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降解高效氯氰菊酯乳酸菌的筛选及其在苜蓿青贮中的应用
Alternative TitleScreening of beta-cypermethrin degrading lactic acid bacteria and its application in alfalfa silage
刘芳
Subtype硕士
Thesis Advisor郭旭生
2021-05-22
Degree Grantor兰州大学
Place of Conferral兰州
Degree Name理学硕士
Degree Discipline微生物学
Keyword苜蓿青贮 降解 高效氯氰菊酯 乳酸菌 多样性
Abstract~高效氯氰菊酯(beta-cyp)又称为2,2-二甲基-3-(2,2-二氯乙烯基)环丙烷羧酸--氰基- (3-苯氧基)苄酯,戊酸氰醚酯,是一种农业杀虫剂,被广泛用于农业中蚜虫,蓟马,棉蚜虫等虫害的预防和控制,易溶于有机溶剂。但随着菊酯类农药的广泛使用,农药残留逐渐成为人们关注的热点,生物降解被认为是一种降解农药的有效方式。本文旨在研究乳酸菌对高效氯氰菊酯的降解。 以335株从全株玉米青贮中分离得到的乳酸菌为研究对象,经筛选后发现一株能够有效降解高效氯氰菊酯的菌株(编号为3-27)。经形态学观察及染色,该菌为革兰氏阳性杆菌。进一步对菌株3-27的16S rRNA基因序列进行了BLAST同源性比对及形态学观察,最终确定菌株3-27为戊糖乳杆菌。本论文研究首次报道了乳杆菌对高效氯氰菊酯的降解特性及其在降解青贮饲料农药残留方面的应用。论文研究结果如下。 1. 菌株3-27在pH3.0-9.0范围内均能对高效氯氰菊酯进行降解,其中在pH7.0条件下的降解效果最好。在不同温度的降解中发现,温度达到30℃时高效氯氰菊酯开始被降解,且降解能力随温度上升而增加,40℃的降解能力最强。该菌株在底物浓度为50mg/l-200mg/l的培养液中均能实现对高效氯氰菊酯的降解。将葡萄糖,蛋白胨,酵母粉分别加入无机盐培养基中均能增强菌株对底物的降解,在培养24h时,添加酵母粉的培养基中降解率为52%,高于其他添加组和未添加组。菌株3-27对高效氯氰菊酯的降解随菌株接种量(1%-5%)的增加而升高,在96h高达94%。 2. 菌株3-27对青贮不同天数苜蓿中高效氯氰菊酯的降解研究结果表明,该菌株对苜蓿青贮中高效氯氰菊酯的降解随发酵天数的延长而逐渐上升,且未添加戊糖乳杆菌和添加戊糖乳杆菌苜蓿样品中的高效氯氰菊酯含量均呈降低趋势。苜蓿在30%干物质(DM)条件下青贮时,添加菌株3-27的苜蓿青贮发酵60d时高效氯氰菊酯的降解率最高,达到57.23%。就发酵品质而言,添加农药不会对不同干物质苜蓿鲜样中的干物质造成影响,但会降低非蛋白氮含量。在30%DM条件下,添加农药处理组中粗蛋白含量显著高于对照组(P <0.001)。在30%和40%DM苜蓿青贮90d样品中,添加戊糖乳杆菌3-27苜蓿样品中的DM含量都高于未添加戊糖乳杆菌组,而氨态氮含量低于未添加戊糖乳杆菌样品。同时,在30%DM加菌处理组中丙酸含量显著降低(P <0.05),但乳酸和乙酸无显著变化;而在40%DM的加菌处理组中,乳酸和WSC含量增加。 3. 本试验采用实时定量单分子测序技术对苜蓿样品中的微生物多样性进行了研究。结果表明,苜蓿鲜样中的微生物多样性高于苜蓿青贮。对细菌门水平和属水平的分析中发现,厚壁菌门和变形菌门的数量明显多于其他门类微生物;在发酵3d的30%DM苜蓿青贮对照中主要由肠球菌属(31.79%),片球菌属(30.59%),乳杆菌属(18.03%),肠杆菌属(12.18%)及乳球菌属(3.76%)组成;随着发酵天数的延长,肠球菌属的相对丰度呈先降低后增加的变化趋势,片球菌属为主导微生物且在7、14、30、60和90 d的相对丰度分别为43.61%、82.68%、79.47%、74.76%和68.08%。接种戊糖乳杆菌的30%DM青贮中乳杆菌属呈显著优势,在青贮过程中其相对丰度变化范围为46.83% 到 95.55%, 其次为其他菌属(1.97%)及片球菌属(1.73%)。在40%DM苜蓿青贮中相对丰度超过1%的有片球菌属(43.61%),肠球菌属(12.64%),乳杆菌属(12.15%),乳球菌属(11.13%),泛菌属(8.16%),肠杆菌属(5.33%)和魏斯氏菌属(1.79%)。
Other Abstract~Beta-cypermethrin(beta-cyp), also known as Cyano(3-phenoxyphenyl)methyl 3-(2,2-dichloroethenyl)-2,2-dimethylcyclopropanecarboxylate and cyano ether valerate, is an agricultural insecticide widely used for the prevention and control of aphids, thrips, cotton aphids and other pests in agriculture. It is easily soluble in organic solvents. However, with the extensive use of pyrethroid pesticides, pesticide residues have become a hot topic of concern, and biodegradation is considered to be an effective way to degrade pesticides. The degradation of beta-cypermethrin by lactic acid bacteria was studied in this paper. With 335 strains of lactic acid bacteria isolated from whole corn silage as the research object, a strain (numbered 3-27) that could effectively degrade beta-cypermethrin was found after screening. Morphological observation and staining showed that the strain was Gram-positive. The 16S rRNA gene sequence of strain 3-27 was further analyzed by BLAST homology. Finally, strain 3-27 was identified as Lactobacillus pentosus. In this paper, the degradation of beta-cypermethrin by Lactobacillus and its application in the degradation of pesticide residues in silage were reported for the first time. The research results are as follows. 