酸性土壤氨氧化微生物及其影响因素研究进展

自然条件变化和人类活动不仅加剧了土壤酸化,扩大了酸性土壤面积,而且严重影响了土壤氮循环。氨氧化过程作为硝化作用的限速步骤,是全球氮循环的核心环节,受到国内外研究者的广泛关注。探究酸性土壤氨氧化作用及其功能微生物对完善氮循环机制和促进土壤养分循环具有重要意义。本文主要综述了土壤中氨氧化代谢途径,对比了氨氧化细菌(ammoniaoxidizing bacteria, AOB)、氨氧化古菌(ammonia-oxidizing archaea, AOA)和全程硝化菌(complete ammoniaoxidizers,Comammox)对酸性土壤氨氧化作用的相对贡献,分析了微生物内源功能差异及pH、底物浓度等外部环境因素对氨氧化微生物丰度、活性和群落结构的影响,最后对氨氧化微生物研究进行了展望,以期为酸性土壤氨氧化作用研究和微生物修复技术应用与实践提供科学参考。     

长期施加氮肥及氧化钙调节对酸性土壤硝化作用及氨氧化微生物的影响

以长期施加氮肥及添加氧化钙调节的酸性土壤为研究对象,运用定量PCR和DGGE技术,探讨了土壤氨氧化微生物及硝化作用对不同施肥处理及氧化钙调节的响应。长期施化学氮肥导致酸性土壤p H(KCl)值(3.35—3.47)和硝化潜势(0.02—0.14μg NO-2-N g-1土壤h-1)进一步降低,而添加Ca O后土壤酸化得以缓解(p H值4.10—4.46),土壤硝化潜势(0.22—0.34μg NO-2-N g-1土h-1)显著增加。同时,添加Ca O处理对氨氧化古菌(AOA)的群落结构无明显影响,但明显提高了各施肥处理土壤中氨氧化细菌(AOB)的群落多样性,加Ca O处理的土壤中,AOA的数量降低而AOB的数量增加。这些结果表明虽然酸性土壤中AOA在数量和活性上占主导优势,AOB在功能上冗余,但当添加Ca O后,AOA和AOB对环境变化迅速作出响应,并根据其不同的生态位需求重新分配优势地位,二者交替作用共同驱动酸性土壤硝化作用。     

Growth of ammonia-oxidizing archaea in soil microcosms is inhibited by acetylene

Autotrophic ammonia-oxidizing bacteria were considered to be responsible for the majority of ammonia oxidation in soil until the recent discovery of the autotrophic ammonia-oxidizing archaea. To assess the relative contributions of bacterial and archaeal ammonia oxidizers to soil ammonia oxidation, their growth was analysed during active nitrification in soil microcosms incubated for 30 days at 30 °C, and the effect of an inhibitor of ammonia oxidation (acetylene) on their growth and soil nitrification kinetics was determined. Denaturing gradient gel electrophoresis (DGGE) analysis of bacterial ammonia oxidizer 16S rRNA genes did not detect any change in their community composition during incubation, and quantitative PCR (qPCR) analysis of bacterial amoA genes indicated a small decrease in abundance in control and acetylene-containing microcosms. DGGE fingerprints of archaeal amoA and 16S rRNA genes demonstrated changes in the relative abundance of specific crenarchaeal phylotypes during active nitrification. Growth was also indicated by increases in crenarchaeal amoA gene copy number, determined by qPCR. In microcosms containing acetylene, nitrification and growth of the crenarchaeal phylotypes were suppressed, suggesting that these crenarchaea are ammonia oxidizers. Growth of only archaeal but not bacterial ammonia oxidizers occurred in microcosms with active nitrification, indicating that ammonia oxidation was mostly due to archaea in the conditions of the present study.     

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.     

