Ammonia-oxidizing bacteria dominates over ammonia-oxidizing archaea in a saline nitrification reactor under low DO and high nitrogen loading

A continuous nitrification reactor treating saline wastewater was operated for almost 1 year under low dissolved oxygen (DO) levels (0.15-0.5 mg/L) and high nitrogen loadings (0.26-0.52 kg-N/(m(3) day)) in four phases. The diversity and abundance of ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB) were analyzed by cloning, terminal restriction fragment length polymorphism (T-RFLP) and quantitative polymerase chain reaction (qPCR). The results showed that there were only one dominant AOA species and one dominant AOB species in the reactor in all of the four experimental phases. The amoA gene of the dominant AOA only had a similarity of 89.3% with the cultured AOA species Nitrosopumilus maritimus SCM1. All of the AOB species detected in the reactor belong to Nitrosomonas genus and it was found that the AOB populations changed with the ammonium loadings and DO levels. The abundance of AOB in the reactor was ～40 times larger than that of AOA, and the ratio of AOB to AOA increased significantly up to ～2,000 to ～4,000 with the increase of ammonium loading, indicating that AOB are much more competitive than AOA in high ammonium environments and probably AOA play a less important role than AOB in the nitrification reactors.     

A rapid DNA extraction method for PCR amplification from wetland soils

Aims: We tested a method of rapid DNA extraction from wetland soil samples for use in the polymerase chain reaction. Methods and Results: The glass bead/calcium chloride/SDS method obtained in the present study was compared with the calcium chloride/SDS/enzymatic extraction method and the UltraClean? Soil DNA Isolation Kit. Rapid DNA extraction could be completed within about two hours without purification steps. Conclusions: This study succeeded in establishing a fast soil DNA extraction protocol that can be applied to various environmental sources that are rich in humic acid content. Significance and Impact of the Study: The method provides a technology with high‐quality DNA extraction from soils for testing the diversity of AOB and AOA.     

Novel application of nitrifying bacterial consortia to ease ammonia toxicity in ornamental fish transport units: trials with zebrafish

Aims: To evaluate whether two commercial nitrifying bacterial consortia can function as biocontrol agents in ornamental fish transporting systems. Methods and Results: The consortia were applied in a simulated set‐up using zebrafish as the model organism in three trials. The efficacy of the bacterial consortia in controlling the ammonia level was validated by measuring water quality parameters such as total ammonia, nitrate and pH of the transport water. The bacterial community structure in the transport unit was studied using denaturing gradient gel electrophoresis. The consortia tested improved the nitrifying activity that in turn facilitated the reduction of ammonia that had accumulated during the transport. Bacterial profiles revealed the presence of both ammonia‐oxidizing and nitrite‐oxidizing bacteria in the transport bags. Conclusions: The application of the consortia during the transportation of zebrafish could profoundly improve the water quality by curbing ammonia accumulation. Significance and Impact of the Study: The potential of applying nitrifying bacteria as a bioremediation practice during the transport of ornamental fish has been demonstrated and this innovative approach contributes to the amelioration of current fish welfare in ornamental fish trade.     

Climate change amplifies gross nitrogen turnover in montane grasslands of Central Europe in both summer and winter seasons

The carbon‐ and nitrogen‐rich soils of montane grasslands are exposed to above‐average warming and to altered precipitation patterns as a result of global change. To investigate the consequences of climatic change for soil nitrogen turnover, we translocated intact plant–soil mesocosms along an elevational gradient, resulting in an increase of the mean annual temperature by approx. 2 °C while decreasing precipitation from approx. 1500 to 1000 mm. Following three years of equilibration, we monitored the dynamics of gross nitrogen turnover and ammonia‐oxidizing bacteria (AOB) and archaea (AOA) in soils over an entire year. Gross nitrogen turnover and gene levels of AOB and AOA showed pronounced seasonal dynamics. Both summer and winter periods equally contributed to cumulative annual N turnover. However, highest gross N turnover and abundance of ammonia oxidizers were observed in frozen soil of the climate change site, likely due to physical liberation of organic substrates and their rapid turnover in the unfrozen soil water film. This effect was not observed at the control site, where soil freezing did not occur due to a significant insulating snowpack. Climate change conditions accelerated gross nitrogen mineralization by 250% on average. Increased N mineralization significantly stimulated gross nitrification by AOB rather than by AOA. However, climate change impacts were restricted to the 2–6 cm topsoil and rarely occurred at 12–16 cm depth, where generally much lower N turnover was observed. Our study shows that significant mineralization pulses occur under changing climate, which is likely to result in soil organic matter losses with their associated negative impacts on key soil functions. We also show that N cycling processes in frozen soil can be hot moments for N turnover and thus are of paramount importance for understanding seasonal patterns, annual sum of N turnover and possible climate change feedbacks.     

