节杆菌发酵培养基的优化及其对海水养殖水体的脱氮作用

发布时间：2018-06-11 12:57

  本文选题：节杆菌 + 筛选鉴定　； 参考：《河北大学》2017年硕士论文

【摘要】：在现代集约化高密度养殖模式下,养殖过程中大量的残饵,粪便排泄物,动植物尸体等含氮有机物污染严重,因此本实验主要以氨氮和亚硝态氮降解能力为主要筛选指标进行目的菌株的筛选。本实验获得一株异养硝化好氧反硝化细菌,并对其进行了脱氮性能的测试、菌种的鉴定、发酵培养基及培养条件的优化,分析了菌株对凡纳滨对虾(Litopenaeus vannamei)在养殖使用中的安全性以及在凡纳滨对虾养殖池塘的脱氮作用。研究主要结果如下:1.异养硝化好氧反硝化细菌菌株的筛选与鉴定将实验室保存的10株菌株进行异养硝化培养48 h,得到4株氨氮和总氮降解效果较好的菌株,然后将4株菌株分别在反硝化培养基A和反硝化培养基B中进行反硝化培养,其中菌株7-3在48 h的降解效果最好,48 h硝态氮去除率为47.37%,亚硝态氮去除率为100.00%。经形态特征、生理生化、16SrRNA序列分析和系统发育树比较,对菌株进行了菌种鉴定,菌株7-3为迈索尔节杆菌(Arthrobacter mysorens)。经生长特性研究表明,菌株7-3的0~6 h处于迟缓期,6 h后进入对数生长期,33 h后菌体达到最大生长量,进入稳定期,48 h后细菌进入衰亡期。2.节杆菌7-3液体发酵培养基配方及条件优化以蔗糖蛋白胨培养基作为基础培养基,采用单因子试验和L9(34)正交试验优化了节杆菌7-3种子培养基的组成,并对其接种量、发酵温度、转速、初始pH值和装液量等发酵条件进行了优化,得到优化后的发酵培养基配方为:玉米粉1%,酵母浸膏1%,K2HPO4 0.2%,KH2PO4 0.2%。确定了最佳培养条件:接种量为7%,发酵温度为25℃,转速为220 r/min,初始p H值8.0,装液量为100 m L。经发酵培养基及发酵条件的优化后,发酵液活菌数达到4.04×1010 CFU/mL。3.菌株对凡纳滨对虾的安全性实验将三株菌的菌液浓度分别设置为104 CFU/mL、106 CFU/mL、108 CFU/m L三个浓度梯度添加到5 L凡纳滨对虾幼虾养殖容器中,观察7 d对虾的死亡情况,结果表明,菌株F1508在水体中的含菌量为108 CFU/m L浓度时,对虾的累计死亡率与对照组无显著差异;菌株7-3和菌株683在水体中的含菌量为106 CFU/mL浓度时,对虾的累计死亡率与对照组无显著差异。菌株F1508在108 CFU/mL浓度以下使用时和菌株7-3和菌株683在含菌量为106 CFU/mL浓度以下在对虾养殖使用时是安全的。4.菌株在对虾养殖水体中对氨氮、亚硝态氮的脱氮作用将复合菌制剂应用到凡纳滨对虾海水养殖池塘。试验周期15 d,每天采样检测氨氮、亚硝态氮的浓度。实验结果表明复合菌剂对养殖水体中的氨氮和亚硝态氮均有一定的降解作用,氨氮最大去除率达到80.31%,亚硝态氮最大去除率达到85.58%,对养殖水体pH的影响均不显著,p H值波动范围在8.1~8.3。
[Abstract]:In the modern intensive high-density culture model, a large number of residual bait, feces, animal and plant carcasses and other nitrogen-containing organic pollutants are seriously polluted during the breeding process. Therefore, the main screening indexes were ammonia nitrogen and nitrite nitrogen degradation ability. In this experiment, a heterotrophic nitrifying aerobic denitrifying bacteria was obtained, and its denitrification performance, strain identification, fermentation medium and culture conditions were optimized. The safety of the strain to Litopenaeus vannamei) in culture and the denitrification in the pond of Penaeus vannamei) were analyzed. The main results are as follows: 1. Screening and identification of heterotrophic aerobic denitrifying bacteria 4 strains with better degradation of ammonia nitrogen and total nitrogen were obtained by heterotrophic nitrification culture of 10 strains stored in laboratory for 48 h. Then, four strains were cultured in denitrification medium A and denitrification medium B, respectively. The best degradation rate of strain 7-3 was 47.37% and 100.00g% respectively at 48 h. Based on the morphological characteristics, physiological and biochemical analysis of 16s rRNA sequence and comparison of phylogenetic tree, the strain 7-3 was identified as Arthrobacter mysorens. The growth characteristics of strain 7-3 were studied. It was found that the bacteria of strain 7-3 reached the maximum growth rate after entering the logarithmic growth stage for 33 h after 6 h of slow growth and 48 h after entering the stable phase. The formulation and conditions of the liquid fermentation medium of Arthrobacter ganglii 7-3 were optimized by using sucrose peptone medium as the basic medium, and the composition of the medium was optimized by single factor test and L9N 34) orthogonal experiment. The inoculation amount and fermentation temperature of the medium were optimized. The fermentation conditions, such as rotation speed, initial pH value and liquid volume, were optimized. The optimized fermentation medium was obtained as follows: corn powder 1, yeast extract 1 K2HPO 4 0.2 and KH 2PO 4 0.2. The optimum culture conditions were determined as follows: inoculation amount was 7, fermentation temperature was 25 鈩，

本文编号：2005326