氨氧化古菌及其在氮循环中的重要作用

氨氧化作用作为硝化过程的第一步,是氮素生物地球化学循环的关键步骤.长期以来,变形菌纲卢和',亚群中的氨氧化细菌一直被认为是氨氧化作用的主要承担者.然而,近年来研究发现氨氧化古菌广泛存在于各种生态系统中,并且在数量上占明显优势,在氮素生物地球化学循环中起着非常重要的作用.本文概述了氨氧化古菌的形态、生理生态特性及其系统发育特征,对比分析了氨氧化古菌和氨氧化细菌的氨单加氧酶及其编码基因的异同,综述了氨氧化古菌在水生和陆地等生态系统氮素循环中的作用,同时就氨氧化古菌在应用生态和环境保护领域今后的研究重点进行了展望.     

氨氧化古菌及其对氮循环贡献的研究进展

硝化作用先将氨氮氧化为亚硝酸盐氮并进一步氧化为硝酸盐氮,这一过程是氮进行全球生物化学循环的重要环节。随着氨氧化古菌(Ammonia-oxidizing archaea,AOA)基因组序列中氨单加氧酶编码基因(amoA)的发现以及AOA在实验室条件下的成功培养(包括分离纯化和富集培养),基于分子生物学的研究表明AOA在各种环境广泛存在,且多数生境中它的数量远远超过氨氧化细菌(Ammonia-oxidizing bacteria,AOB)。AOA相对于AOB在氮循环中的贡献也引起了多方面的论证和争论。本文就氨氧化古菌的生态分布、系统进化、生境存在丰度及参与硝化作用等进行综述,指出不同生境AOA的活性及其对氮循环的重要性仍需做进一步的研究。     

海洋氮循环中细菌的厌氧氨氧化

细菌厌氧氨氧化过程是在一类特殊细菌的厌氧氨氧化体内完成的以氨作为电子供体硝酸盐作为电子受体的一种新型脱氮反应.厌氧氨氧化菌的发现,改变人们对传统氮的生物地球化学循环的认识:反硝化细菌并不是大气中氮气产生的唯一生物类群.而且越来越多的证据表明,细菌厌氧氨氧化与全球的氮物质循环密切相关,估计海洋细菌的厌氧氨氧化过程占到全球海洋氮气产生的一半左右.由于氮与碳的循环密切相关,因此可以推测,细菌的厌氧氨氧化会影响大气中的二氧化碳浓度,从而对全球气候变化产生重要影响.另外,由于细菌厌氧氨氧化菌实现了氨氮的短程转化,缩短了氮素的转化过程,因此为开发更节约能源、更符合可持续发展要求的废水脱氮新技术提供了生物学基础.     

厌氧氨氧化菌驱动脱氮途径的研究进展

厌氧氨氧化菌(anaerobic ammonia-oxidizing bacteria, AnAOB)的代谢多样性,使得该菌群能够在海洋、湿地和陆地等不同的自然生态系统中广泛分布,甚至在一些极热和极寒环境中也检测到了该菌群的存在。本文回顾并总结了厌氧氨氧化菌在不同生态系统中的发现、分布及脱氮贡献等方面的研究,分析了厌氧氨氧化菌分布的主要环境影响因素。该综述将帮助我们更好地理解全球氮循环中厌氧氨氧化菌的实际角色和功能,并基于厌氧氨氧化(anaerobicammoniaoxidation,anammox)过程,探究能与其进行协作的新型生物脱氮工艺,以期为这些工艺的研发和推广提供生态学基础和新的思考,从而实现脱氮工艺的技术变革。     

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

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-1h-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—78gN m-2a-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。     

氨氧化微生物生态学与氮循环研究进展

氮的生物地球化学循环主要由微生物驱动,除固氮作用、硝化作用、反硝化作用和氨化作用外,近年还发现厌氧氨氧化是微生物参与氮循环的一个重要过程.同时,随着宏基因组学等分子生物技术的快速发展和应用,参与氮循环的新的微生物类群--氨氧化古菌也逐渐被发现.这两个重要的发现大大改变了过去人们对氮循环的认识,就近年有关厌氧氨氧化细菌、氨氧化古菌和氨氧化细菌的生态学研究进展作一简要综述.     