Geospatial variation in co‐occurrence networks of nitrifying microbial guilds

Microbial communities transform nitrogen (N) compounds, thereby regulating the availability of N in soil. The N cycle is defined by interacting microbial functional groups, as inorganic N‐products formed in one process are the substrate in one or several other processes. The nitrification pathway is often a two‐step process in which bacterial or archaeal communities oxidize ammonia to nitrite, and bacterial communities further oxidize nitrite to nitrate. Little is known about the significance of interactions between ammonia‐oxidizing bacteria (AOB) and archaea (AOA) and nitrite‐oxidizing bacterial communities (NOB) in determining the spatial variation of overall nitrifier community structure. We hypothesize that nonrandom associations exist between different AO and NOB lineages that, along with edaphic factors, shape field‐scale spatial patterns of nitrifying communities. To address this, we sequenced and quantified the abundance of AOA, AOB, and Nitrospira and Nitrobacter NOB communities across a 44‐hectare site with agricultural fields. The abundance of Nitrobacter communities was significantly associated only with AOB abundance, while that of Nitrospira was correlated to AOA. Network analysis and geostatistical modelling revealed distinct modules of co‐occurring AO and NOB groups occupying disparate areas, with each module dominated by different lineages and associated with different edaphic factors. Local communities were characterized by a high proportion of module‐connecting versus module‐hub nodes, indicating that nitrifier assemblages in these soils are shaped by fluctuating conditions. Overall, our results demonstrate the utility of network analysis in accounting for potential biotic interactions that define the niche space of nitrifying communities at scales compatible to soil management.     

Higher Abundance of Ammonia Oxidizing Archaea than Ammonia Oxidizing Bacteria and Their Communities in Tibetan Alpine Meadow Soils under Long-term Nitrogen Fertilization

Increasing usage of nitrogen fertilizer for food production has resulted in severely environmental problems of nutrients enrichment. This study aimed to examine the response of ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA) to a long-term nitrogen fertilization in Tibetan alpine meadow. The abundance and composition of both AOB and AOA were assessed using quantitative real-time PCR, cloning and sequencing techniques based on amoA gene under different fertilization gradient (0, 30, 60, 90, and 120 g m^?2 year^?1). Our results showed that, abundances of AOA amoA genes (ranging from 1.48 × 10⁹ to 2.00 × 10⁹ copies per gram of dry soil) were significantly higher than those of AOB amoA genes (1.25 × 10⁷ to 2.62 × 10⁸ copies per gram of dry soil) under fertilization scenario. The abundance of AOB amoA genes increased with increasing nitrogen fertilization, whereas fertilization had little effect on AOA abundance. Sequences of clone libraries of the different treatments revealed that AOB communities were dominated by representatives of Cluster 4, constituting 48.94–64.44% in each clone library. Sequences of Clusters 9, 1 and 2 were prevalent in soils under higher fertilization. All archaeal amoA sequences recovered were affiliated with the soil/sediment clade and marine sediment clade, and no significant difference was observed on the community structure among different fertilization treatments. Variations in the AOB community structure and abundance were linked to ammonium-N and soil pH induced by different fertilization treatments. These results showed that the abundance and structure of the AOB community respond to the fertilization gradient, not AOA.     

Changes in rhizosphere bacterial populations during phytoextraction of heavy metal contaminated soil with poplar

Abstract

The objective of this paper was to study the response of rhizosphere ammonia‐oxidizing bacterial (AOB) populations during phytoextraction. Hybrid poplars were grown in compartmented root containers with an aged heavy metal (HM)‐contaminated soil for 13 weeks. Bulk and poplar rhizosphere soils were analyzed by denaturing gradient gel electrophoresis (DGGE) of amoA gene fragments. DGGE patterns revealed that amoA‐containing populations in the contaminated soils were markedly different from those in the uncontaminated soils. AmoA profiles appeared to be stable over time in the bulk soils. In contrast, contaminated rhizosphere soils revealed a clear shift of populations with removal of HMs. Rhizosphere AOB populations of the HM‐contaminated soils became similar to the populations of the uncontaminated soils during phytoextraction. The effect of phytoextraction was, however, not evident in the bulk samples, which still contained large amounts of HMs. This study suggests that rhizosphere AOB populations are able to recover after the relief of HM stress by phytoextraction practices.     

