Characterization of heme protein expressed by ammonia-oxidizing bacteria under low dissolved oxygen conditions

We have recently reported that expression of an unidentified heme protein is enhanced in a nitrifying activated sludge community under low (0.1 mg O₂/L) dissolved oxygen (DO) conditions. A preliminary assessment suggested it may be a type of hemoglobin (Hb) or a lesser-known component of the energy-transducing pathways of ammonia-oxidizing bacteria (AOB) (particularly an oxidase or peroxidase). Here, additional work was done to characterize this protein. Due to the unfeasibility of identifying the protein using gene-based methods, our approach was to carry out assays that target the activity and function of the protein, its location in the cell, and determination of the organisms that express it. Using CO-difference spectra, it was shown that the protein is expressed by AOB preferentially in the cytoplasm, while the pyridine hemochromogen method demonstrated that it has heme c as its prosthetic group. Peroxidase and oxidase assays were carried out on the soluble fraction of the low DO-grown cells; neither the peroxidase nor oxidase activities matched those of the CO-binding heme protein detected. Even though it is not possible to conclusively identify the protein detected as a Hb, all other known possibilities have been ruled out. Further work is needed to verify the identity of the heme protein as a Hb and to determine its type and biochemical role under low oxygen conditions.     

Nitrospira community composition in nitrifying reactors operated with two different dissolved oxygen levels

Nitrospira is a dominant member of nitrite-oxidizing bacteria (NOB) in nitrifying bioreactors as well as in natural habitats. In this study, Nitrospira NOB were investigated in the two nitrifying reactors operated with high and low dissolved oxygen (DO) concentrations for a period of 300 days. Phylogenetic and terminal restriction fragment length polymorphism analyses based on 16S rRNA gene sequences revealed that the Nitrospira community compositions of the two reactors during the early period related to group 1 and half of the Nitrospira community composition shifted to group 2 in the high-DO reactor after day 179, although there was no significant change in the low-DO reactor. These results suggested that DO was an important factor affecting Nitrospira community compositions in the nitrifying reactors.     

Effects of ammonium and nitrite on communities and populations of ammonia-oxidizing bacteria in laboratory-scale continuous-flow reactors

This study investigated the effects of ammonium and nitrite on ammonia-oxidizing bacteria (AOB) from an activated sludge process in laboratory-scale continuous-flow reactors. AOB communities were analyzed using specific PCR followed by denaturing gel gradient electrophoresis, cloning and sequencing of the 16S rRNA gene, and AOB populations were quantified using real-time PCR. To study the effect of ammonium, activated sludge from a sewage treatment system was enriched in four reactors receiving inorganic medium containing four different ammonium concentrations (2, 5, 10 and 30 mM NH(4) (+)-N). One of several sequence types of the Nitrosomonas oligotropha cluster predominated in the reactors with lower ammonium loads (2, 5 and 10 mM NH(4) (+)-N), whereas Nitrosomonas europaea was the dominant AOB in the reactor with the highest ammonium load (30 mM NH(4) (+)-N). The effect of nitrite was studied by enriching the enriched culture possessing both N. oligotropha and N. europaea in four reactors receiving 10-mM-ammonium inorganic medium containing four different nitrite concentrations (0, 2, 12 and 22 mM NO(2) (-)-N). Nitrosomonas oligotropha comprised the majority of AOB populations in the reactors without nitrite accumulation (0 and 2 mM NO(2) (-)-N), whereas N. europaea was in the majority in the 12- and 22-mM NO(2) (-)-N reactors, in which nitrite concentrations were 2.1-5.7 mM (30-80 mg N L(-1)).     

