亚热带松林中强酸性土壤自养和异养硝化作用的研究

The occurrence of nitrification in some acidic forest soils is still a subject of debate. Identification of main nitrification pathways in acidic forest soils is still largely unknown. Acidic yellow soil (Oxisol) samples were selected to test whether nitrification can occur or not in acidic subtropical pine forest ecosystems. Relative contributions of autotrophs and heterotrophs to nitrification were studied by adding selective nitrification inhibitor nitrapyrin. Soil NH₄⁺-N concentrations decreased, but NO₃^--N concentrations increased significantly for the no-nitrapyrin control during the first week of incubation, indicating that nitrification did occur in the acidic subtropical soil. The calculated net nitrification rate was 0.49 mg N kg^-1 d^-1 for the no-nitrapyrin control during the first week of incubation. Nitrapyrin amendment resulted in a significant reduction of NO₃^--N concentration. Autotrophic nitrification rate averaged 0.28 mg N kg^-1 d^-1 and the heterotrophic nitrification rate was 0.21 mg N kg^-1 d^-1 in the first week. Ammonia-oxidizing bacteria (AOB) abundance increased slightly during incubation, but nitrapyrin amendment significantly decreased AOB amoA gene copy numbers by about 80%. However, the ammonia-oxidizing archaea (AOA) abundance showed significant increases only in the last 2 weeks of incubation and it was also decreased by nitrapyrin amendment. Our results indicated that nitrification did occur in the present acidic subtropical pine forest soil, and autotrophic nitrification was the main nitrification pathway. Both AOA and AOB were the active biotic agents responsible for autotrophic nitrification in the acidic subtropical pine forest soil.     

Humic acids stimulate growth and activity of in vitro tested axenic cultures of soil autotrophic nitrifying bacteria

In the present study, the effect of humic acids on activity and growth of Nitrosomonas europaea and Nitrobacter agilis was investigated in vitro under axenic conditions. Humates from compost-stabilized vegetable waste or leonardite were added to the chemolithotrophic culturing medium at concentrations of 0, 5, 50 and 100 mg l^–1. It was found that both types of humic acids increased either NH₄ ⁺ or NO₂ ^– oxidation and cell growth of nitrifying bacteria in a dose-independent manner. By combining these results with data from a comparative growth evaluation of N. agilis based on possible utilization of humates or pyruvate in heterotrophic conditions, evidence was obtained that nitrifiers cannot use humic acids as an alternative carbon and energy source. Thus, the stimulating effect of this fraction of soil organic matter on chemolithotrophic ammonia and nitrite oxidizers might be attributed to an increase in microbial membrane permeability favouring a better utilization of nutrients. Received: 15 April 1996     

不同温度下尿素对水稻土甲烷产生及相关古菌群落的影响

水稻田是大气甲烷的重要排放源。尿素氮肥施用是提升水稻产量和品质的重要措施,但其对稻田土壤中产甲烷古菌的影响规律仍不清楚。通过模拟水稻生长季节可能的田间温度变化,本文对水稻土进行施加尿素（400 mg N/kgdry soil）与未施加尿素（0 mg N/kgdry soil）两个处理,并在15℃、25℃、37℃以及50℃四个温度下进行为期100d的厌氧培养,定期测定了培养过程中产甲烷累积量以及土壤理化因子pH、铵氮以及有机碳的变化。并运用基于16S rRNA基因的T-RFLP（末端限制性片段多态性分析）技术分析了产甲烷过程中古菌群落结构随时间的变化情况。结果表明,在中低温范围内（15℃-37℃）,尿素对水稻土产甲烷有抑制作用,但在50℃高温下尿素对水稻土产甲烷量没有显著影响。尿素可能通过改变产甲烷古菌群落结构来影响产甲烷,在15℃-37℃范围内,尿素降低了水稻土产甲烷古菌群落的稳定性,增大了其在不同时间的差异性;而在50℃高温时,尿素对水稻土产甲烷古菌稳定性和差异性的影响不明显。不同温度下,尿素均降低了甲烷八叠球菌（Methanosarcinaceae）的丰度,且随着温度的变化,尿素对水稻土产甲烷机制的改变可能没有影响。     

Main environmental drivers of abundance, diversity and community structure of comammox Nitrospira in paddy soils