Responses of community structure of amoA‐encoding archaea and ammonia‐oxidizing bacteria in ammonia biofilter with rockwool mixtures to the gradual increases in ammonium and nitrate

Aims

To investigate community shifts of amoA‐encoding archaea (AEA) and ammonia‐oxidizing bacteria (AOB) in biofilter under nitrogen accumulation process.

Methods and Results

A laboratory‐scale rockwool biofilter with an irrigated water circulation system was operated for 436 days with ammonia loading rates of 49–63 NH₃ g m^?3 day^?1. The AEA and AOB communities were investigated by denaturing gradient gel electrophoresis, sequencing and real‐time PCR analysis based on amoA genes. The results indicated that changes in abundance and community compositions occurred in a different manner between archaeal and bacterial amoA during the operation. However, both microbial community structures mainly varied when free ammonia (FA) concentrations in circulation water were increasing, which caused a temporal decline in reactor performance. Dominant amoA sequences after this transition were related to Thaumarchaeotal Group I.1b, Nitrosomonas europaea lineages and one subcluster within Nitrosospira sp. cluster 3, for archaea and bacteria, respectively.

Conclusions

The specific FA in circulation water seems to be the important factor, which relates to the AOB and AEA community shifts in the biofilter besides ammonium and pH.

Significance and Impact of the Study

One of the key factors for regulating AEA and AOB communities was proposed that is useful for optimizing biofiltration technology.     

3 次连续重复提取DNA 能较好反映土壤微生物丰度

【目的】研究同一个土壤需要反复提取几次才能在最大程度上反映土壤微生物的丰度,探讨风干土壤代替新鲜土壤用于微生物丰度研究的可行性。【方法】针对两种理化性质具有较大差异的旱地和稻田新鲜土壤及其风干土壤,分别对土壤微生物进行5次连续裂解提取DNA。通过实时荧光定量PCR技术分析连续反复提取对土壤古菌和细菌16S rRNA gene数量、氨氧化古菌和细菌功能基因amoA数量的影响。【结果】3次连续提取DNA占5次提取DNA总量的76%以上,氨氧化古菌、氨氧化细菌、古菌和细菌4类微生物的3次连续提取最低回收率为77.5%;与新鲜土壤相比,风干处理导致氨氧化古菌、氨氧化细菌、古菌、细菌的数量分别降低84.3%、81.2%、12.5%和90.3%,然而,2种土壤风干过程中主要微生物类群的数量变化规律基本一致,表明土壤微生物对风干处理的响应可能受土壤类型的影响较小。【结论】土壤微生物连续3次裂解能较好反映微生物丰度。与新鲜土壤相比,风干过程显著降低了土壤微生物丰度,然而,通过风干土壤中微生物丰度的变化趋势反映新鲜土壤中微生物数量变化规律具有一定的可行性。     

Activity and composition of ammonia oxidizing bacterial communities and emission dynamics of NH3 and N2O in a compost reactor treating organic household waste

Aims:  To monitor emissions of NH₃ and N₂O during composting and link these to ammonia oxidation rates and the community structure of ammonia oxidizing bacteria (AOB).
Methods and Results:  A laboratory-scale compost reactor treating organic household waste was run for 2 months. NH₃ emissions peaked when pH started to increase. Small amounts of N₂O and CH₄ were also produced. In total, 16% and less than 1% of the initial N was lost as NH₃-N and N₂O-N respectively. The potential ammonia oxidation rate, determined by a chlorate inhibition assay, increased fourfold during the first 9 days and then remained high. Initially, both Nitrosospira and Nitrosomonas populations were detected using DGGE analysis of AOB specific 16S rRNA fragments. Only Nitrosomonas europaea was detected under thermophilic conditions, but Nitrosospira populations re-established during the cooling phase.
Conclusions:  Thermophilic conditions favoured high potential ammonia oxidation rates, suggesting that ammonia oxidation contributed to reduced NH₃ emissions. Small but significant amounts of N₂O were emitted during the thermophilic phase. The significance of different AOBs detected in the compost for ammonia oxidation is not clear.
Significance and Impact of Study:  This study shows that ammonia oxidation occurs at high temperature composting and therefore most likely reduces NH₃ emissions.     