亚高山/高山森林土壤有机层氨氧化细菌和氨氧化古菌丰度特征

为了解季节性冻融作用对川西亚高山/高山地区土壤氨氧化微生物群落的影响,采用qPCR技术,以氨单加氧酶基因的α亚基(amoA)为标记,在生长阶段、冻结阶段、融化阶段中的9个关键时期调查了该地区不同森林群落：岷江冷杉(Abies faxoniana)原始林(PF)、岷江冷杉(A. faxoniana)和红桦(Betula albosinensis)混交林(MF)、岷江冷杉次生林(SF)土壤有机层的氨氧化细菌(ammonia-oxidizing bacteria, AOB)和氨氧化古菌(ammonia-oxidizing archaea, AOA)丰度的特征。结果表明,三个森林群落土壤有机层中都具有相当数量的氨氧化细菌和古菌,均表现出从生长阶段至冻结阶段显著降低,在冻结阶段最低,但冻结阶段后显著增加,在融化阶段为全年最高的趋势。土壤氨氧化微生物类群结构(AOA/AOB)受负积温影响明显。冻结后期三个森林群落土壤负积温最大时,AOA数量明显高于AOB,但其他关键时期土壤氨氧化微生物类群结构与群落类型密切相关。高海拔的PF群落土壤有机层表现为AOA>AOB(冻结初期除外),低海拔的SF群落中表现为AOB>AOA(冻结后期除外),而MF群落则仅在融冻期和生长季节末期表现为AOB>AOA。这些结果为认识亚高山/高山森林及其相似区域的生态过程提供了一定的科学依据。     

好氧氨氧化菌的种群生态学研究进展

好氧氨氧化菌是一类能够在好氧条件下将NH4^＋转化为NO2^-的化能无机自养型细菌,其活动将直接或间接影响土壤养分循环、水体富营养化、温室气体（N2O）和生态系统的功能。现代分子生物学技术的发展促进了人们对好氧氨氧化菌种群生态学的研究。介绍了近年来基于16SrRNA和氨单加氧酶amoA基因序列分析的好氧氨氧化菌的系统发育研究,比较了两种基因序列分析在好氧氨氧化菌遗传多样性研究中存在的差异;概述了环境条件诸如铵浓度、酸度、氧的可利用性、温度、盐度等对好氧氨氧化菌种类、数量及其种群生态分布的影响;阐述了好氧氨氧化菌对铵、氧饥饿的响应特征及其在酸性环境中的生存机制;并对今后好氧氨氧化菌的应用生态学研究及其主要方向进行了展望。     

攀枝花不同海拔高度烤烟农田红壤中氨氧化细菌与氨氧化古菌的群落结构分析

为探究攀枝花干热河谷区农田土壤氨氧化古菌（Ammonia oxidizing archaea,AOA）与氨氧化细菌（Ammonia oxidizing bacteria,AOB）群落对海拔高度的响应特征,深入认识该区域的氮素循环过程。以攀枝花米易县不同海拔（1600 m、1800 m和2000 m）农田红壤为研究对象,运用化学分析和末端限制性片段长度多态性（Terminal restriction fragment length polymorphism,T-RFLP）分别测定土壤理化性质、AOA和AOB群落组成及多样性,研究不同海拔农田土壤中AOA和AOB群落变异及其驱动因子。研究结果显示,不同海拔农田土壤pH均小于7,土壤有机碳（SOC）、全氮（TN）、速效钾（AK）和铵态氮（NH₄⁺-N）含量随海拔升高而降低,碱解氮（AN）、有效磷（AP）和硝态氮（NO₃^--N）含量随海拔升高先增加后降低;随海拔升高,AOA群落多样性指数增加,而AOB群落多样性指数先增加后降低;AOA以亚硝基球菌属（Nitrososphaera）为优势菌群,AOB以亚硝化螺菌属（Nitrosospira）为优势菌群;土壤有机碳（SOC）、速效钾（AK）和硝态氮（NO₃^--N）是影响该区域农田土壤AOA和AOB群落发育的主要因子。总体而言,攀枝花干热河谷区不同海拔农田土壤AOA和AOB群落结构变化明显,土壤硝态氮、速效钾和有机碳是影响AOA和AOB群落结构变异的主要因子;研究结果可为揭示干热河谷区农田红壤氮循环相关微生物的海拔分布格局提供理论依据。     

污水短程硝化体系氨氧化菌与亚硝酸盐氧化菌的生态学研究进展

短程硝化(partial nitrification, PN)是一种绿色低碳的生物脱氮创新技术,伴随厌氧氨氧化(anaerobic ammonia oxidation, Anammox)污水脱氮技术的进一步推广,短程硝化作为提供其电子受体的重要环节,已成为了污水脱氮领域的研究热点。氨氧化菌(ammonia-oxidizing bacteria,AOB)和亚硝酸盐氧化菌(nitrite-oxidizing bacteria, NOB)是该技术的核心竞争微生物,掌握这两类微生物的生态学特征,借助生态学理论和手段调控AOB淘汰NOB,提高种群的可预测性,对于实现稳定高效的短程硝化具有重要意义。本文基于生态学角度介绍了AOB和NOB基础分类、生理性能及生态位分离,重点综述了短程硝化系统中AOB和NOB的生长动力学、群落构建、环境因素和相互作用,最后对这两类微生物的未来研究重点和研究方法进行了展望,为短程硝化工艺的快速启动和稳定运行提供理论指导。     

