水质净化乳酸菌的分离鉴定及发酵参数优化

Isolation, identification and fermentation optimization of lactic acid bacteria for aquaculture water purification

摘要:

[目的]从水产养殖环境和养殖生物体中选育具有水质净化功能的乳酸菌，以期为水产养殖提供专用高效的菌种资源。[方法]在低温和常温条件下从皮皮虾、南美白对虾肠道及养殖池底质活性污泥中分离具有水质净化功能的乳酸菌，对筛选的优良菌株采用形态、生理生化实验及16S rRNA序列分析进行鉴定，并对菌株的发酵参数进行了研究。[结果]低温和常温条件下从3种介质中共分离到乳酸菌136株，经水质净化能力筛选，发现常温分离的r13对模拟水体中亚硝态氮去除效果较强，72 h能将11.5 mg/L的亚硝态氮彻底去除，且对13.0 mg/L氨氮的去除率达到29.1%。经形态特征、生理生化特性及16S rRNA基因序列分析，鉴定菌株r13为植物乳杆菌Lactobacillus plantarum。发酵参数研究结果表明，该菌最适培养基组成为：酵母膏6.0 g/L，葡萄糖20.0 g/L，乙酸钠4.0 g/L，柠檬酸氢二铵2.0 g/L，K₂HPO₄ 2.0 g/L，番茄汁50 mL/L；培养条件为：初始pH 6.0，接种量5%，装液量45/50，培养温度为34℃；在上述优化培养条件下发酵72 h，菌液的生物量达28.4 g/L湿重，有效活菌浓度达4.4×10⁹ CFU/mL。

Abstract:

[Objective] In order to get excellent strains for aquiculture water purification, we screened lactic acid bacteria from the aquaculture environment and intestinal tract of shrimp.[Methods] The potential water purification ability of lactic acid bacteria at normal and low temperature was evaluated in the simulated wastewater. Morphological physio-biochemical characteristics, 16S rRNA gene sequence analysis were used to identify strain r13. Single factor test and orthogonal-design experiment were applied to optimize fermentation for r13.[Results] In total 136 lactic acid bacteria strains were isolated from 3 samples. The results of water purification test suggested r13 had higher removal ability of nitrite and ammonia from water. After 72 h treatment by r13, nitrite with 11.5 mg/L in the water was completely removed and ammonia degradation rate was 29.1% with 13.0 mg/L original concentration. According to morphological, physio-biochemical characteristics and 16S rRNA gene sequence analysis, r13 was identified as Lactobacillus plantarum. The optimal fermentation condition for r13 was 6.0 g/L yeast extract, 20.0 g/L glucose, 4.0 g/L sodium acetate, 2.0 g/L diammonium hydrogen citrate, 2.0 g/L monopotassium phosphate, 50 mL tomato juice, with inoculation rate 5% (V/V), at pH 6.0 and 34℃. Under this condition cultured for 72 h, the bacterial biomass reached 28.4 g/L wet weight and cell counting reached 4.4×10⁹ CFU/mL.[Conclusion] Considering high nitrite removal ability, we suggested that r13 would be promising microorganism for water purification in aquiculture.