厌氧氨氧化菌的研究进展

厌氧氨氧化技术是一种新型生物脱氮技术,在废水处理中具有广泛的应用前途,对全球海洋的氮循环起着重要作用。由于反应中不需另加有机物、不消耗氧气、不会产生二次污染等优点,厌氧氨氧化技术受到格外关注。通常认为,厌氧氨氧化的机理在于厌氧氨氧化菌使氨和亚硝酸反应生成氮气。通过16SrRNA分子生物学方法已鉴定出该菌群属于分枝很深的浮霉菌,由于至今未能成功分离到纯的菌株,未正式命名,对其微生态环境以及生理生化特征也未能取得一致的意见。本文综述了国内外对厌氧氨氧化微生物的作用、分布、种类、生理生化特征等研究进展,认为厌氧氨氧化菌的分离纯化、生物特性、小生境等是今后的主要研究方向。     

若尔盖高原湿地土壤氨氧化古菌的多样性

【目的】研究自然界中氨氧化古菌(ammonia-oxidizing archaea,AOA)对于理解全球氮循环起着至关重要的作用,但人们对高原湿地AOA种群生态还知之甚少。本研究旨在了解若尔盖高原湿地土壤AOA群落组成及多样性。【方法】从若尔盖高原阿西(A'xi)、麦西(Maixi)和分区(Fenqu)3个典型牧区采集土壤样品,提取土壤总DNA,利用AOA氨单加氧酶(ammonia monooxygenase,amoA)基因通用引物扩增amoA基因,构建amoA基因克隆文库。从每个克隆文库中随机挑选80个阳性克隆子用于后续限制性酶切片段长度多态性(restriction fragment length polymorphism,RFLP)分析,挑选不同酶切类型的克隆子进行测序、比对,利用MEGA 5.0软件构建amoA基因系统发育树。【结果】从3个克隆文库共240个AOA amoA基因阳性克隆中得到15条代表序列,通过Mothur软件进行OTUs(operational taxonomic units)分类得到7个不同的分类单元。其中OTU 6为优势类群,在3个克隆文库均有发现,约占所有特异性克隆子的27%。15条amoA基因序列分属于Zoige Wetland Clade 1(4 OTUs)、Zoige Wetland Clade 2(2 OTUs)和Zoige Wetland Clade 3(1OTU)3个系统发育分支。BLAST分析显示所有OTUs均归于泉古菌门(Crenarchaeota)。相关性分析表明,若尔盖高原湿地AOA多样性指数与土壤铵态氮和硝态氮含量存在显著的相关性(P0.05)。【结论】若尔盖高原湿地中AOA多样性较低,均属于泉古菌,且与土壤中氨态氮和硝态氮密切相关。     

厌氧氨氧化菌的研究进展

近年来,有关厌氧氨氧化过程这一特殊的生化机制以及微生物类群的研究引起了人们的极大关注,尤其是这类微生物的生态生境可能比人们预想的范围更加广泛,因而在自然界N循环中可能具有重要意义。对这类菌结构特征、系统发育地位以及厌氧氨氧化小体和厌氧氨氧化机制的更深入认识将大大促进它们在污水处理工程中的应用。综述了近年来有关厌氧氨氧化菌的生理特性、生化机制、结构特点、生态生境以及工程应用等方面的最新进展。     

厌氧氨氧化菌特性及其在生物脱氮中的应用

在无分子氧环境中,同时存在NH4^＋和NO2^-时,NH4^＋作为反硝化的无机电子供体,NO2^-作为电子受体,生成氮气,这一过程称为厌氧氨氧化。目前已经发现了3种厌氧氨氧化菌（Brocadia anammoxidans,Kuenenia stuttgartiensis,Scalindua sorokinii）;对厌氧氨氧化菌的细胞色素、营养物质、抑制物、结构特征和生化反应机理的研究表明,厌氧氨氧化菌具有多种代谢能力。基于部分硝化至亚硝酸盐,然后与氨一起厌氧氨氧化,以及厌氧氨氧化菌与好氧氨氧化菌或甲烷菌的协同耦合作用,提出了几种生物脱氮的新工艺（ANAMMOX、SHARON—ANAMMOX、CANON和甲烷化与厌氧氨氧化耦合工艺）。     