Evaluating four mathematical models for nitrous oxide production by autotrophic ammonia‐oxidizing bacteria

There is increasing evidence showing that ammonia‐oxidizing bacteria (AOB) are major contributors to N₂O emissions from wastewater treatment plants (WWTPs). Although the fundamental metabolic pathways for N₂O production by AOB are now coming to light, the mechanisms responsible for N₂O production by AOB in WWTP are not fully understood. Mathematical modeling provides a means for testing hypotheses related to mechanisms and triggers for N₂O emissions in WWTP, and can then also become a tool to support the development of mitigation strategies. This study examined the ability of four mathematical model structures to describe two distinct mechanisms of N₂O production by AOB. The production mechanisms evaluated are (1) N₂O as the final product of nitrifier denitrification with NO as the terminal electron acceptor and (2) N₂O as a byproduct of incomplete oxidation of hydroxylamine (NH₂OH) to NO. The four models were compared based on their ability to predict N₂O dynamics observed in three mixed culture studies. Short‐term batch experimental data were employed to examine model assumptions related to the effects of (1) NH concentration variations, (2) dissolved oxygen (DO) variations, (3) NO accumulations and (4) NH₂OH as an externally provided substrate. The modeling results demonstrate that all these models can generally describe the NH, NO, and NO data. However, none of these models were able to reproduce all measured N₂O data. The results suggest that both the denitrification and NH₂OH pathways may be involved in N₂O production and could be kinetically linked by a competition for intracellular reducing equivalents. A unified model capturing both mechanisms and their potential interactions needs to be developed with consideration of physiological complexity. Biotechnol. Bioeng. 2013; 110: 153–163. © 2012 Wiley Periodicals, Inc.     

Expression of merA, amoA and hao in continuously cultured Nitrosomonas europaea cells exposed to zinc chloride additions

The effects of ZnCl2 additions on a mercuric reductase, merA, ammonia monooxygenase, amoA, and hydroxylamine (NH2OH) oxidoreductase, hao, gene expression were examined in continuously cultured Nitrosomonas europaea cells. The reactor was operated for 85 days with a 6.9 d hydraulic retention time and with four successive additions of ZnCl2 achieving maximum concentrations from 3 to 90 microM Zn2+. Continuously cultured N. europaea cells were more resistant to Zn2+ inhibition than previously examined batch cultured cells due to the presence of Mg2+ in the growth media, suggesting that Zn2+ enters the cell through Mg2+ import channels. The maximum merA up-regulation was 45-fold and expression increased with increases in Zn2+ concentration and decreased as Zn2+ concentrations decreased. Although Zn2+ irreversibly inactivated ammonia oxidation in N. europaea, the addition of either 600 microM CuSO4 or 2250 microM MgSO4 protected N. europaea from ZnCl2 inhibition, indicating a competition between Zn2+ and Cu2+/Mg2+ for uptake and/or AMO active sites. Since ZnCl2 inhibition is irreversible and amoA was up-regulated at 30 and 90 microM additions, it is hypothesized that de novo synthesis of the AMO enzyme is needed to overcome inhibition. The up-regulation of merA during exposure to non-inhibitory Zn2+ levels indicates that merA is an excellent early warning signal for Zn2+ inhibition.     

硝化作用研究的新发现:单步硝化作用与全程氨氧化微生物

硝化作用是氨被微生物氧化为硝酸盐的过程,分别由氨氧化微生物(AOB和AOA)和亚硝酸盐氧化细菌(NOB)主导完成.一个世纪以来,我们把这个分步硝化过程当成唯一的硝化途径来学习和研究.虽然根据动力学理论推测,环境中应该存在单步硝化作用,即由一种微生物单独完成整个硝化过程,将NH₃氧化为NO₃^-,但一直没有研究能直接证明该种微生物的存在.直到2015年底,3个科研团队分别在不同环境中发现了3种不同的经过纯培养的细菌(Candidatus Nitrospira nitrosa、Candidatus Nitrospira nitrificans和Candidatus Nitrospira inopinata)和一种未经过纯培养的细菌(类Nitrospira),它们都具备单独将氨氧化为硝酸盐的能力,这些微生物被定义为全程氨氧化微生物(Comammox).单步硝化作用和全程氨氧化微生物的发现终结了传承百年的理论,并引发了众多关于全球氮素循环的重要科学问题,如这些微生物在环境中的生态位点及其在硝化作用中的相对贡献等.本文就单步硝化作用及全程氨氧化微生物的发现作了简要概述.     

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.     