Autotrophic growth of nitrifying community in an agricultural soil

The two-step nitrification process is an integral part of the global nitrogen cycle, and it is accomplished by distinctly different nitrifiers. By combining DNA-based stable isotope probing (SIP) and high-throughput pyrosequencing, we present the molecular evidence for autotrophic growth of ammonia-oxidizing bacteria (AOB), ammonia-oxidizing archaea (AOA) and nitrite-oxidizing bacteria (NOB) in agricultural soil upon ammonium fertilization. Time-course incubation of SIP microcosms indicated that the amoA genes of AOB was increasingly labeled by ¹³CO₂ after incubation for 3, 7 and 28 days during active nitrification, whereas labeling of the AOA amoA gene was detected to a much lesser extent only after a 28-day incubation. Phylogenetic analysis of the ¹³C-labeled amoA and 16S rRNA genes revealed that the Nitrosospira cluster 3-like sequences dominate the active AOB community and that active AOA is affiliated with the moderately thermophilic Nitrososphaera gargensis from a hot spring. The higher relative frequency of Nitrospira-like NOB in the ¹³C-labeled DNA suggests that it may be more actively involved in nitrite oxidation than Nitrobacter-like NOB. Furthermore, the acetylene inhibition technique showed that ¹³CO₂ assimilation by AOB, AOA and NOB occurs only when ammonia oxidation is not blocked, which provides strong hints for the chemolithoautotrophy of nitrifying community in complex soil environments. These results show that the microbial community of AOB and NOB dominates the nitrification process in the agricultural soil tested.     

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。     

不同放牧对滇西北高原泥炭沼泽土壤氨氧化微生物群落的影响

氨氧化由氨氧化细菌(AOB)和氨氧化古菌(AOA)共同执行,是土壤硝化过程的第一步和限速步骤。放牧过程中,动物啃食、排泄和践踏等行为将影响土壤氨氧化微生物群落,但目前关于不同类型放牧对湿地氨氧化微生物群落结构及其多样性的影响尚不清楚。利用Illumina Mise高通量测序技术,对比研究牦牛放牧和藏香猪放养两种放牧类型对泥炭沼泽土壤氨氧化微生物群落结构及其多样性的影响。结果表明,牦牛放牧显著增加土壤容重,显著降低土壤pH、TN、TOC、NH⁺₄-N和NO^-₃-N含量;藏香猪放养显著增加土壤NO^-₃-N含量和硝化潜势(PNR)。牦牛放牧显著降低土壤AOA的丰富度和AOB的α多样性,藏香猪放养降低土壤AOA的α多样性和AOB的丰富度。放牧显著降低泉古菌门(Crenarchaeota)的相对丰度。AOA的α多样性与土壤NO^-₃-N含量和PNR呈显著负相关。AOB的α多样性与pH、TOC、TN和NH⁺     

Isotopic signatures of N2O produced by ammonia-oxidizing archaea from soils

N₂O gas is involved in global warming and ozone depletion. The major sources of N₂O are soil microbial processes. Anthropogenic inputs into the nitrogen cycle have exacerbated these microbial processes, including nitrification. Ammonia-oxidizing archaea (AOA) are major members of the pool of soil ammonia-oxidizing microorganisms. This study investigated the isotopic signatures of N₂O produced by soil AOA and associated N₂O production processes. All five AOA strains (I.1a, I.1a-associated and I.1b clades of Thaumarchaeota) from soil produced N₂O and their yields were comparable to those of ammonia-oxidizing bacteria (AOB). The levels of site preference (SP), δ¹⁵N^bulk and δ¹⁸O -N₂O of soil AOA strains were 13–30%, −13 to −35% and 22–36%, respectively, and strains MY1–3 and other soil AOA strains had distinct isotopic signatures. A ¹⁵N-NH₄⁺-labeling experiment indicated that N₂O originated from two different production pathways (that is, ammonia oxidation and nitrifier denitrification), which suggests that the isotopic signatures of N₂O from AOA may be attributable to the relative contributions of these two processes. The highest N₂O production yield and lowest site preference of acidophilic strain CS may be related to enhanced nitrifier denitrification for detoxifying nitrite. Previously, it was not possible to detect N₂O from soil AOA because of similarities between its isotopic signatures and those from AOB. Given the predominance of AOA over AOB in most soils, a significant proportion of the total N₂O emissions from soil nitrification may be attributable to AOA.     

