单细胞水平微流体技术筛选北极沉积物中的联苯降解菌

Isolation of biphenyl-degrading bacteria in arctic sediments by single-cell level isolation of microfluidics(SLIM)technology

极地环境微生物具有独特而丰富的多样性,是亟待开发的资源宝库.随着人类影响范围的扩大,极地环境中持久性有机污染物与石油资源开采等造成的环境污染不容忽视,而利用土著微生物进行生物修复是一种理想的方法.为克服传统的基于富集驯化的稀释平板法分离降解菌存在耗时长、筛选结果单一等缺点,建立了一种单细胞水平微流体筛选(Single-cell Level Isolation with Microfluidics,SLIM)技术.以北极沉积物为菌源,以联苯为底物,利用该技术成功筛选得到9株菌株,分属于4个菌属:Streptomyces、Micrococcus、Dermacoccus和Aspergillus;通过稀释平板法筛选得到3株菌株,分属于Acidovorax、Chryseobacterium和Nocardia.通过两种方法得到的菌株完全不同.系统发育分析表明,SLIM技术筛选得到的菌株具有更加丰富的微生物多样性.通过对沉积物及各代富集驯化培养物的16S rRNA基因高通量测序发现,并非所有富集的菌属都能被稀释平板法分离,通过SLIM技术筛选得到的菌属在所有样品中的相对丰度都很低.本文建立的SLIM技术从单细胞水平实现了目标菌种的筛选,相比于稀释平板法,具有效率更高的优点,同时避免了菌种间竞争等造成的分离困难.本研究为极地环境中有机污染的生物修复及“微生物暗物质”资源的挖掘提供了新的思路与方法.

Microbial community in arctic regions owns unique and abundant biodiversity.It is a tremendous and copious reservoir of microbial resources.With the expanding impacts induced by human activities,pollution caused by persistent organic pollutants and excavation of petroleum in polar regions should not be ignored.Utilizing indigenous bacteria to perform bioremediation seems to be an ideal method.The traditional dilution-plate method based on enrichment and acclimatization for organic pollutant degrader isolation is often time-consuming and results in limited number of cultures.To overcome these disadvantages,the technology Single-cell Level Isolation with Microfluidics(SLIM)was developed.With the help of this technology,by choosing biphenyls as sole carbon source,we successfully isolated nine strains from arctic sediments,which belong to four genera Streptomyces,Micrococcus,Dermacoccus and Aspergillus.By performing the dilution-plate method,three strains were obtained,belonging to Acidovorax,Chryseobacterium and Nocardia,respectively.Strains isolated by two methods were completely different.By phylogenetic analysis,it was found that cultures obtained by SLIM owned a higher microbial diversity.16S rRNA gene high-throughput sequencing of all samples indicated that not all enriched genera could be obtained by diluted-plate method.It was also found that genera isolated by SLIM had a very low relative abundance in all samples.The SLIM technology used in this study makes isolating target bacteria from a single-cell level possible.Compared with the dilution-plate method,it is more effective.Most importantly,it avoids microbial competition during enrichment.This study provides illuminating insights into the bioremediation of organic pollution and the mining of“microbial dark matter”.