The recently discovered complete ammonia oxidizers comammox Nitrospira contain clades A and B that can establish an independent one-step nitrification process; however, little is known about their environmental drivers or habitat distributions in agricultural soils. Previous studies on comammox Nitrospira in paddy soils have mainly focused on small-scale samples, and there is a lack of multisite research on comammox Nitrospira in paddy soils. In this study, we conducted a survey of 36 paddy soils to understand the community structure, abundance, and diversity of comammox Nitrospira and the degree to which they are affected by environmental factors at a large scale. Comammox Nitrospira were found to be widely distributed among the paddy soils. The abundance of comammox Nitrospira clade A was mostly lower than that of clade B, whereas its diversity was mostly higher than that of clade B. Correlation analysis showed that multiple factors affected (P < 0.05) the abundance of comammox Nitrospira, including soil pH, organic matter, total carbon, and total nitrogen, latitude, mean annual temperature, and mean annual precipitation. Moreover, there was a clear relationship between the comammox Nitrospira community and habitat, indicating that some amplicon sequence variants (ASVs) had a unique dominant position in specific habitats. Phylogenetic analysis showed that the ASVs of comammox Nitrospira clade A clustered with the known sequences in the paddy soils and were significantly different from the known sequences in other habitats, which may be related to the unique paddy field habitat. In contrast, comammox Nitrospira clade B showed no clear habitat dependence. These results support the wide distribution and high abundance of comammox Nitrospira in paddy soils and provide novel insights into nitrogen cycling and nutrient management in agricultural ecosystems.     

A positive relationship between the abundance of ammonia oxidizing archaea and natural abundance δ15N of ecosystems

We present a significant relationship between the natural abundance isotopic composition of ecosystem pools and the abundance of a microbial gene. Natural abundance ¹⁵N of soils and soil DNA were analysed and compared with archaeal ammonia oxidizer abundance along an elevation gradient in northern Arizona and along a substrate age gradient in Hawai'i. There was a significant positive correlation between the abundance of archaeal amoA genes and natural abundance δ¹⁵N of total soil or DNA suggesting that ammonia oxidizing archaea play an important role in ecosystem N release.     

三种农田土壤中氨氧化细菌amoA基因多样性比较分析

比较分析中国东部不同季风气候区中海伦黑土(HL)、封丘潮土(FQ)和鹰潭红壤(YT)3种土壤氨氧化细菌amoA基因多样性。采用非培养方法直接从土壤中提取微生物总DNA,用氨氧化细菌amoA基因特异引物扩增总DNA,构建了3种土壤amoA基因文库,并对文库进行限制性长度多态性(RFLP)分析。HL、FQ和YT的amoA基因文库克隆数量分别为49、50和48个,相应的RFLP类型数为10、10和14个OTUs,其中有4个OTUs为三种土壤共有;YT中氨氧化细菌amoA基因多样性指数最高,FQ最低;HL和FQ群落的相似为70%,HL与YT的相似度为50%,而FQ和YT之间仅为42%,说明氨氧化细菌具有地理分布的规律:17个amoA基因序列可以被聚成6个cluster,分属Nitrosospira和Nitrosomonas两个属。三种农田土壤中均存在丰富的氨氧化细菌,表明氨氧化细菌在农田土壤氮素循环中具有重要作用。     

Effects of a blend of garlic oil,nitrate and fumarate on in vitro ruminal fermentation and microbial population

Although garlic oil and nitrate can effectively suppress ruminal methane (CH₄) production in vitro, the application of these compounds is associated with suppressed total volatile fatty acid (VFA) concentration. On the other hand, the effectiveness of fumarate as a ruminal CH₄ mitigating agent is variable but its application increases total VFA concentration. We therefore hypothesized that the different characteristics of the compounds can compensate for the shortcomings of the other. The objective of this study was to develop an optimal blend of garlic oil, nitrate and fumarate that can suppress in vitro ruminal CH₄ without affecting total VFA concentration. Three ruminal in vitro fermentation experiments were carried out. The first one, a one factor at a time experiment was employed to investigate the effective concentration of each of the compounds on CH₄ and VFA production by ruminal bacteria. We then applied the fractional factorial design and response surface methodology in the second experiment to determine optimal concentrations of the compounds in the blend. The optimal blending of garlic oil, fumarate and nitrate was determined to be 50 mg/l, 15 mm and 20 mm , respectively. This simulated optimal blend was verified in a 48 h in vitro batch fermentation experiment. The blend achieved the intended goal of suppressing CH₄ whilst maintaining total VFA concentration. The blend and nitrate suppressed archaea populations (p < 0.001) but did not affect the total microbial population (p = 0.945). The observed results could be explained by additive effects of the agents making up the blend. Supplementing a high concentrate diet with the blend can significantly decrease ruminal CH₄ and maintain total VFAin vitro. These findings however, need to be verified in vivo using the optimized ratio of combining the three methane inhibitors as a guide.     