抑制剂在氨氧化微生物研究中的应用

在氨氧化微生物的相关研究中经常使用各类抑制剂,包括针对硝化作用的抑制剂和针对微生物生长的抑制剂。自发现氨氧化古菌以来,人们在氨氧化细菌抑制剂的基础上重新筛选和使用不同的抑制剂来满足氨氧化微生物研究的需求。抑制剂既可以加速氨氧化古菌的富集,也可以帮助研究者区分古菌与细菌对硝化作用的贡献以及它们自身合成代谢能力的差别。本文综述了各类抑制剂的使用浓度和抑制效果,包括双氰胺(DCD)、3,4-二甲基吡啶磷酸盐(DMPP)、丙烯基硫脲(ATU)等传统抑制剂,乙炔和辛炔等炔烃类抑制剂,一氧化氮清除剂以及抗生素等对氨氧化微生物的活性和生长有特异性或通用抑制能力的抑制剂。通过对氨氧化微生物抑制剂的归纳总结,可为氨氧化微生物研究过程中抑制剂的选择提供参考。     

轮作模式对冬小麦土壤氨氧化微生物群落多样性和组成的影响

农田温室气体减排已成为农业绿色发展的重要内容,驱动温室气体氧化亚氮(N₂O)产生的氨氧化微生物受到了研究者们的广泛关注。为探究轮作模式对土壤氨氧化微生物群落的影响,基于田间定位试验,研究了夏红小豆-冬小麦、夏绿豆-冬小麦、夏花生-冬小麦、夏大豆-冬小麦和夏玉米-冬小麦5种轮作模式中冬小麦根际和非根际土壤氨氧化古菌(AOA)和氨氧化细菌(AOB)的群落组成和多样性变化特征。结果表明:与夏玉米-冬小麦模式相比,豆禾轮作模式增加了根际土中有机碳和硝态氮含量,以及非根际土中全氮和铵态氮含量。豆禾轮作模式降低了非根际土壤中AOA群落的ACE指数和Chao1指数,并显著降低根际土中AOB群落的ACE指数和Chao1指数(P<0.05)。豆禾轮作显著增加AOA群落中泉古菌门(Crenarchaeota)和AOB群落中变形菌门(Proteobacteria)某些类群的相对丰度(P<0.05)。根际土中豆禾轮作模式与麦玉模式的AOA群落结构发生明显分离,而非根际土中豆禾轮作模式与麦玉模式的AOB群落发生分离(P<0.05)。研究结果表明:豆禾轮作种植改变了AOA和AOB的群落结构和多样性,土壤pH值和速效氮含量是驱动AOA和AOB群落结构变化的重要因子,且根际与非根际土壤中氨氧化微生物存在生态位分离。     

Nitrification rates in Arctic soils are associated with functionally distinct populations of ammonia-oxidizing archaea

The functioning of Arctic soil ecosystems is crucially important for global climate, and basic knowledge regarding their biogeochemical processes is lacking. Nitrogen (N) is the major limiting nutrient in these environments, and its availability is strongly dependent on nitrification. However, microbial communities driving this process remain largely uncharacterized in Arctic soils, namely those catalyzing the rate-limiting step of ammonia (NH₃) oxidation. Eleven Arctic soils were analyzed through a polyphasic approach, integrating determination of gross nitrification rates, qualitative and quantitative marker gene analyses of ammonia-oxidizing archaea (AOA) and bacteria (AOB) and enrichment of AOA in laboratory cultures. AOA were the only NH₃ oxidizers detected in five out of 11 soils and outnumbered AOB in four of the remaining six soils. The AOA identified showed great phylogenetic diversity and a multifactorial association with the soil properties, reflecting an overall distribution associated with tundra type and with several physico-chemical parameters combined. Remarkably, the different gross nitrification rates between soils were associated with five distinct AOA clades, representing the great majority of known AOA diversity in soils, which suggests differences in their nitrifying potential. This was supported by selective enrichment of two of these clades in cultures with different NH₃ oxidation rates. In addition, the enrichments provided the first direct evidence for NH₃ oxidation by an AOA from an uncharacterized Thaumarchaeota–AOA lineage. Our results indicate that AOA are functionally heterogeneous and that the selection of distinct AOA populations by the environment can be a determinant for nitrification activity and N availability in soils.     

