一株稻田土壤梭状芽孢杆菌的分离及其固氮产氢能力研究

Isolation of Clostridium Strain from Rice Paddy Soil and Its Synergistic Nitrogen Fixation and Hydrogen Production Ability

为研究水稻土壤中协同固氮产氢菌的固氮产氢特性,拓展协同固氮产氢菌菌种资源,利用厌氧微生物富集培养、亨盖特厌氧滚管等技术,从华南稻田土壤中筛选到一株同时具有产氢和固氮能力的菌株BZ-1.经形态学观察及16S rRNA基因序列系统发育分析,鉴定菌株BZ-1属于梭状芽孢杆菌属(Clostridium sp.).通过测定产氢量、生物量、发酵产物及固氮酶酶活等,对菌株BZ-1的产氢能力及协同固氮产氢特性进行分析.结果表明:菌株BZ-1发酵34.55 mmol/L葡萄糖可产生42.19 mmol/L H_(2),主要副产物为丁酸(15.96 mmol/L)、乙酸(7.14 mmol/L)和乳酸(5.09 mmol/L);菌株BZ-1具有固氮酶活性,能够以N_(2)为唯一氮源进行生长;菌株BZ-1固氮产氢时,相比于添加7 mmol/L氯化铵的试验组,产氢量提高了14.71%,最大生物量降低了33.33%,乙酸产量提升了61.49%.研究显示,菌株BZ-1在协同固氮产氢条件下固氮能力的提升、生物量的降低以及核心碳代谢途径的改变可能是其产氢量提升的原因.固氮产氢菌株BZ-1的获得将为提高土壤肥力以及缓解铅、镉等重金属对农作物的胁迫作用提供新的思路.

In order to explore the nitrogen-fixing and hydrogen-producing characteristics of bacteria in paddy soil and to expand their resources,a strain BZ-1 with simultaneous hydrogen production and nitrogen fixation ability was isolated from paddy soil in South China by anaerobic microbial enrichment and Hungate tube rolling technology.The strain was identified as Clostridium sp.by morphological observation and phylogenetic analysis based on 16S rRNA gene sequence.The hydrogen production capacity and simultaneous nitrogen fixation and hydrogen production characteristics of strain BZ-1 were analyzed by determining hydrogen production,nitrogenase activity,fermentation products and biomass.The results show strain BZ-1 fermented 34.55 mmol/L glucose to produce 42.19 mmol/L hydrogen,and the main by-products were butyric acid(15.96 mmol/L),acetic acid(7.14 mmol/L)and lactic acid(5.09 mmol/L).Strain BZ-1 exhibited nitrogenase activity and was able to grow with N_(2) as the sole nitrogen source.Under the nitrogen fixation condition,the hydrogen production of strain BZ-1 increased by 14.71%,the maximum biomass decreased by 33.33%,and the acetic acid yield increased by 61.49%compared with the experimental group supplemented with 7 mmol/L NH_(4)Cl.These results indicated that there was a synergistic effect between nitrogen fixation and hydrogen production by strain BZ-1.The increase of nitrogen fixation capacity,decrease of biomass and change of core carbon metabolic pathway of strain BZ-1 might be responsible for the increase in hydrogen production under nitrogen fixation condition.Obtaining nitrogen-fixing and hydrogen-producing strain BZ-1 will provide new ideas for improving soil fertility and alleviating heavy metal stress in crops.