Dynamics of ammonia-oxidizing archaea and bacteria populations and contributions to soil nitrification potentials

It is well known that the ratio of ammonia-oxidizing archaea (AOA) and bacteria (AOB) ranges widely in soils, but no data exist on what might influence this ratio, its dynamism, or how changes in relative abundance influences the potential contributions of AOA and AOB to soil nitrification. By sampling intensively from cropped-to-fallowed and fallowed-to-cropped phases of a 2-year wheat/fallow cycle, and adjacent uncultivated long-term fallowed land over a 15-month period in 2010 and 2011, evidence was obtained for seasonal and cropping phase effects on the soil nitrification potential (NP), and on the relative contributions of AOA and AOB to the NP that recovers after acetylene inactivation in the presence and absence of bacterial protein synthesis inhibitors. AOB community composition changed significantly (P⩽0.0001) in response to cropping phase, and there were both seasonal and cropping phase effects on the amoA gene copy numbers of AOA and AOB. Our study showed that the AOA:AOB shifts were generated by a combination of different phenomena: an increase in AOA amoA abundance in unfertilized treatments, compared with their AOA counterparts in the N-fertilized treatment; a larger population of AOB under the N-fertilized treatment compared with the AOB community under unfertilized treatments; and better overall persistence of AOA than AOB in the unfertilized treatments. These data illustrate the complexity of the factors that likely influence the relative contributions of AOA and AOB to nitrification under the various combinations of soil conditions and NH₄⁺-availability that exist in the field.     

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。     

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.     

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

Little information is available on the ecology of ammonia-oxidizing bacteria (AOB) and archaea (AOA) in flooded rice soils. Consequently, a microcosm experiment was conducted to determine the effect of nitrogen fertilizer on the composition of AOB and AOA communities in rice soil by using molecular analyses of ammonia monooxygenase gene (amoA) fragments. Experimental treatments included three levels of N (urea) fertilizer, i.e. 50, 100 and 150 mg N kg⁻¹ soil. Soil samples were operationally divided into four fractions: surface soil, bulk soil deep layer, rhizosphere and washed root material. NH₄⁺-N was the dominant form of N in soil porewater and increased with N fertilization. Cloning and sequencing of amoA gene fragments showed that the AOB community in the rice soil consisted of three major groups, i.e. Nitrosomonas communis cluster, Nitrosospira cluster 3a and cluster 3b. The sequences related to Nitrosomonas were predominant. There was a clear effect of N fertilizer and soil depth on AOB community composition based on terminal restriction fragment length polymorphism fingerprinting. Nitrosomonas appeared to be more abundant in the potentially oxic or micro-oxic fractions, including surface soil, rhizosphere and washed root material, than the deep layer of anoxic bulk soil. Furthermore, Nitrosomonas increased relatively in the partially oxic fractions and that of Nitrosospira decreased with the increasing application of N fertilizer. However, AOA community composition remained unchanged according to the denaturing gradient gel electrophoresis analyses.     

黑土区水田化肥氮去向的研究

选取东北北部黑土地区水稻生产上施氮量、施肥方法和主要氮肥品种(尿素)为参数,采用示踪元素微区法和常规尿素小区法,连续2年系统地研究了水田化肥氮的去向。结果表明,化肥氮22．2％～46．1％进入了水稻体内,平均为37．7％,当年进入土壤中的残留氮12．7％～25．4％,氨挥发为8．8％～17．2％,作物对化肥氮的利用高低决定于施氮方法,化肥氮深施、混施均比表层施用利用率高,低施肥量化肥氮利用率比高施肥量利用率高,土壤残留量与施肥方法有关,深施和高施氮量均增加土壤残留,^15N试验证明,由于东北北部黑土比较粘重和土体构型的原因,在土层深度为80cm以下未检测出土壤残留化肥氮,示踪试验和小区试验证明,化肥氮的激发效应(PE)量和土壤残留氮量从该地区总体估算为大致平衡。     

Effects of simulated warming on soil ammonia-oxidizing bacteria and archaea communities in an alpine forest of western Sichuan,China

