study investigated the start-up of anammox process by a laboratory sequential batch reactor (SBR) for 218days and subsequently modified the reactor as a membrane bioreactor (MBR) for 178days. Modification of a SBR as MBR with installation of an external membrane module resulted in acceleration of specific anammox activity by 19 times. The acceleration of specific anammox activity with MBR was further confirmed by starting-up another MBR for a 242day period. Molecular microbial analyses showed that Candidatus “Brocadia anammoxidans” and Candidatus “Kuenenia stuttgartiensis” were the dominant species in the inocula and biomass developed in the reactor. The start-up with MBR appeared to be more effective than SBR for the enrichment of anammox bacteria due to high sludge retention property of MBR configuration.

Physiological characteristics of the anaerobic ammonium-oxidizing bacterium ‘Candidatus Brocadia sinica’

The present study investigated the phylogenetic affiliation and physiological characteristics of bacteria responsible for anaerobic ammonium oxidization (anammox); these bacteria were enriched in an anammox reactor with a nitrogen removal rate of 26.0 kg N m(-3) day(-1). The anammox bacteria were identified as representing 'Candidatus Brocadia sinica' on the basis of phylogenetic analysis of rRNA operon sequences. Physiological characteristics examined were growth rate, kinetics of ammonium oxidation and nitrite reduction, temperature, pH and inhibition of anammox. The maximum specific growth rate (μ(max)) was 0.0041 h(-1), corresponding to a doubling time of 7 days. The half-saturation constants (K(s)) for ammonium and nitrite of 'Ca. B. sinica' were 28±4 and 86±4 µM, respectively, higher than those of 'Candidatus Brocadia anammoxidans' and 'Candidatus Kuenenia stuttgartiensis'. The temperature and pH ranges of anammox activity were 25-45 °C and pH 6.5-8.8, respectively. Anammox activity was inhibited in the presence of nitrite (50 % inhibition at 16 mM), ethanol (91 % at 1 mM) and methanol (86 % at 1 mM). Anammox activities were 80 and 70 % of baseline in the presence of 20 mM phosphorus and 3 % salinity, respectively. The yield of biomass and dissolved organic carbon production in the culture supernatant were 0.062 and 0.005 mol C (mol NH (+)(-4))(-1), respectively. This study compared physiological differences between three anammox bacterial enrichment cultures to provide a better understanding of anammox niche specificity in natural and man-made ecosystems.

Anammox bacteria community and nitrogen removal in a strip-like wetland in the riparian zone

A strip-like wetland was constructed in the riparian zone for investigation of ammonium nitrogen (NH3-N) removal in the Peach River. An inner zeolite layer was set in the wetland to adsorb NH3-N and further to remove total nitrogen (TN). An oxygen-deficient condition with dissolved oxygen of 0.87–1.60 mg L−1 was observed in the zeolite layer, which benefits anaerobic ammonium oxidation (anammox) bacteria survival. The community structure of anammox bacteria was analyzed in the zeolite layer. The analysis shows that the anammox bacterial sequences are grouped into three known distinct clusters: Candidatus Brocadia fulgida, Candidatus Brocadia anammoxidans and Candidatus Jettenia asiatica. The intensified test driven by artificial pumping shows that average removal rates of NH3-N and TN are 41.6 mg m−3d−1 and 63.2 mg m−3d−1, respectively. The normal test driven by natural hydrodynamics also verifies that NH3-N removal mainly happens in the zeolite layer. Microbial mechanism of TN removal in the wetland involves both the autotrophic and heterotrophic process. These results suggest that the strip-like RW can be a cost-effective approach for NH3-N removal and can potentially be extended to similar rivers as no extra energy is required to maintain the wetland operation.

Nitrogen removal of anammox cultures under different enrichment conditions

Anammox bacteria in sludge from an anoxic tank of a municipal wastewater treatment plant at Nongkhaem, Bangkok, Thailand were enriched in two sequencing batch reactors (SBRs; SBR-1 and SBR-2), under different conditions. SBR-1 was open to the atmosphere, while SBR-2 was closed and flushed with a mixture of 95% argon and 5% CO2 during the fill period in order to provide strict anaerobic conditions. The specific nitrogen removal rates of SBR-1 and SBR-2 were 0.43 g N/g VSS-d and 2.59 g N/g VSS-d, respectively. Denaturing gradient gel electrophoresis (DGGE) analyses showed differences in band patterns among the Nongkhaem sludge and the two enrichment cultures. Based on fluorescent in situ hybridization (FISH), the anammox bacteria in both systems were either “Candidatus Brocadia anammoxidans” or “Candidatus Kuenenia stuttgartiensis”. The results from this study demonstrate the potential of alternative anammox systems for nitrogen removal and provide information on the microbial communities of anammox cultures under different enrichment conditions.