Strategies of aerobic ammonia-oxidizing bacteria for coping with nutrient and oxygen fluctuations

In most natural environments as well as in engineered environments, such as wastewater treatment plants, ammonia-oxidizing bacteria (AOB) experience fluctuating substrate concentrations. Several physiological traits, such as low maintenance energy demand and decay rate, cell-to-cell communication, cell mobility, stable enzymes and RNAs, could allow AOB to maintain themselves under unfavourable circumstances. This review examines whether AOB possess such traits and how these traits might offer advantages over competing organisms such as heterotrophic bacteria during periods of starvation. In addition, within the AOB groups, differences exist in adaptation to and competitiveness under conditions of high or low ammonia or oxygen concentrations. Because these findings are of importance with regard to the ecology and activity of AOB in natural and engineered environments, concluding remarks are directed towards future research objectives that may clarify unanswered questions, thereby contributing to the general knowledge of the ecology and activity of ammonia oxidizers.     

Ammonia-oxidizing archaea have more important role than ammonia-oxidizing bacteria in ammonia oxidation of strongly acidic soils

Increasing evidence demonstrated the involvement of ammonia-oxidizing archaea (AOA) in the global nitrogen cycle, but the relative contributions of AOA and ammonia-oxidizing bacteria (AOB) to ammonia oxidation are still in debate. Previous studies suggest that AOA would be more adapted to ammonia-limited oligotrophic conditions, which seems to be favored by protonation of ammonia, turning into ammonium in low-pH environments. Here, we investigated the autotrophic nitrification activity of AOA and AOB in five strongly acidic soils (pH<4.50) during microcosm incubation for 30 days. Significantly positive correlations between nitrate concentration and amoA gene abundance of AOA, but not of AOB, were observed during the active nitrification. ¹³CO₂-DNA-stable isotope probing results showed significant assimilation of ¹³C-labeled carbon source into the amoA gene of AOA, but not of AOB, in one of the selected soil samples. High levels of thaumarchaeal amoA gene abundance were observed during the active nitrification, coupled with increasing intensity of two denaturing gradient gel electrophoresis bands for specific thaumarchaeal community. Addition of the nitrification inhibitor dicyandiamide (DCD) completely inhibited the nitrification activity and CO₂ fixation by AOA, accompanied by decreasing thaumarchaeal amoA gene abundance. Bacterial amoA gene abundance decreased in all microcosms irrespective of DCD addition, and mostly showed no correlation with nitrate concentrations. Phylogenetic analysis of thaumarchaeal amoA gene and 16S rRNA gene revealed active ¹³CO₂-labeled AOA belonged to groups 1.1a-associated and 1.1b. Taken together, these results provided strong evidence that AOA have a more important role than AOB in autotrophic ammonia oxidation in strongly acidic soils.     

内蒙古呼伦贝尔草原土壤氨氧化细菌多样性及群落结构

采用聚合酶链式反应-变性梯度凝胶电泳技术及扩增产物序列分析方法,研究了呼伦贝尔5种草地类型(线叶菊草原、贝加尔针茅草原、羊草草原、大针茅草原、克氏针茅草原)土壤氨氧化细菌多样性及群落结构特征.研究表明:不同草地类型间土壤氨氧化细菌群落结构组成差异显著,相似性均低于50%.线叶菊草原土壤氨氧化细菌群落多样性最高,其次是贝加尔针茅草原、羊草草原和克氏针茅草原,大针茅草原最低.5种草地类型土壤氨氧化细菌均以Nitrosospira cluster 3为优势种群,此外还发现有Nitrosospira cluster 1、2、4和Nitrosomonas.线叶菊草原土壤氨氧化细菌群落组成较其他草地类型复杂,而羊草草原和大针茅草原群落组成较简单.经相关性分析,土壤含水量、土壤全氮、有机碳、土壤C/N与土壤氨氧化细菌群落多样性显著正相关(P<0.05).     

