【摘要】：土壤鉛污染已嚴(yán)重威脅人類的健康,利用微生物修復(fù)鉛是目前治理土壤鉛污染的方法之一,原生質(zhì)體融合技術(shù)可以選育出優(yōu)良的菌種。因此本研究將一株耐鉛能力強(qiáng)的菌(CGMCC13007)和一株修復(fù)土壤鉛污染能力強(qiáng)的菌(CGMCC5515)進(jìn)行原生質(zhì)體融合,目的是篩選出同時耐鉛能力強(qiáng)和修復(fù)土壤鉛能力強(qiáng)的融合子。本論文首先對這兩株菌進(jìn)行菌種鑒定:通過培養(yǎng)特征與菌體形態(tài)觀察、生理生化特性鑒定和遺傳特性鑒定綜合分析出兩株菌分別為蠟樣芽胞桿菌(Bacillus cereus)和枯草芽孢桿菌(Bacillus subtilis)。然后確定原生質(zhì)體的制備及融合條件:溶菌酶濃度為4mg/mL,酶解溫度為36℃,酶解時間為60min,PEG 6000濃度為36%,融合條件為冰浴5min,原生質(zhì)體形成率可分別達(dá)68%和73%,再生率分別達(dá)23.73%和58.23%。融合子的篩選條件為:初篩用含1mg/L慶大霉素、1mg/L利福平和1mg/L 2,3,5-氯化三苯基四氮唑(TTC)的高滲再生培養(yǎng)基,復(fù)篩用含1800mg/L鉛的高滲再生培養(yǎng)基。最終獲得四株融合子。將四株融合子接種至含1mg/L慶大霉素、1mg/L利福平、1mg/L TTC和1800mg/L鉛的營養(yǎng)瓊脂平板上,但均未傳代成功,原因有可能這四株融合子均為異核體。最后確定了白菜種子發(fā)芽的最佳土壤含鉛濃度為800mg/kg,發(fā)芽率為50%;用這兩株菌分別浸種后在含鉛土壤上的發(fā)芽率分別為65%和78.5%;石墨爐原子吸收法分析出兩株混合菌使含鉛500mg/kg土壤一年后降低到46.48mg/kg,使土壤達(dá)到國家標(biāo)準(zhǔn)《GB15618-1995》中土壤對鉛的二級標(biāo)準(zhǔn)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 .
【學(xué)位授予單位】：河北經(jīng)貿(mào)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：X53;X172
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本文編號：2208946

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