Enriquecimento de bactérias anaeróbias oxidadoras de amônia - anammox

Bactérias anaeróbias oxidadoras de amônia (bactérias Anammox, do inglês anaerobic ammonium oxidizing bacteria) foram enriquecidas em reator em batelada sequencial (RBS), a partir de lodo proveniente de um sistema convencional de lodos ativados tratando esgoto doméstico de Belo Horizonte (MG). Após três meses de cultivo, atividade Anammox foi detectada no sistema pelo consumo de quantidades estequiométricas de NO2- e NH4+. Análises de hibridação in situ fluorescente (FISH, do inglês fluorescent in situ hybridization) confirmaram a presença de bactérias Anammox, provavelmente Candidatus Brocadia anammoxidans, e revelaram que estas representavam 53% do total de células (após 6 meses de cultivo). O desempenho do reator ao longo dos sete meses de operação demonstrou remoção quase que total de nitrito, baseada em concentração afluente de 61 a 95 mg N-NO2-/L. A eficiência máxima de remoção de amônia alcançada foi de 95%, a partir de concentração afluente de 55 a 82 mg N-NH4+/L.

Microbial nitrate-dependent cyclohexane degradation coupled with anaerobic ammonium oxidation

An anaerobic nitrate-reducing enrichment culture was established with a cyclic saturated petroleum hydrocarbon, cyclohexane, the fate of which in anoxic environments has been scarcely investigated. GC-MS showed cyclohexylsuccinate as a metabolite, in accordance with an anaerobic enzymatic activation of cyclohexane by carbon-carbon addition to fumarate. Furthermore, long-chain cyclohexyl-substituted cell fatty acids apparently derived from cyclohexane were detected. Nitrate reduction was not only associated with cyclohexane utilization but also with striking depletion of added ammonium ions. Significantly more ammonium was consumed than could be accounted for by assimilation. This indicated the occurrence of anaerobic ammonium oxidation (anammox) with nitrite from cyclohexane-dependent nitrate reduction. Indeed, nitrite depletion was stimulated upon further addition of ammonium. Analysis of 16S rRNA genes and subsequent cell hybridization with specific probes showed that approximately 75% of the bacterial cells affiliated with the Geobacteraceae and approximately 18% with Candidatus 'Brocadia anammoxidans' (member of the Planctomycetales), an anaerobic ammonium oxidizer. These results and additional quantitative growth experiments indicated that the member of the Geobacteraceae reduced nitrate with cyclohexane to nitrite and some ammonium; the latter two and ammonium added to the medium were scavenged by anammox bacteria to yield dinitrogen. A model was established to quantify the partition of each microorganism in the overall process. Such hydrocarbon oxidation by an alleged 'denitrification' ('pseudo-denitrification'), which in reality is a dissimilatory loop through anammox, can in principle also occur in other microbial systems with nitrate-dependent hydrocarbon attenuation.

Nitrogen removal performance and microbial community analysis of an anaerobic up-flow granular bed anammox reactor

We investigated nitrogen removal performance and responsible microbial community in an anaerobic up-flow granular bed anammox reactor. The anammox reactor was operated more than 1 year. Biomass in the reactor formed granules after about 2 months of operation, and a sufficient amount of the granules was retained in the reactor with a metallic net to avoid biomass washout during the entire operation. The average diameter of the granules was 3.6 mm at day 310. After 8 months of operation, stable nitrogen removal (60%) was achieved at an average total inorganic nitrogen removal rate of 14 kg-N m −3 d −1. The phylogenetic analysis and fluorescence in situ hybridization results revealed that the anammox granules consisted of mono species of anammox bacteria, “ Candidatus Brocadia–like species”, affiliated with “ Candidatus Brocadia anammoxidans” with 16S rRNA gene sequence similarity of 95.7%. The relative abundance of the anammox bacteria in the granules was more than 80% of the total bacteria stained with 4′,6-diamidino-2-phenylindole. The anammox bacteria were present throughout the granules whereas the other bacterial groups, Chloroflexi-like filamentous bacteria and betaproteobacterial ammonia-oxidizing bacteria, were mainly present on the surface of the anammox granules and around the anammox bacterial clusters. The in situ anammox activity was detected mainly from near the surface of granules to the upper 800 μm of the granules with microsensors. The granular anammox biomass tolerated higher concentrations of nitrite (400 mg-N L −1) than did the homogenized biomass (200 mg-N L −1) probably due to substrate diffusion limitation.

