刺参池塘低温有机污染物降解细菌的分离筛选及其降解特性

Isolation,selection and characterization of low-temperature organic-pollutant-degrading bacteria in sea cucumber ponds

利用刺参(Apostichopus japonicus)饵料培养基,在低温(16±1)℃下,从刺参养殖池塘环境中富集驯化分离有机污染物低温降解细菌;根据菌株生长情况、好氧性和生物安全性进行了初步筛选,然后在菌体接种密度1×106 CFU/mL、(16±1)℃、pH 8.0、160 r/min条件下,通过检测菌株对饵料中有机物(COD)和氨态氮(NH4+-N)成分的去除率来最终筛选获得优良低温有机污染物降解细菌,并对其降解特性进行研究。菌株筛选结果表明,从分离的17株低温细菌中共获得3株菌株DR7、DR8和DR11,它们能同时高效降低和去除饵料浸出物中COD和NH4+-N,5 d COD去除率分别为37.7%、50.0%和21.0%,NH4+-N去除率分别为95.0%、98.0%和99.8%。根据菌株的形态特征、生理生化特性以及16S rRNA序列分析,初步鉴定DR7和DR11属于假单胞菌属细菌(Pseudomonas sp.),DR8属于副球菌属细菌(Paracoccus sp.)。菌株降解特性研究结果表明,菌株培养时间对饵料降解有显著影响(P<0.05),在0～6 d内3菌株对饵料中有机物和氨态氮的去除过程明显,其中DR8降解力最强;各组合菌株的饵料降解效果差异显著(P<0.05),但并不完全优于对照单菌株,其中最佳组合为DR8-DR11。研究认为,筛选的3株低温有机污染物降解细菌为良好的菌种资源,在刺参养殖池塘污染环境调控及刺参微生态制剂的研制方面具有较好的潜在应用前景。

Low-temperature organic-pollutant-degrading bacteria were isolated from sea cucumber （Apostichopus japonicus） outdoor rearing ponds after enriching the liquid sea cucumber feed-medium and acclimating at （16± 1）℃. All of the bacterial strains were preliminarily selected by evaluating their aerobic growth on sea cucumber feed-medium and verifying their specificity for the sea cucumber. The bacteria were finally selected by evaluating their ability to degrade organic pollutants （COD） and ammonia nitrogen （NH4＋-N） in the feed-medium, with an inoculation of 1.0× 106 cfu/mL. Culture conditions were （16±1）℃, pH 8.0 and a shaking rate of 160 r/min. After screening of 17 bacterial isolates, three bacterial strains （DR7, DR8 and DR11） were selected. These strains were able to degrade the feed over 5 d with COD removal rates of 37.7%, 50.0% and 21.0%, and NHa＋-N removal rates of 95.0%, 98.0% and 99.8%, respectively. From morphological and physio-biochemical analyses, and 16S rRNA sequence similarity analysis, strains DR7 and DR11 were identified as Pseudomonas sp., and strain DR8 as Para- coccus sp. The time course of degradation of the feed was observed by examination of the concentrations of the organic pollutants and ammonia-nitrogen in the liquid sea cucumber-feed-medium between 0 d and 6 d. Bacterial cultivation time had a significant effect on the feed-degrading activities of the three strains （P〈0.05）. Based on the dynamics of feed degradation, strain DR8 was considered to be the most effective of the three strains. Significant differences （P〈0.05） were also observed in the combined feed-degrading capacities of mixtures of the strains. The optimal combination was considered to be DR8 ＋ DRI 1. However, the overall feed-degrading abilities of the combined strains were not much better than that of the single strain. We conclude that the 3 low-temperature or- ganic-pollutant-degrading bacteria screened in this study are useful bacterial resources, which could show good potential for pollution control in sea cucumber culture ponds and the development of probiotics for sea cucumber culture.