Rapid detection of ammonia-oxidizing bacteria in activated sludge based on 16S-rRNA gene by using PCR and fluorometry

To detect whole ammonia-oxidizing bacteria in the activated sludge, group-specific primers targeting the 16S-rRNA gene of ammonia-oxidizing bacteria were used. The electrophoresis pattern of the PCR products seemed to produce a single band of approximately 1.0 k bp for the bacteria in activated sludge andNitrosomonas europaea. No band was observed for nitrite-oxidizerNitrobacter winogradskyi and heterotrophs such asPseudomonas putida. Then direct measurement of the PCR product was made by fluorometry using the reagent Hoechist 33258, so that the fluorescent intensity was in proportional to the cell number of the sample up to 240. Total time required for the test was about 4 h including DNA extraction. The DNA fragments produced were cloned and their sequences showed high similarity to those ofNitrosomonas spp. This study showed the feasibility to detect ammonia-oxidizing bacteria and to estimate their population rapidly for the control of the nitrogen elimination process.     

三种利用方式对羊草草原土壤氨氧化细菌群落结构的影响

本文以内蒙古呼伦贝尔羊草草原为研究对象,利用聚合酶链式反应－变性梯度凝胶电泳(PCR－DGGE)技术及扩增产物序列分析方法比较研究了刈割、放牧和围栏封育三种利用方式对羊草草原土壤氨氧化细菌多样性和系统发育的影响,结果表明：羊草草原土壤中氨氧化细菌的优势类群为亚硝化螺旋菌属和亚硝化单孢菌属的细菌,分别占氨氧化细菌总数的72.4%和28.6%,Cluster 4和Cluster 6在各个样地中均为优势类群但所占比例不同,放牧样地中氨氧化细菌的多样性最高,逐步回归分析的结果表明,影响多样性指数的关键理化因子是土壤硝态氮含量。放牧样地中脲酶活性和硝化率都显著高于其他两种利用方式,其中,脲酶活性与土壤硝态氮和全磷含量表现出极显著的相关性,与速效磷含量表现出显著相关性;而硝化率仅与硝态氮含量表现出显著相关性。不同利用方式土壤氨氧化细菌群落与土壤理化因子的典范对应分析结果表明铵态氮的含量对氨氧化细菌群落的影响显著。     

Effects of nitrobenzene contamination and of bioaugmentation on nitrification and ammonia-oxidizing bacteria in soil

Bioaugmentation of nitrobenzene-contaminated soil was performed by inoculation with Pseudomonas putida ZWL73, which can grow on nitrobenzene as carbon and nitrogen sources and release free ammonium from the aromatic ring via a partial-reductive pathway. Removal of nitrobenzene was effectively enhanced with concurrent accumulation of ammonium in the bioaugmented soil. Moreover, the negative impact of nitrobenzene contamination on culturable bacterial types and soil nitrification was reduced by strain ZWL73. Changes in the community structure of ammonia-oxidizing bacteria, determined by denaturing gradient gel electrophoresis, were associated with changes in environmental factors in nitrobenzene-contaminated soil, including concentrations of nitrobenzene, ammonium, nitrite and nitrate, but their influence was attenuated in the bioaugmented soil. Overall, P. putida ZWL73 shows promising abilities for effective removal of nitrobenzene and for attenuating the negative effects of nitrobenzene contamination on soil functioning.     

Community survey of ammonia-oxidizing bacteria in full-scale activated sludge processes with different solids retention time

AIMS: To study the effects of different solids retention time (SRT) on the nitrification activity and community composition of ammonia-oxidizing bacteria (AOB) in two full-scale activated sludge processes during a 5-month period. METHODS AND RESULTS: The AOB community composition was analysed using fluorescence in situ hybridization (FISH) and denaturing gradient gel electrophoresis (DGGE), and the identified populations were enumerated by quantitative FISH. Potential nitrification rates were determined in batch tests and the in situ rates were calculated from mass balances of nitrogen in the plants. Increased SRT reduced the nitrification activity, but neither the number per mixed liquor suspended solids nor community composition of AOB were affected. Two dominant AOB populations related to Nitrosomonas europaea and Nitrosomonas oligotropha were identified by FISH, whereas only the latter could be detected by DGGE. CONCLUSIONS: The effect of a longer SRT on the activity was probably because of physiological changes in the AOB community rather than a change in community composition. SIGNIFICANCE AND IMPACT OF THE STUDY: Physiological alterations of a stable AOB community are possible and may stabilize activated sludge processes. The commonly used FISH probes designed to target all beta-proteobacterial AOB does not detect certain Nitrosomonas oligotropha populations, leading to an underestimation of AOB if a wider set of probes is not used.