土壤氨氧化古菌适应酸性胁迫的ATPase基因分子进化研究

摘 要：分子生态研究发现,中性/碱性土壤中常见的氨氧化古菌DNA序列也存在于pH酸性土壤中,我们最近的研究发现中性氨氧化古菌可能通过V-ATPase适应性进化获得了耐酸或嗜酸生理生长能力。但这一现象是否具有普适性,尚不清楚。针对5个不同种植年限的马尾松人工林酸性土壤（15年、24年、45年、55年、63年）为研究对象,通过深度宏基因组测序获得7360亿碱基对,重构了氨氧化古菌AOA氨单加氧酶amoA基因和ATPase基因,研究了不同生态型氨氧化古菌AOA的嗜酸机制。结果表明,基于amoA基因的系统发育分析发现,AOA主要包括嗜酸Ca. Nitrosotaleales生态型、中碱性Nitrososphaerales生态型。所有酸性森林土壤中均存在pH中性的生态型AOA（Nitrososphaerales）,并且ATPase主要是V-ATPase,其分类结果及变化规律与amoA基因相同,表明ATPase可能是马尾松森林土壤中AOA适应酸性胁迫的分子进化机制。随林龄的增加,Ca. Nitrosotaleales丰度先减少后增加,而Nitrososphaerales丰度先增加后减少,速效钾是显著影响AOA群落结构的重要环境因子。这些结果表明,人工林种植极可能促进了酸性AOA的种群分化,目前尚未获得纯培养的Nitrososphaerales生态型极可能在马尾松林原位土壤硝化过程中发挥了重要作用。     

石灰和双氰胺对红壤酸化和硝化作用的影响及其机制

施用石灰是改良酸性土壤的重要措施,但其对土壤硝化作用的增强不仅加速土壤酸化,也增加硝态氮流失风险.传统的硝化抑制剂双氰胺(Dicyandiamide,DCD)能否在石灰改变pH的条件下始终有效抑制硝化是当前红壤区生产中亟需解决的问题.采用短期土壤培养试验,探讨了不同用量石灰与DCD配合施用对土壤酸化和硝化作用的影响及其...     

Effects of herbicide butachlor application on the growth of periphytic biofilms and nitrogen loss in paddy systems

The application of butachlor as an herbicide in paddy fields is widely practiced,aiming to increase rice yield by directly or indirectly influencing the paddy environment.Periphytic biofilms,which form at the soil-water interface in paddy fields,are complex bioaggregates that play an important role in nitrogen (N) cycling.The objective of this study was to investigate the effect of butachlor on periphytic biofilm growth and N cycling under both light and dark conditions in the laboratory.The res...     

长期施肥对黄土旱塬黑垆土氨氧化古菌群落多样性和丰度的影响

 【目的】研究长期不同施肥制度下黄土旱塬黑垆土氨氧化古菌群落多样性和丰度的变化,为提高黄土高原地区氮素利用效率、检测土壤质量变化提供重要依据。【方法】 利用PCR-RFLP技术和Real-time PCR技术分析无肥（CK）、有机肥（M）、氮肥+有机肥（NM）、磷肥+有机肥（PM）、氮磷肥+有机肥（NPM）等5种长期施肥处理对土壤氨氧化古菌群落结构和丰度的影响。【结果】从5种施肥处理的氨氧化古菌氨单加氧酶A（amoA）的克隆文库中分别得到25、18、29、20、30种RFLP酶切类型。α多样性指数表明各施肥处理土样之间氨氧化古菌多样性差异显著,NPM处理产生的氨氧化古菌多样性最多,而M处理多样性最少。欧氏距离聚类结果表明各施肥处理与对照（CK）处理土壤中氨氧化古菌群落类型趋同性系数最大,说明长期施肥处理使氨氧化古菌群落类型产生了较大变化。各施肥处理土壤中氨氧化古菌amoA基因拷贝数不同,NPM处理的氨氧化古菌丰度最高,与其它施肥处理呈显著性差异。基于amoA氨基酸序列构建的系统发育树将5种施肥处理的主要的优势氨氧化古菌与来自土壤/淡水沉积物的未培养氨氧化古菌聚为一类。【结论】长期不同施肥改变了土壤中氨氧化古菌群落多样性和丰度。     