Development of batch-culture enrichment coupled to molecular detection for screening of natural and man-made environments in search of anammox bacteria for N-removal bioreactors systems

This investigation probed for anammox bacterial populations that would be suitable to start-up a biological reactor for N-removal. Samples of sludge and sediments from different environments were screened and used as inoculum for enrichment of anammox bacteria in batch cultures. Enrichments were monitored in order to detect anammox bacteria or their potential activity. Candidatus “Brocadia anammoxidans” was successfully enriched, detected, and identified in five of the twelve batch cultures. Furthermore, this organism was retrieved for the first time from a brackish environment. Wide-range primers used in several Polymerase Chain Reaction (PCR) attempts were unable to successfully amplify the 16S rRNA sequence from anammox populations, but were used to search for hypothetical ecological partners of anammox. A nested PCR approach with specific primers was also employed since conventional PCR was unable to amplify anammox DNA from the inocula of the successful enrichments. However, it was impossible to obtain optimal results with the different strategies utilized to improve PCR performance (higher annealing temperature or more specific primers), and only the primer set Amx368F-Amx820R resulted in an acceptable balance between both specificity and sensitivity. Although the enrichment process is relatively slow and requires lengthy incubation periods, it proved to be useful as the molecular analyses were not sensitive enough to detect anammox in the original samples or even after short enrichment periods. Therefore, a batch enrichment procedure coupled with molecular techniques was an appropriate approach to achieve successful inocula for starting-up anammox biological reactor.

16S rRNA gene and lipid biomarker evidence for anaerobic ammonium-oxidizing bacteria (anammox) in California and Nevada hot springs

Anammox, the oxidation of ammonium with nitrite to dinitrogen gas under anoxic conditions, is an important process in mesophilic environments such as wastewaters, oceans and freshwater systems, but little is known of this process at elevated temperatures. In this study, we investigated anammox in microbial mats and sediments obtained from several hot springs in California and Nevada, using geochemical and molecular microbiological methods. Anammox bacteria-specific ladderane core lipids with concentrations ranging between 0.3 and 52 ng g(-1) sediment were detected in five hot springs analyzed with temperatures up to 65 degrees C. In addition, 16S rRNA gene analysis showed the presence of genes phylogenetically related to the known anammox bacteria Candidatus Brocadia fulgida, Candidatus Brocadia anammoxidans and Candidatus Kuenenia stuttgartiensis (96.5-99.8% sequence identity) in three hot springs with temperatures up to 52 degrees C. Our data indicate that anammox bacteria may be able to thrive at thermophilic temperatures and thus may play a significant role in the nitrogen cycle of hot spring environments.

Anaerobic ammonium oxidation by Nitrosomonas spp. and anammox bacteria in a sequencing batch reactor

A sequencing batch reactor (SBR) was inoculated with mixed nitrifying bacteria from an anoxic tank at the conventional activated sludge wastewater treatment plant in Nongkhaem, Bangkok, Thailand. This enriched nitrifying culture was maintained under anaerobic conditions using ammonium (NH 4 +) as an electron donor and nitrite (NO 2 −) as an electron acceptor. Autotrophic ammonium oxidizing bacteria survived under these conditions. The enrichment period for anammox culture was over 100 days. Both ammonium and nitrite conversion rates were proportional to the biomass of ammonium oxidizing bacteria; rates were 0.08 g N/g VSS/d and 0.05 g N/g VSS/d for ammonium and nitrite, respectively, in a culture maintained for 3 months at 42 mg N/L ammonium. The nitrogen transformation rate at a ratio of NH 4 +–N to NO 2 −–N of 1:1.38 was faster, and effluent nitrogen levels were lower, than at ratios of 1:0.671, 1:2.18, and 1:3.05. Fluorescent in situ hybridization (FISH) was used to identify specific autotrophic ammonium oxidizing bacteria ( Nitrosomonas spp., Candidatus Brocadia anammoxidans, and Candidatus Kuenenia stuttgartiensis). The ammonium oxidizing culture maintained at 42 mg N/L ammonium was enriched for Nitrosomonas spp. (30%) over Candidati B. anammoxidans and K. stuttgartiensis (2.1%) while the culture maintained at 210 mg N/L ammonium was dominated by Candidati B. anammoxidans and K. stuttgartiensis (85.6%). The specific nitrogen removal rate of anammox bacteria (0.6 g N/g anammox VSS/d) was significantly higher than that of ammonium oxidizing bacteria (0.4 g N/g Nitrosomonas VSS/d). Anammox bacteria removed up to 979 mg N/L/d of total nitrogen (ammonium:nitrite concentrations, 397:582 mg N/L). These results suggest significant promise of this approach for application to wastewater with high nitrogen but low carbon content, such as that found in Bangkok.