施氮肥对华北平原土壤氨氧化细菌和古菌数量及群落结构的影响

利用荧光定量PCR、末端限制性片段长度多样性(T-RFLP)和基因克隆文库技术,比较了4种施氮水平(不施氮肥,0 kg N/hm~2,CK;施低水平氮肥,75 kg N/hm~2,N1;施中水平氮肥,150 kg N/hm~2,N2;施高水平氮肥,225 kg N/hm~2,N3)下华北平原地区小麦季表层(0—20 cm)土壤总细菌、氨氧化细菌(AOB)和氨氧化古菌(AOA)的丰度和群落结构。结果表明,土壤总细菌、AOB和AOA数量分别在每克干土5.74×10~9—7.50×10~9、8.89×10~6—2.66×10~7和3.83×10~8—7.78×10~8之间。不同施氮量土壤AOA数量均高于AOB数量,AOA/AOB值在81.72—14.38之间。增施氮肥显著显著提高AOB数量(P0.05),对总细菌和AOA数量的影响不显著(P0.05)。与CK相比,处理N1、N2和N3中AOB数量分别提高了0.64、1.50和1.99倍。增施氮肥显著改变了AOB和AOA的群落结构,且不同施氮量处理中AOB群落结构差异更大。系统进化分析显示,施氮肥小麦土壤AOB主要为Nitrosospira属类群,分布在Cluster 3的两个分支中;AOA分布在Cluster S的4个分支中。相关性分析显示,AOB数量与全氮和铵态氮含量呈显著正相关关系,与土壤pH和碳氮比呈显著负相关关系(P0.05);AOA数量与硝态氮含量和土壤pH呈显著正相关关系,与铵态氮含量呈显著负相关关系(P0.05)。研究结果表明:增施氮肥可显著改变华北平原地区碱性土壤AOB数量与群落结构,该地区小麦土壤中AOB比AOA对氮肥响应更敏感。     

从典型硝化细菌到全程氨氧化微生物：发现及研究进展

生物硝化过程在全球氮循环中起关键性作用,被认为由氨氮氧化成亚硝酸盐和亚硝酸盐氧化成硝酸盐两个步骤组成,分别由氨氧化微生物(Ammonia oxidizing microorganisms,AOM)和硝化细菌(Nitrite oxidizing bacteria,NOB)催化完成。AOM包括氨氧化细菌(Ammonia oxidizing bacteria,AOB)和氨氧化古菌(Ammonia oxidizing archaea,AOA),AOB与AOA分布广泛,两者的相对丰度和氨氮浓度密切相关。2015年底,3个硝化螺菌属(Nitrospira)谱系Ⅱ的NOB被证实含有AOM的特征功能酶,包括氨单加氧酶(AMO)和羟胺脱氢酶(HAO),并证明NOB同时具有氨氧化和亚硝酸盐氧化的能力,命名为全程氨氧化微生物(Complete ammonia oxidizer,Comammox)。根据AMO的α亚基基因amoA的相似性将Comammox分为两大分支clade A和clade B。它们广泛分布于自然环境和人工系统,包括土壤(稻田、森林)、淡水(湿地、河流、湖泊沉积物、蓄水层)、污水处理厂和自来水厂等。本文综述了Comammox的发现及其最新的研究进展,并展望了Comammox作为氮循环关键功能菌群的研究方向和应用前景。     

Using respirometric techniques and fluorescent in situ hybridization to evaluate the heterotrophic active biomass in activated sludge

The separation and accurate quantification of active biomass components in activated sludge is of paramount importance in models, used for the management and design of waste water (WW) treatment plants. Accurate estimates of microbial population concentrations and the direct, in situ determination of kinetic parameters could improve the calibration and validation of existing models of biological nutrient removal activated sludge systems. The aim of this study was to obtain correlations between heterotrophic active biomass (Z(BH)) concentrations predicted by mathematical models and quantitative information obtained by Fluorescent in situ hybridizations (FISH). Respirometric batch test were applied to mixed liquors drawn from a well-defined parent anoxic/aerobic activated sludge system to quantify the Z(BH) concentrations. Similarly fluorescent labeled, 16S rRNA-targeted oligonucleotide probes specific for ammonia and nitrite oxidizers were used in combination with DAPI staining to validate the Z(BH) active biomass component in activate sludge respirometric batch tests. For the direct enumeration and simultaneous in situ analysis of the distribution of nitrifying bacteria, in situ hybridization with oligonucleotide probes were used. Probes (NSO 1225, NSR 1156, and NIT3) were used to target the nitrifiers and the universal probe (EUB MIX) was used to target all Eubacteria. Deducting the lithoautotrophic population from the total bacteria population revealed the Z(BH) population. A conversion factor of 8.49 x 10(-11) mg VSS/cell was applied to express the Z(BH) in terms of COD concentration. Z(BH) values obtained by molecular probing correlated closely with values obtained from the modified batch test. However, the trend of consistently poor correspondence of measured and theoretical concentrations were evident. Therefore, the focus of this study was to investigate alternative technology, such as FISH to validate or replace kinetic parameters which are invariably incorporated into models.