氟磺胺草醚及其降解菌对大豆生长及生物固氮的影响

从生长于氟磺胺草醚污染土壤的大豆根瘤中筛选出的Sinorhizobium sp.W16菌株,能高效降解氟磺胺草醚并能缓解氟磺胺草醚的生物负效应。以大豆（苏C1008）为研究对象,采用盆栽试验,探究Sinorhizobium sp.W16对大豆生长、氮累积量、根瘤固氮酶活性及根际土壤氮循环相关微生物功能基因丰度等的影响。结果表明：氟磺胺草醚的施用量（以有效成分计）超过450 g·hm^-2时显著降低了大豆生物量,抑制了大豆根瘤固氮酶活性和土壤脲酶活性,降低了土壤固氮细菌（nifH）、氨氧化古菌（AOA）、氨氧化细菌（AOB）基因丰度,限制了植物-根际系统的生物固氮及有机氮素转化;接种Sinorhizobium sp.W16降解菌显著提高土壤中氟磺胺草醚的降解率至81.97%,且显著提高了大豆根瘤干质量、根瘤固氮酶活性和土壤nifH基因丰度,增强了大豆的固氮作用,同时刺激了脲酶活性,提升土壤AOA和AOB的基因丰度,增加了土壤有效氮素的供应,从而使大豆植株氮含量提高了15.85%~24.93%。研究表明,氟磺胺草醚的施用抑制了大豆-根际系统的生物固氮作用,但接种Sinorhizobium sp.W16降解菌不仅能有效降低土壤中氟磺胺草醚的残留量、缓解氟磺胺草醚对大豆的持续药害,还增强了植物-根际系统中生物固氮能力、土壤有效氮素供应及大豆的氮素累积,对修复氟磺胺草醚污染土壤、增强大豆固氮具有较好的应用价值和市场前景。     

Responses of ammonia-oxidizing bacteria and archaea to nitrogen fertilization and precipitation increment in a typical temperate steppe in Inner Mongolia

As the first and rate-limiting step of nitrification, ammonia oxidation can be realized either by ammonia-oxidizing bacteria (AOB) or archaea (AOA). However, the key factors driving the abundance, community structure and activity of ammonia oxidizers are still unclear, and the relative importance of AOA and AOB in ammonia oxidation is unresolved. In the present study, we examined the effects of long-term (6 years) nitrogen (N) addition and simulated precipitation increment on the abundance and community composition of AOA and AOB based on a field trial in a typical temperate steppe of northern China. We used combined approaches of quantitative PCR, terminal-restriction fragment length polymorphism (T-RFLP) and clone library analyses of amoA genes. The study objective was to determine (1) AOA and AOB diversity and activity in response to N addition and increased precipitation and (2) the relative contributions of AOA and AOB to soil ammonia oxidation in the typical temperate steppe. The results showed that the potential nitrification rate (PNR) increased with N addition, but decreased with increased precipitation. Both N addition and increased precipitation significantly increased AOB but not AOA abundance, and a significant correlation was only observed between PNR and AOB amoA gene copies. The T-RFLP analysis showed that both N and precipitation were key factors in shaping the composition of AOB, while AOA were only marginally influenced. Phylogenetic analysis indicated that all AOA clones fell within the soil and sediment lineage while all AOB clones fell within the Nitrosospira. The study suggested that AOA and AOB had distinct physiological characteristics and ecological niches. AOB were shown to be more sensitive to N and precipitation than AOA, and the ammonia oxidation process was therefore supposed to be mainly driven by AOB in this temperate steppe.     

共固定化亚硝化-反硝化细菌工艺及特性研究

以亚硝化细菌、反硝化细菌为研究对象,采用共固定化细胞技术,以海藻酸钠共固定化亚硝化-反硝化细菌,研究了共固定化工艺条件及其在模拟污水中的脱氮效果。结果表明,共固定化亚硝化-反硝化细菌最佳工艺条件为4.5%海藻酸钠和2.1%氯化钙共固定化细胞,接种量为3个/m L培养基,接种于装有140 m L模拟污水液体培养液的250 m L三角瓶中,最佳p H为8,最佳培养温度30℃,110~140 r/min培养。54 h时氨氮去除率为95.95%,78 h时亚硝态氮去除率为95.82%。共固定化小球可重复使用3次、低温对共固定化后菌种脱氮性能的影响较小。     