Another multiheme protein, hydroxylamine oxidoreductase, abundantly produced in an anammox bacterium besides the hydrazine-oxidizing enzyme

A hydroxylamine oxidoreductase (HAO) was purified from anammox sludge in which an anammox bacterium, strain KSU-1, was dominant. The enzyme was a 118-kDa homodimer composed of a 53-kDa subunit. With phenazine methosulfate and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide as electron acceptors, the V(max) and K(m) for hydroxylamine were determined as 9.6+/-0.2 micromol/min x mg and 33+/-2 microM, while those for hydrazine were 0.54+/-0.0 micromol/min x mg and 25+/-2 microM, respectively. The HAO had a P468 chromophore. These enzymatic properties were different from those of the hydrazine-oxidizing enzyme (HZO), a multiheme protein abundantly produced by the KSU-1 strain, but were similar to those of the HAO purified from Candidatus Brocadia anammoxidans. The hao gene exists upstream of the hzoB gene, which codes for the HZO. The sequence deduced from the hao gene indicated eight c-type heme binding motifs and showed 87% identity with a polypeptide encoded by an open reading frame (kustc1061) in the genome of an anammox bacterium Candidatus Kuenenia stuttgartiensis. These suggested that the HAO is an indispensable enzyme and well conserved in anammox bacteria, similar to the HZO. This enzyme might therefore be a specific hydroxylamine oxidoreductase for anammox bacteria.

Start‐up and enrichment of a granular anammox SBR to treat high nitrogen load wastewaters

AbstractBACKGROUND: Landfill leachate is characterized by low biodegradable organic matter that presents difficulties for the complete biological nitrogen removal usually performed by conventional biological nitrification/denitrification processes. To achieve this, the anaerobic ammonium oxidation (anammox) process is a promising biological treatment. This paper presents an anammox start‐up and enrichment methodology for treating high nitrogen load wastewaters using sequencing batch reactor (SBR) technology.RESULTS: The methodology is based on the gradual increase of the nitrite‐to‐ammonium molar ratio in the influent (from 0.76 to 1.32 mole NO2−‐N mole−1NH4+‐N) and on the exponential increase of the nitrogen loading rate (NLR, from 0.01 to 1.60 kg N m−3 d−1). 60 days after start‐up, anammox organisms were identified by polymerase chain reaction (PCR) technique as Candidatus Brocadia anammoxidans. After one year of operation, NLR had reached a value of 1.60 kg N m−3 d−1 with a nitrogen (ammonium plus nitrite) removal efficiency of 99.7%. The anammox biomass activity was verified by nitrogen mass balances with 1.32 ± 0.05 mole of nitrite removed per mole of ammonium removed and 0.23 ± 0.05 mole of nitrate produced per mole of ammonium removed. Also, enrichment of anammox bacteria was quantified by fluorescence in situ hybridization (FISH) analysis as 85.0 ± 1.8%.CONCLUSIONS: This paper provides a methodology for the enrichment of the anammox biomass in a SBR to treat high nitrogen loaded wastewaters. Copyright © 2007 Society of Chemical Industry

Diversity and abundance of aerobic and anaerobic ammonium‐oxidizing bacteria in freshwater sediments of the Xinyi River (China)

Here we report on the biodiversity and abundance of aerobic and anaerobic ammonium-oxidizing bacteria in sediment samples from the Xinyi River, Jinagsu Province (China). The biodiversity of aerobic ammonium-oxidizing bacteria in the sediment was assessed using the amoA gene as functional marker. The retrieved amoA clones were affiliated to environmental sequences from freshwater habitats. The closest cultivated relative was Nitrosomonas urea. Anaerobic ammonium-oxidizing (anammox) bacteria were studied using anammox and planctomycete-specific 16S rRNA gene primers. The sediments contained 16S rRNA genes and bacterial cells closely related to the known anammox bacterium Candidatus'Brocadia anammoxidans'. Anaerobic continuous flow reactors were set up to enrich anammox organisms from the sediments. After an adaptation period of about 25 days the reactors started to consume ammonium and nitrite, indicating that the anammox reaction was occurring with a rate of 41-58 nmol cm(-3) h(-1). Community analysis of the enrichments by quantitative fluorescence in situ hybridization showed an increase in the abundance of anammox bacteria from < 1% to 6 +/- 2% of the total population. Analysis of the 16S rRNA genes showed that the enriched anammox organisms were related to the Candidatus'Scalindua' genus.