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。     

长期不同施肥制度对水稻土氨氧化微生物数量和群落结构的影响

由氨氧化微生物驱动的氨氧化过程是硝化作用的限速步骤,在土壤氮素循环过程中扮演着重要角色.以湖南省宁乡县长达30 a定位试验水稻土壤为研究对象,采用荧光定量PCR和Illumina MiSeq高通量测序分析方法,以amoA基因为靶标,研究了4种施肥制度[不施肥(CK)、化肥(CF)、70%化肥+30%有机肥(CFM₁)和40%化肥+60%有机肥(CFM₂)]水稻土壤氨氧化微生物的数量和群落结构变化.结果表明: 不同施肥处理氨氧化古菌(AOA)和氨氧化细菌(AOB) amoA基因拷贝数分别为3.09×10⁷～8.37×10⁷和1.04×10⁷～7.03×10⁷ copies·g^-1干土.施肥显著提高了AOA和AOB数量,但处理CFM₂中AOB数量与CK差异不显著.有机肥配施比例对AOB群落α多样性指数的影响强于AOA,处理CFM₁中AOA群落的多样性指数(Shannon)和AOB群落的丰富度指数(ACE和Chao1)均显著高于CK.奇古菌门和泉古菌门是AOA群落的优势门类群,占AOA amoA基因总序列的83.4%;亚硝化螺菌属、environmental_samples_norank、Bacteria_unclassified和Nitrosomonadales_unclassified是AOB群落的优势属类群,占AOB amoA基因总序列的97.8%.维恩分析结果显示,有机肥配施比例对AOB群落操作分类单元(OTU)数量的影响强于AOA,但对各处理共有AOA和AOB amoA基因序列条数的影响均较小.冗余分析结果显示,不同施肥处理AOB群落结构差异强于AOA,且所有土壤理化性质均与AOA和AOB群落结构存在显著相关关系.综上可知:有机肥配施比例显著改变了AOA和AOB数量、多样性和群落结构,配施30%有机肥时,AOA群落的Shannon指数最高,AOB群落数量、ACE和Chao1指数均最高.研究结果可为进一步探讨农业系统中氨氧化微生物对不同施肥制度的响应机制及其在氮素转化中的作用提供科学依据.     

Diversity and abundance of ammonia-oxidizing archaea and bacteria in polluted mangrove sediment

Ammonia oxidation by microorganisms is a critical process in the nitrogen cycle. Recent research results show that ammonia-oxidizing archaea (AOA) are both abundant and diverse in a range of ecosystems. In this study, we examined the abundance and diversity of AOA and ammonia-oxidizing beta-proteobacteria (AOB) in estuarine sediments in Hong Kong for two seasons using the ammonia monooxygenase A subunit gene (amoA) as molecular biomarker. Relationships between diversity and abundance of AOA and AOB and physicochemical parameters were also explored. AOB were more diverse but less abundant than AOA. A few phylogenetically distinct amoA gene clusters were evident for both AOA and AOB from the mangrove sediment. Pearson moment correlation analysis and canonical correspondence analysis (CCA) were used to explore physicochemical parameters potentially important to AOA and AOB. Metal concentrations were proposed to contribute potentially to the distributions of AOA while total phosphorus (TP) was correlated to the distributions of AOB. Quantitative PCR estimates indicated that AOA were more abundant than AOB in all samples, but the ratio of AOA/AOB (from 1.8 to 6.3) was smaller than most other studies by one to two orders. The abundance of AOA or AOB was correlated with pH and temperature while the AOA/AOB ratio was with the concentrations of ammonium. Several physicochemical factors, rather than any single one, affect the distribution patterns suggesting that a combination of factors is involved in shaping the dynamics of AOA and AOB in the mangrove ecosystem.     

Altitude ammonia-oxidizing bacteria and archaea in soils of Mount Everest

To determine the abundance and distribution of bacterial and archaeal ammonia oxidizers in alpine and permafrost soils, 12 soils at altitudes of 4000–6550 m above sea level (m a.s.l.) were collected from the northern slope of the Mount Everest (Tibetan Plateau), where the permanent snow line is at 5800–6000 m a.s.l. Communities were characterized by real-time PCR and clone sequencing by targeting on amo A genes, which putatively encode ammonia monooxygenase subunit A. Archaeal amo A abundance was greater than bacterial amo A abundance in lower altitude soils (≤5400 m a.s.l.), but this situation was reversed in higher altitude soils (≥5700 m a.s.l.). Both archaeal and bacterial amo A abundance decreased abruptly in higher altitude soils. Communities shifted from a Nitrosospira amo A cluster 3a-dominated ammonia-oxidizing bacteria community in lower altitude soils to communities dominated by a newly designated Nitrosospira ME and cluster 2-related groups and Nitrosomonas cluster 6 in higher altitude soils. All archaeal amo A sequences fell within soil and sediment clusters, and the proportions of the major archaeal amo A clusters changed between the lower altitude and the higher altitude soils. These findings imply that the shift in the relative abundance and community structure of archaeal and bacterial ammonia oxidizers may result from selection of organisms adapted to altitude-dependent environmental factors in elevated soils.