Improvement of media for thermophilic ammonia-oxidizing bacteria in compost

Thermophilic ammonia-oxidizing bacteria (AOB) have the potential to reduce ammonium emissions from commercial-scale animal waste composting operations. Two new media have been designed to culture and enumerate thermophilic AOB species. C-1 medium was designed to reflect compost composition. C-2 medium and CQ-2 medium were modified synthetic organic wastewater for the cultivation and enumeration of AOB, respectively. These new media have several advantages over the conventional medium. C-2 medium is effective for nitrification at 50°C, and C-1 and C-2 media support reasonable growth of thermophilic AOB. Furthermore, C-1 and CQ-2 media allow enumeration of thermophilic AOB during the composting process. This is the first report of the successful cultivation and enumeration of thermophilic AOB in compost. These results suggest the possibility of isolation and manipulation of novel thermophilic AOB species for environmental bioremediation.     

施肥和淹水管理对水稻土氨氧化微生物数量的影响

全程氨氧化细菌(Completeammoniaoxidizers,Comammox)的发现被认为是氮循环研究的重要进展,但复杂土壤中Comammox与氨氧化细菌(Ammonia-oxidizing bacteria, AOB)和氨氧化古菌(Ammonia-oxidizingarchaea,AOA)共存的环境驱动机制尚不清楚。针对紫色水稻土长期定位试验的植稻淹水(夏季植稻施肥并且全年淹水)管理、休耕冬干(不植稻、不施肥,仅在夏季植稻期间淹水,冬季排水落干)管理,研究了施肥和水分管理对水稻土硝化潜势及氨氧化微生物类群丰度的影响。结果表明,植稻淹水土壤的硝化潜势显著高于休耕冬干,分别为25.0 mg·kg~(-1)·d~(-1)、2.11mg·kg~(-1)·d~(-1),相差可达12倍之多。实时荧光定量PCR分析表明,两种管理方式下水稻土中均能检测到Comammox、AOA和AOB,并且其数量均为ComammoxAOAAOB。植稻淹水中Comammox丰度分别为AOA的8.5倍、AOB的77.3倍,而休耕冬干中Comammox丰度分别为AOA的4.1倍、AOB的490.3倍。相比于休耕冬干管理,植稻淹水刺激了Comammox分支A(Clade A)、AOA和AOB的生长,三者增长倍数分别为9、3、42,但Comammox分支B(Clade B)的降幅高达两倍之多。这些结果表明,与休耕冬干管理相比,28年长期植稻淹水,可能导致水稻土氨氧化微生物类群长期处于低O_2胁迫,并选择性促进了Comammox Clade A和AOA的生长,高强度施肥则显著促进了AOB生长,而Comammox Clade A和AOA则具有更广的铵态氮底物适应范围。未来应通过稳定性同位素示踪DNA技术,明确水稻土中数量上占优势的Comammox的功能意义及其与AOA和AOB的相对重要性。     

Chronic Nitrogen Fertilization Modulates Competitive Interactions Among Microbial Ammonia Oxidizers in a Loess Soil

Nitrogen(N) application may lead to niche segregation of soil ammonia-oxidizing archaea(AOA) and bacteria(AOB), thereby reducing the competitive interactions between AOA and AOB due to higher ammonium substrate availability. However, the adaptive mechanisms of AOA and AOB under N enrichment remain poorly understood. Stable isotope probing(SIP) microcosm incubation was employed to reveal community changes of active AOA and AOB in a loess soil from a field experiment growing potatoes that received no N(control, CK), low N(LN, 75 kg N ha~(-1)), and high N(HN, 375 kg N ha~(-1)). The results showed that the soil potential nitrification rate(PNR) was measured by culturing of the soil samples from the field experiment. Soil PNR was significantly increased in HN by87.5% and 67.5% compared with CK and LN, respectively. Compared with CK, the~(13)C-amoA genes of soil AOA and AOB in HN had 2.58 × 10~4 and 1.55 × 10~6 copies, representing 1.6-and 16.2-fold increase respectively. It was indicated that AOB dominated soil ammonia oxidation. A phylogenetic analysis of the~(13)C-amoA gene showed that N application significantly increased the proportion of54 d9-like AOA up to 90% in HN, while the Nitrososphaera gargensis-like and Nitrososphaera viennensis-like AOA were inhibited and completely disappeared. Nitrogen application also resulted in the community shift of active AOB-dominant group from Nitrosospira briensis-like to Nitrosospira sp. TCH711-like. Our study provides compelling evidence for the emergence and maintenance of active nitrifying communities under the intensified N input to an agricultural ecosystem.