污染河流底泥亚铁氧化硝酸盐还原菌分离及代谢特性

Isolation and characterization of metabolic activity of Fe(Ⅱ)-oxidizing denitrifying bacteria from anaerobic river sediments

采用常规分离培养方法,从富含亚铁厌氧还原态底泥中分离得到多株亚铁氧化硝酸还原菌,并从中选取1株:FX-Fe5菌株进行详细分析研究.经革兰氏染色、光学显微镜及扫描电镜观察,该菌为革兰氏染色阴性长杆菌,经16Sr RNA测序分析确定为:拉乌尔菌属种类(Raoultella sp.).利用亚铁作为单独电子供体和亚铁与不同比例乙酸作为共同电子供体反硝化反应液,研究了FX-Fe5菌株对亚铁、硝酸盐氮和有机物的代谢特点.结果表明,FX-Fe5在24h内可对亚铁进行较大幅度的去除,去除率(%)分别为:89.77±0.47a(亚铁与乙酸盐比例1:1)、87.01±0.22b(亚铁与乙酸盐比例3:1)、86.96±0.07b(仅亚铁)、72.97±0.53c(亚铁与乙酸盐比例1:3),高于文献报道大部分纯菌的亚铁氧化率;亚铁和乙酸盐为共同电子供体时,乙酸和亚铁在硝酸还原过程中同步减少;重复反应的结果发现FX-Fe5菌株在不提供乙酸盐时,不能持续地利用亚铁进行硝酸还原反应.FX-Fe5菌株利用亚铁对硝酸还原过程中没有出现明显的亚硝酸盐氮和氨的积累,各反应体系反应气体中都含有N2O的存在,所占比例都不高,最高的情况不超过0.5%(5000×10-6),但不同电子供体间差别非常大.利用Biology对碳源代谢特点分析表明,FX-Fe5菌株但对单糖/糖苷/聚合糖类优先利用且利用程度较高.

Several Fe（II）-oxidizing denitrifying bacteria （FODBs） were isolated from iron-rich anaerobic river sediments using the conventional cultivation method, and one of the FODBs named strain FX-FeSwas selected for further study. Based on the observation of gram staining, optical microscope and scanning electron microscope, and identification by 16s rRNA sequencing, strain FX-Fe5 was as gram-negative bacillus and was most closely related to Raoultella sp., which was the first time to be reported as FODBs. Strain FX-Fe5could efficiently oxide ferrous iron within 24h, when ferrous and acetic acid were supplied as electron donor separately or jointly. The oxidation rates of FeⅡ （%） in different electron donor systems were 89.77±0.47（ratio of ferrous to acetate was 1：1）, 87.01±0.22 （ratio of ferrous to acetate was 3：1）, 86.96±0.07 （ferrous only） and 72.97±0.53（ratio of ferrous to acetate was 1：3）, respectively. The oxidation rates of Fe（Ⅱ） were all higher than those of pure isolates reported by other studies. In addition, the concentration of ferrous and acetic acid decreased simultaneously, when both of them were supplied as electron donor during the process of denitrification. However, the Fe oxidation process was limited when ferrous was used as a single electron. Furthermore, during the denitrification process driven by strain FX-Fe5, only N2O gas was observed, without nitrite and ammonium accumulation in the reaction system. Otherwise, the highest content of N2O gas was less than 0.5% （5000×10^-6）, and varied largely with different electron donor systems. Carbon source metabolism analysis using Biolog EcoplateTM confirmed that strain FX-Fe5 had a higher preference to use monosaccharide, indican and oolvmerization sugars as carbon source.