一株反硝化光合细菌的生物学特性及系统发育分析

摘要

[目的]养殖水体中亚硝酸盐的过量积累会对养殖生物产生毒害作用,应用脱氮细菌去除亚硝酸盐是养殖水质调控的重要手段之一,本文意在得到一株高效去除亚硝酸盐的光合细菌.[方法]采用软琼脂稀释法分离纯化光合细菌菌株,通过电镜观察、生理生化试验研究其生物学特性、依据16S rDNA和光合反应中心M亚基基因(Gene coding for photosynthetic reaction center subunit M,pufM)序列对其做系统发育分析.[结果]从淡水养殖塘中分离筛选到一株高效还原亚硝氮的光合细菌菌株wps.该菌株为革兰氏阴性菌,细胞杆状,大小为0.4-0.6μm×1.5-4.0 μm,极生丛生鞭毛,片层状光合内膜,兼性厌氧光照条件生长,单菌落及液体培养物呈红色,含细菌叶绿素a和类胡萝卜素.最适生长pH范围为5.5-8.5,最适生长盐度范围为0-2%,最适生长温度范围为25℃-38℃.菌株wps与Rhodopseudomonas palustris的16S rDNA序列相似性为98.9%,光合反应中心M亚基基因序列的相似性为94.9%,但是二者在生物学性质上有较大差异,如菌株wps在pH5.5生长,不能光自养生长,不利用柠檬酸盐、甲酸盐进行光异养生长,需盐酸硫胺素和泛酸钙做生长因子等.[结论]菌株wps可能为Rhodopseudomonas属的一个新种,且在养殖水体水质调控中具有重要应用前景.%[Objective]Nitrite accumulation in aquaculture water is toxic to reared animals.One of the solutions to this problem is to apply denitrifying bacteria.This paper is intended to get a strain of phototrophic bacteria for efficient removal of nitrite from aquaculture water.[Methods]We used soft agar to isolate and purify phototrophic bacteria.We investigated biological characteristics of the isolate by means of light and electronic observations, physical and chemical tests.We analyzed its phylogenetical position based on the sequences of 16S rDNA and the gene that codes for photosynthetic reaction center subunit M ( pufM).[Results]A photosynthetic bacterial strain, named wps, showing high removal efficiency of nitrite, was isolated from the freshwater ponds.Cells were Gram-negative, rod-shaped, slightly curved,0.4 -0.6 × 1.5 -4.0 μm,motile by means of polar multiple flagella.Intracellular membranes were of the lamellar type.It grew under facultative anaerobic conditions in the light with bacteriochlorophyll a and carotenoid of sppirilloxanthin series as photosynthetic pigment.The optimum growth was obtained at pH 5.5 - 8.5 , in a range of 0 - 2％ salinity and at 25 -38℃.The similarity of 16S rDNA between strain wps and Rhodopseudomonas palustris was 98.9％ and 94.9％ for pufM gene.However,there are significant differences between them in the morphological and physiological characteristics,i, e.grew at pH 5.5 ; no growth photoautotrophicaly with sodium hydrogen carbonate; could not utilize citrate or formate as only carbon source;required thiamine hydrochloride and calcium pantothenate as growth factors.[Conclusion]Strain wps may represent a novel species in genus Rhodopseudomonas and possibly find its application in the bioremediation of polluted aquaculture water.