铁还原菌株P4的碳源利用特征及其系统发育学分析

Characterization of iron reduction using different carbon sources and phylogenetic analysis of Fe(Ⅲ)-reducing microorganism P4

【目的】从水稻土中分离铁还原微生物,研究不同碳源对其铁还原特征的影响,对分离的铁还原微生物进行系统发育学分析。【方法】对采自四川的水稻土进行厌氧恢复培养,通过PTYG选择性夹层培养基分离纯化及LB液体培养基扩繁后,接种到Fe（OH）3培养基中进行铁还原能力鉴定,筛选出具有铁还原功能的铁还原菌株。通过对6种碳源利用的纯培养比较,确定分离菌株利用不同碳源时的铁还原特征。采用通用引物进行PCR扩增,对PCR产物进行16S rDNA序列分析,建立系统发育进化树。【结果】分离得到1株具有铁还原功能的革兰氏阳性杆状厌氧菌株P4,其在培养的10-30 h达到对数生长期。菌株P4利用不同碳源时,铁还原的最大反应速率（Vmax）大小顺序为：葡萄糖〉丙酮酸盐〉乳酸盐〉琥珀酸盐〉丙酸盐〉乙酸盐。菌株P4利用葡萄糖和丙酮酸盐的Fe（Ⅲ）还原率分别为63.79%和22.19%,而其他几种碳源的铁还原率均在10%以下。采用PCR技术获得了1 325 bp的铁还原菌株P4的部分16S rDNA序列。菌株P4与多种不可培养的厌氧菌株及丁酸梭状芽孢杆菌具有98%的同源性。【结论】从四川水稻土中分离得到了G^＋、杆状、圆末端的铁还原菌P4,其在培养10-30 h能够达到对数生长期。葡萄糖和丙酮酸盐可作为菌株P4的优势碳源。菌株P4可归属为厌氧丁酸梭状芽胞杆菌。

【Objective】 Fe（Ⅲ）-reducing microorganism was separated from paddy soil,the effect of strain on Fe（OH）3 reduction using different carbon sources was analyzed and physiological characterization of strain was also obtained.【Method】 The soil solution collected from Sichuan paddy soil was incubated under anoxic atmosphere.Through separation and purification from PTYG selective medium and multiplication from LB medium,and then identification of the function of Fe（Ⅲ） reduction in Fe（OH）3 medium,the Fe（Ⅲ）-reducting organism was isolated.Through comparison of effects on carbon utilization with six carbon sources in pure culture,the characteristic of strain on Fe（Ⅲ） reduction using different carbon sources was investigated.With the PCR and 16S rDNA sequence analysis,the phylogenetic tree was established.【Result】 An anaerobic,G＋,rod-shaped organism P4 with function of Fe（Ⅲ） reduction was isolated.It attained a logarithms growth period within 10~30 h.With different carbon sources,the maximal reaction rate（Vmax） of iron reduction was in proper order for：glucose〉pyruvate〉lactate〉succinate〉propionat〉acetate.With glucose and pyruvate as carbon source,iron reduction rate were 63.79% and 22.19%,the others were all below 10%,displaying glucose and pyruvate be the optimal carbon source of strain P4.The adoption of the PCR acquired parts of 16S rDNA sequences of Fe（Ⅲ）-reducting strain P4 with 1 325 bp.【Conclusion】 A G＋,rod-shaped Fe（Ⅲ）-reducting organism P4 was isolated from Sichuan paddy soil.It attained a logarithms growth period within 10~30 h.Glucose and pyruvate were the optimal carbon sources of strain P4.Homology analysis and phylogenetic comparison of strain P4 and relative strains based on 16S rDNA showed that strain P4 belonged to Clostridium butyricum,most to several uncultured bacterium and Clostridium butyricum with 98% similarity.