土壤铅修复菌的鉴定及原生质体融合技术选育
发布时间：2018-08-28 09:12
【摘要】：土壤铅污染已严重威胁人类的健康,利用微生物修复铅是目前治理土壤铅污染的方法之一,原生质体融合技术可以选育出优良的菌种。因此本研究将一株耐铅能力强的菌(CGMCC13007)和一株修复土壤铅污染能力强的菌(CGMCC5515)进行原生质体融合,目的是筛选出同时耐铅能力强和修复土壤铅能力强的融合子。本论文首先对这两株菌进行菌种鉴定:通过培养特征与菌体形态观察、生理生化特性鉴定和遗传特性鉴定综合分析出两株菌分别为蜡样芽胞杆菌(Bacillus cereus)和枯草芽孢杆菌(Bacillus subtilis)。然后确定原生质体的制备及融合条件:溶菌酶浓度为4mg/mL,酶解温度为36℃,酶解时间为60min,PEG 6000浓度为36%,融合条件为冰浴5min,原生质体形成率可分别达68%和73%,再生率分别达23.73%和58.23%。融合子的筛选条件为:初筛用含1mg/L庆大霉素、1mg/L利福平和1mg/L 2,3,5-氯化三苯基四氮唑(TTC)的高渗再生培养基,复筛用含1800mg/L铅的高渗再生培养基。最终获得四株融合子。将四株融合子接种至含1mg/L庆大霉素、1mg/L利福平、1mg/L TTC和1800mg/L铅的营养琼脂平板上,但均未传代成功,原因有可能这四株融合子均为异核体。最后确定了白菜种子发芽的最佳土壤含铅浓度为800mg/kg,发芽率为50%;用这两株菌分别浸种后在含铅土壤上的发芽率分别为65%和78.5%;石墨炉原子吸收法分析出两株混合菌使含铅500mg/kg土壤一年后降低到46.48mg/kg,使土壤达到国家标准《GB15618-1995》中土壤对铅的二级标准要求。
[Abstract]:Soil lead pollution has been a serious threat to human health. Microbial remediation of lead is one of the methods to control soil lead pollution. Good strains can be selected by protoplast fusion technology. The aim of somatic fusion is to screen Fusants with high tolerance to lead and strong ability to repair soil lead. Firstly, the two strains were identified: Bacillus cereus and Bacillus cereus, respectively, were identified by culture characteristics and morphological observation, physiological and biochemical characteristics identification and genetic characteristics identification. Bacillus subtilis. Then the conditions of protoplast preparation and fusion were determined: lysozyme concentration was 4mg/mL, enzymatic hydrolysis temperature was 36, enzymatic hydrolysis time was 60 min, PEG 6000 concentration was 36%, fusion condition was 5 min in ice bath, protoplast formation rate was 68% and 73%, regeneration rate was 23.73% and 58.23%, respectively. In order to obtain four fusants, four Fusants were inoculated into the medium containing 1 mg/L gentamicin, 1 mg/L rifampicin, 1 mg/L TTC and 1 800 mg/L lead. The optimum concentration of Pb in soil for seed germination of Chinese cabbage was 800mg/kg and the germination rate was 50%. The germination rates of the two strains were 65% and 78.5% respectively after soaking in the soil containing Pb. Two mixed bacteria were isolated by graphite furnace atomic absorption spectrometry. The content of Pb in 500 mg/kg soil decreased to 46.48 mg/kg after one year, which made the soil meet the secondary standard of Pb in the national standard.
【学位授予单位】：河北经贸大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：X53;X172
，
本文编号：2208946
[Abstract]:Soil lead pollution has been a serious threat to human health. Microbial remediation of lead is one of the methods to control soil lead pollution. Good strains can be selected by protoplast fusion technology. The aim of somatic fusion is to screen Fusants with high tolerance to lead and strong ability to repair soil lead. Firstly, the two strains were identified: Bacillus cereus and Bacillus cereus, respectively, were identified by culture characteristics and morphological observation, physiological and biochemical characteristics identification and genetic characteristics identification. Bacillus subtilis. Then the conditions of protoplast preparation and fusion were determined: lysozyme concentration was 4mg/mL, enzymatic hydrolysis temperature was 36, enzymatic hydrolysis time was 60 min, PEG 6000 concentration was 36%, fusion condition was 5 min in ice bath, protoplast formation rate was 68% and 73%, regeneration rate was 23.73% and 58.23%, respectively. In order to obtain four fusants, four Fusants were inoculated into the medium containing 1 mg/L gentamicin, 1 mg/L rifampicin, 1 mg/L TTC and 1 800 mg/L lead. The optimum concentration of Pb in soil for seed germination of Chinese cabbage was 800mg/kg and the germination rate was 50%. The germination rates of the two strains were 65% and 78.5% respectively after soaking in the soil containing Pb. Two mixed bacteria were isolated by graphite furnace atomic absorption spectrometry. The content of Pb in 500 mg/kg soil decreased to 46.48 mg/kg after one year, which made the soil meet the secondary standard of Pb in the national standard
【学位授予单位】：河北经贸大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：X53;X172
，
本文编号：2208946
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