Ongoing climate change, characterized by winter warming, snow cover decline and extreme weather events, is changing terrestrial ecosystem processes in high altitude and latitude regions. Winter soil processes could be particularly sensitive to climate change. In fact, winter warming and snow cover decline are interdependent in cold biomes, and have a synergistic effect on soil processes. Soil microorganisms not only play crucial roles in material cycling and energy flow, but also act as sensitive bio-indicators of climate change. However, little information is available on the effect of winter warming on forest soil ammonia-oxidizing bacteria (AOB) and archaea (AOA). The alpine and subalpine forest ecosystems on the eastern Tibet Plateau have important roles in conserving soil, holding water, and maintaining biodiversity. To understand the changes in AOB and AOA communities under climate change scenarios, an altitudinal gradient experiment in combination with soil column transplanting was conducted at the Long-term Research Station of Alpine Forest Ecosystems, which is situated in the Bipeng Valley of Lixian County, Sichuan, China. Thirty intact soil columns under an alpine forest at an altitude of 3582 m were transplanted and incubated at 3298 m and 3023 m forest sites, respectively. Compared with the 3582 m, we expected air temperature increases of 2 °C and 4 °C at the 3298 m and 3023 m, respectively. However, the temperatures in the soil organic layer (OL) and mineral soil layer (ML) increased by 0.27 °C and 0.13 °C, respectively, at 3023 m and ? 0.36 °C and ? 0.35 °C at 3298 m. Based on a previous study and with simultaneous monitoring of soil temperature, the abundances of AOB and AOA communities in both the OL and ML were measured by qPCR in December 2010 (i.e., the onset of the frozen soil period) and March 2011 (i.e., the late frozen soil period). The soil columns incubated at 3023 m had relatively higher AOB abundances and lower AOA/AOB ratios than those at 3298 m, while higher AOA abundances and AOA/AOB ratios were observed at 3298 m. The abundance of the microbial community at the late frozen period was higher than that at the onset of frozen soil, and the changes in microbial community abundance at the late frozen period were more substantial. Furthermore, the nitrate nitrogen (N) concentrations in both the OL and ML were significantly higher than ammonia N concentrations, implying that soil nitrate N is the primary component of the inorganic N pool in the alpine forest ecosystem. Additionally, the responses of AOA and AOB in the soil OL to soil column transplanting were more sensitive than the responses of those in ML. In conclusion, climate warming alters the abundance of the ammonia-oxidizing microbial community in the alpine forest ecosystem, which, in turn, might affect N cycling.     

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。     

黄土区深层土壤干燥化与土壤水分循环特征

深层土壤干燥化是黄土高原地区植被建设过程中出现的重大生态环境问题。采用人工和天然降雨试验,研究了黄土高原沟壑区荒草地和裸地的土壤水分循环特征,并分析和探讨了深层土壤干燥化的成因。2002年天然降雨量为459.9mm(多年平均降雨量为584.1mm),属干旱年,土壤水分观测期间(2002年6月13日至11月24日)天然和人工降雨试验小区的天然降雨量分别为305.2mm和236.8mm。人工降雨试验主要在2002年6~8月进行,土壤水分观测期间荒草地和裸地的人工降雨量分别为360.7mm和418.5mm。试验结果表明:干旱年,荒草地和裸地土壤储水量处于负补偿,入渗雨量全为蒸发蒸腾作用所消耗。在强烈的蒸发蒸腾作用下,剖面内(0~200cm)土壤水分的整体移动性能较强,最大蒸发蒸腾作用层深度很快形成。荒草地土壤水分循环强度大于裸地,表现为荒草地最大蒸发蒸腾作用层深度较大,两者分别为200cm和180cm。雨季量少且分散的降雨极易为强烈的蒸发蒸腾作用所消耗,深层土壤由于缺乏降雨入渗的补给而逐渐干燥化。丰水年,荒草地和裸地土壤储水量处于正补偿,但入渗雨量的大部分(80%以上)为强烈的蒸发蒸腾作用所消耗。在相同的降雨量条件下,荒草地土壤水分循环强度高于裸地,表现为荒草地降雨入渗补给深度较小。连续降雨有利于土壤水分向深层的运移,从而部分缓减深层土壤的干燥化进程。近50a来黄土高原地区气候变暖和降雨减少可能是土壤干层形成的直接原因,而植被类型选择失当、群落密度过大和生产力过高则会加剧深层土壤的干燥化进程。     

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。