长期咸水滴灌对土壤氨氧化微生物丰度和群落结构的影响

为探讨长期咸水滴灌对棉田土壤氨氧化细菌（AOA）和氨氧化古菌（AOB）的丰度和群落结构的影响,于2018年采集已经过10年咸水滴灌的棉田土壤,通过实时荧光定量PCR和高通量测序技术测定土壤AOA和AOB的丰度和群落结构。试验设3个灌溉水盐度水平：0.35、4.61 dS·m^-1和8.04 dS·m^-1（分别代表淡水、微咸水和咸水）。结果表明：微咸水、咸水灌溉显著降低土壤NO₃^--N含量和潜在硝化势（PNR）,但显著增加土壤盐分和NH₄⁺-N含量。不同处理AOA和AOB的amoA基因拷贝数分别为2.2×10⁶~3.6×10⁶copies·g^-1和1.9×10⁵~3.2×10⁵ copies·g^-1干土;微咸水、咸水处理AOA和AOB amoA基因拷贝数均显著低于淡水处理,且微咸水处理显著降低AOA/AOB。PNR与AOA丰度（P<0.001）和AOB丰度（P<0.001）均呈显著正相关关系。此外,不同灌溉水盐度下AOA群落操作分类单元（OTUs）的数量大于AOB,微咸水、咸水灌溉显著降低AOB群落的OTUs。与淡水处理相比,咸水、微咸水处理显著增加AOA群落的香农指数,咸水处理显著降低AOB群落的香农指数。AOA和AOB群落的优势类群分别为Candidatus Nitrosocaldus和Nitrosospira;咸水、微咸水处理抑制AOA群落的Betaproteobacteria生长,而咸水处理中Candidatus Nitrosocaldus显著高于淡水和微咸水处理。AOB群落中Nitrosomonas的相对丰度随着灌溉水盐度的增加而显著降低。LEf Se分析显示,AOA在咸水灌溉下仅有1个差异物种,而AOB在微咸水灌溉时有5个差异物种。冗余分析结果显示：AOA群落结构的改变与土壤NO₃^--N、pH和盐度的变化密切相关,而AOB群落结构的改变仅与NO₃^--N和pH显著相关。盐分是影响氨氧化微生物生长及群落结构的主导因素,AOA和AOB共同参与土壤硝化作用,淡水、微咸水灌溉条件下AOB可能是硝化作用主导微生物种群,而咸水灌溉条件下AOA可能是主导微生物种群。     

瘤胃古菌的多样性研究进展

反刍动物瘤胃内由古茼驱动的甲烷生成不仅消耗了6%～10%的日粮能量摄入,而且生成甲烷气体是重要的温室效应气体.在全球气候变化的背景下,瘤胃古茵甲烷代谢调控成为关系畜牧业可持续性发展的重要课题.全面认识瘤胃中古菌多样性是寻找广谱、长效的瘤胃产甲烷调控方法的基础.通过对公共数据库中收录的3 112条瘤胃古茵16S rRNA基因序列进行分类和多样性预测分析,发现现有的古茵特异性引物与一些古菌种属的16S rRNA基因序列之间存在许多错配.瘤胃古茵群落在种属水平上的多样性较低,甲烷短杆茵(Methanobrevibacter)占到总数的70.1%,嗜酸球茵(Picrophilus)和甲烷球形茵(Methanosphaera)分别占13.5%和5.8%.瘤胃产甲烷古菌群落的组成也因受到宿主类型、日粮和环境等多重因素的影响而表现出较大的差异.这些结果拟为寻找一种高效、广谱的瘤胃古菌产甲烷活性的调控方法提供依据.     

用DGGE和Real-Time PCR对低温沼气池中产甲烷古菌群落的研究

利用DGGE及Real-Time PCR技术对不同地区低温沼气池中沼泥行进发酵前期、后期产甲烷古菌群落变化进行了研究.DGGE技术用于分析发酵前后产甲烷古菌优势群落的变化;Real-Time PCR技术用于分析发酵前期、后期样品产甲烷古菌的数量变化.结果表明,沼气低温发酵的过程中,不同沼泥样品发酵前期、后期产甲烷古菌的优势群落差异明显且数量上也存在较大差异;主要变化的产甲烷古菌的优势群落为甲烷粒菌属(Methanocorpusculum),甲烷八叠球菌属(Methanosarcina),甲烷鬃毛菌属(Methanosaeta).甲烷八叠球菌属(Methanosarcina)在黑龙江沼泥样品(A1)发酵后期成为优势群落,甲烷鬃毛菌属(Methanosaeta)在山东发酵前期(B1),后期样品(B2)及安徽发酵前期样品(C1)中占有绝对优势.产甲烷古菌的数量介于104 ～ 105个·mL-1之间,其中黑龙江样品中产甲烷菌数量最多,而安徽样品中产甲烷菌数量最少.