镉及四环素污染土壤耐性菌株的筛选及其根际促生能力研究

Screening of resistant strains from soil contaminated with cadmium and tetracycline hydrochloride and their rhizosphere growth promotion ability

重金属和抗生素是土壤中关注度渐高的两类典型污染物,其在土壤环境中很可能形成各种形式和不同浓度构成的复合污染物,而复合污染物的生物积累和联合毒性在相互作用下具有更大的危害。因此,为修复被重金属和抗生素污染的土壤,采用含重金属镉(CdCl 2)和抗生素盐酸四环素的培养基,从土壤中初筛出重金属镉和盐酸四环素的抗性菌,接着利用一系列不同浓度梯度的重金属和抗生素对抗性菌进行复筛,分离纯化出高耐性菌株,并同步检测高耐性菌株的根际促生能力。最后,针对高耐性菌和根促生能力强的菌株进行16S rRNA测序以及系统发育树,进一步鉴定优势菌种。研究表明:筛选得到的8株耐性菌(编号为B1,B2,…,B8),其中B8耐受镉浓度高达1 500 mg/L,B4在盐酸四环素浓度达到3 000 mg/L时仍能大量生长;在促生能力检测中,B1、B2有较强的产生长素能力,尤其B3的产生长素和产氨能力皆较显著,B6利用柠檬酸盐的能力最为突出;经鉴定B4、B8两种优势耐性菌株分别为粘质沙雷氏菌(Serratia marcescens)和蜡样芽孢杆菌(Bacillus cereus),根促生能力较强的B2、B3分别为弗氏柠檬酸杆菌、恶臭假单胞菌。筛选出的优势耐性菌B4和B8以及根促生能力较强的菌株B2和B3的联合使用将在土壤重金属和抗生素复合污染的微生物-植物修复技术方面有良好的应用前景。

Heavy metals and antibiotics with increasing attention are two kinds of typical pollutants in the soil environment and are likely to form compound pollutants in a variety of forms and concentrations.The compond pollutants are of biological accumulation and joint toxicity interaction and have greater harms.Therefore,for the repair of soil polluted by heavy metals and antibiotics,medium containing heavy metal cadmium(CdCl 2,Cd)and tetracycline antibiotics hydrochloric acid(TET)was used to screen the resistant bacteria of Cd and TET from the soil.Then,a series of different concentration gradient of Cd and TET was used to re-screen,isolate and purfy the high-tolerant strains.The growth-promoting ability of these strains was also investigated.Finally,16S rRNA sequence and phylogenetic tree were performed on strains with high tolerance and strong ability of root growth to further identify the dominant strains.The results showed that all the screened eight strains(number as B1,B2,…,B8)were Gram-positive.Among them,B8 could tolerate more than 1 500 mg/L of Cd and B4 could still grow in large quantities when TET reached 3 000 mg/L.In the growth-promoting ability test,B1 and B2 had strong ability to produce auxin,B3 had significant ability to produce auxin and ammonia,and B6 had the most outstanding ability to use citrate.Two dominant resistant strains B2,B3,B4 and B8 were identified as Citrobacter freundii,Pseudomonas putida,Serratia marcescens and Bacillus cereus,respectively.The combination of dominant tolerant bacteria B4 and B8,and strains B2 and B3 with strong root-promoting ability screened in this experiment will have a good prospect in the application of microbial-phytoremediation technology of soil with combined contamination of heavy metal and antibiotic.