一株铜抗性细菌的分离鉴定及其耐铜机制

Isolation and identification of a Cu-resistant bacterial strain and its Cu resistance mechanism

利用微生物修复铜污染环境是当前研究的热点之一,筛选铜污染环境生物修复的菌种,研究其耐铜机制对铜污染土壤修复具有重要的意义.从铜矿附近的土壤中分离得到一株具有较强铜抗性的细菌,经形态观察、生理生化实验及系统发育分析,鉴定该菌株为不动杆菌属,命名为Acinetobacter sp. MA9.为探索MA9对铜的耐性、富集及其可能机制,研究不同浓度铜处理下菌株的生长和细菌对培养基铜的去除效率,同时分析铜胁迫下MA9胞外聚合物的含量、菌体表面形貌和官能团的变化.结果表明,铜处理细菌36 h后,以培养基铜浓度表征的半数效应浓度(EC_(50))值为251mg/L,细菌对溶液铜的去除量最高达到了溶液铜浓度的68%.与对照相比,铜胁迫使得MA9产生了更多的胞外聚合物,其中多糖增加62%,蛋白增加185%;利用扫描电镜观察发现,与无铜处理的菌株相比,铜处理菌体表面存在大量颗粒物;能谱分析也显示,MA9细胞表面检测到铜,这说明铜可能与细菌代谢物结合产生胞外吸附;傅里叶红外光谱分析结果表明,菌株在铜处理后细胞表面与醛基官能团相关的吸收峰消失,说明铜主要和菌体表面的醛基官能团结合.本研究表明胞外吸附和胞外沉淀是不动杆菌MA9主要的耐铜机制,醛基是参与MA9和铜反应的主要基团;结果可为MA9在铜污染环境修复中的应用提供数据和基础.(图6表2参36)

Microbial remediation of copper (Cu) pollution is one of the key research topics at present and has received a great deal of attention. Screening bacterial strains that can remediate a Cu contaminated environment and uncoupling the underlying Cu resistance mechanisms are of great significance for engineering practices to clean up Cu polluted soils. A bacterial strain with strong Cu resistance was isolated from soil near a Cu mine. This bacterium was identified and named Acinetobacter sp. MA9, based on morphological, physiological, and biochemical experiments, as well as 16S rRNA gene sequence analysis. To explore the Cu tolerance, enrichment, and the possible resistance mechanism, MA9 growth and its Cu removal efficiency from the culture medium were studied under different Cu treatments. Variations of the extracellular polymeric substances (EPS), surface morphology, and functional groups of MA9 under Cu stress were also analyzed. The results showed that the concentration for 50% of maximal effect EC50 value of the MA9 treated with Cu for 36 h was 251 mg/L, and the highest Cu removal rate was 68% of the Cu concentration in solution. Compared with the control, Cu stress resulted in more EPS production by MA9, in which polysaccharide increased by 62% and protein increased by 185%. From analysis with a scanning electronic microscope, a large number of particles were formed on the surface of the bacterial cells treated with Cu. Energy dispersive spectrometry analysis also showed that Cu was detected on the surface of MA9 cells, suggesting that Cu might be bound extracellularly with bacterial metabolites. Fourier transform infrared spectroscopy demonstrated that the absorption peaks related to aldehyde functional groups disappeared after Cu treatment, indicating that these groups on the cell surface were involved in Cu binding. In conclusion, extracellular adsorption and precipitation may be the main Cu resistance mechanism of Acinetobacter sp. MA9. The aldehyde group may be the major group involved in the reaction of MA9 and Cu. The results provide a foundation for the application of MA9 in the remediation of Cu polluted environments.