1. Strain 3-27 could degrade beta-cypermethrin in a large pH range, and the strain showed the strongest degradation ability at pH 7.0 after 72 h culture. Within the temperature range of 20℃–40℃, the strain could degrade beta-cypermethrin within the temperature range of 30℃–40℃, and the degradation ability increased with the temperature, and reached the highest at 40℃. The strain tolerated substrates at concentrations ranging from 50 mg/L to 200 mg/L and was not degraded at 250 mg/L. The addition of nutrients such as glucose, peptone, and yeast extract to the mineral salt medium could enhance the degradation of the substrate by the strain. When the strain was cultured for 24 h, the degradation rate in the medium with yeast extract was 52%, which was higher than that in other groups with or without yeast extract. The degradation of beta-cypermethrin by strain 3-27 increased with the increase of inoculation amount of the strain, and reached 94% at 96 h. 2. The degradation of beta-cypermethrin in alfalfa silage for different days was studied by strain 3-27. The results showed that the degradation of beta-cypermethrin in alfalfa silage increased gradually with the prolongation of fermentation days, and decreased slightly after 90 days of fermentation. During the silage process, the degradation of beta-cypermethrin in the non-inoculated control group was also changed along with the variation of fermentation days. The degradation rate of beta-cypermethrin in the 30% inoculated treatment was always the highest among the 30% and 40% dry matter and reached the highest at 60 days, 57.23%. Compared with the control without pesticide, the dry matter content of fresh alfalfa samples with different dry matter was not affected by pesticide addition, but the non-protein nitrogen content was decreased. At the same time, in 30% dry matter, the crude protein content of pesticide treatment group was significantly higher than that of control group (P <0.001). The chemical composition and fermentation quality of alfalfa silage were tested for 90 days. The results showed that the dry matter content in the addition group of strain 3-27 was higher than that in the non-addition group and the ammonia nitrogen content was lower than that in the control group at two different dry matter levels. Among them, propionic acid content was significantly reduced in the 30% dry matter inoculation group (P <0.05), while lactic acid and acetic acid showed no significant changes. The lactic acid and WSC contents were increased in the 40% dry matter inoculation group. 3. In this study, the microbial diversity in alfalfa samples was studied by real-time quantitative monomolecule sequencing. The results showed that the microbial diversity of fresh alfalfa samples was higher than that of silage alfalfa. The number of Firmicutes and Proteobacteria was significantly higher than that of other phyla at the level and genus level. In the control of 30% DM alfalfa silage fermented for 3 days, it was mainly composed of Enterococcus (31.79%), Pediococcus (30.59%), Lactobacillus (18.03%), Enterobacter (12.18%) and Lactococcus (3.76%). With the prolongation of fermentation days, the relative abundance of Enterococcus was firstly decreased and then increased, and the relative abundance of Pediococcus was the dominant microorganism, and the relative abundance at 7, 14, 30, 60 and 90 days were 43.61%, 82.68%, 79.47%, 74.76% and 68.08%, respectively. In silage inoculated with 30%DM of Lactobacillus pentosus, the relative abundance of Lactobacillus varied from 46.83% to 95.55% during silage, followed by other genera (1.97%) and Pediococcus (1.73%). In 40% DM alfalfa silage, the relative abundance of more than 1% in 40% dry matter alfalfa was in the genera Pediococcus (43.61%), Enterococcus (12.64%), Lactobacillus (12.15%), Lactococcus (11.13%), Panbacteria (8.16%), Enterobacter (5.33%) and Weissella (1.79%).
Pages72
URL查看原文
Language中文
Document Type学位论文
Identifierhttps://ir.lzu.edu.cn/handle/262010/459410
Collection生命科学学院
Affiliation
生命科学学院
Recommended Citation
GB/T 7714
刘芳. 降解高效氯氰菊酯乳酸菌的筛选及其在苜蓿青贮中的应用[D]. 兰州. 兰州大学,2021.
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