明亮发光杆菌培养的响应面优化及其对常见金属离子的毒性评价研究  

Optimization of culture conditions of Photobacterium phosphoreum using response surface methodology and toxicity evaluation on metal ions

作　　者：王丹 周彦宏 白林明 延卫[1] 李珊珊[1] WANG Dan;ZHOU Yanhong;BAI Linming;YAN Wei;LI Shanshan(Department of Environmental Science&Engineering,School of Energy and Power Engineering,Xi’an Jiaotong University,Xi’an 710049)

机构地区：[1]西安交通大学能源与动力工程学院环境科学与工程系,西安710049

出　　处：《环境科学学报》2022年第5期454-464,共11页Acta Scientiae Circumstantiae

基　　金：国家重点研发计划项目(No.2019YFB2103003)。

摘　　要：随着人们对金属毒性认识的不断深入,对生物毒性检测方法的灵敏度提出了更高的要求.以发光度为响应值,通过响应面法对传统的明亮发光杆菌(Photobacteriumphosphoreum)培养基中主要组分的浓度进行了优化,得到了最佳培养基组成:氯化钠(25.00 g·L^(-1))、胰蛋白胨(4.010 g·L^(-1))、酵母浸出液(1.200 g·L^(-1))、甘油(2.475 g·L^(-1)),在此条件下培养14 h后,发光细菌的发光度达到1.7×10^(9)RLU,比优化前增加了1倍,菌浓度达到0.85,较优化前增加了5%.在优化培养基组分浓度的基础上,对其他条件(接种量、pH值、温度)进行了单因素发光度影响分析,发现在接种量1%(初始种子液OD595为0.003),测定环境22~25℃,样品pH值为6~8的条件下,利用优化培养基进行急性毒性测定基本不会影响发光度.随后,基于优化后的培养条件对常见金属离子的急性毒性进行了评价,结果表明:除Fe^(2+)、Zn^(2+)、K^(+)外,优化条件培养的明亮发光杆菌对其余金属离子的灵敏度都有所提高,其中对Cr^(6+)毒性的灵敏度提高最大,其检出限从1.0 mg·L^(-1)提高到0.15 mg·L^(-1),IC_(50)下降了47.13%(由7.818 mg·L^(-1)下降到4.133 mg·L^(-1)),能更好地满足不同行业对Cr^(6+)检出限的要求.本实验提出了一种通过优化培养基来提高金属离子毒性测定灵敏度的方法.Increasing awareness and concerns about the toxicity of metals is driving demand for a higher sensitivity biological toxicity detection method.In this study,the concentration of main components in traditional medium of Photobacterium phosphoreum was optimized by Response Surface Methodology(RSM)with the luminescent intensity as response value.The optimum medium composition was NaCl(25.00 g·L^(-1)),Tryptone(4.010 g·L^(-1)),yeast extract(1.200 g·L^(-1))and glycerin(2.475 g·L^(-1)).After incubation for 14 h in the optimum culture,the biomass(the value of OD595 was 0.85)was increased by 5%.Based on the optimization of the medium components,the influence of inoculation amount,temperature and pH value on luminescent intensity was carried out by single factor analysis.It was concluded that luminescent intensity would not be affected significantly under the conditions of 1%inoculation amount(the initial OD_(595) was 0.003),temperature of 22~25℃ and pH value of 6~8.Subsequently,the acute toxicity of common metal ions was evaluated in the optimized culture conditions,and the results showed that except Fe^(2+),Zn^(2+),K^(+),the sensitivity of P.phosphoreum to other heavy metal ions was improved,among which the sensitivity to Cr^(6+) increased most significantly with the detection limit increasing from 1.00 mg·L^(-1) to 0.15 mg·L^(-1) and IC_(50) decreasing from 7.818 mg·L^(-1) to 4.133 mg·L^(-1).The improved detection limit could better meet the requirements of different industries for the detection limit of Cr^(6+).In this study,a method to improve the toxicity sensitivity of common metals by optimizing the culture composition and condition was proposed.

关 键 词：六价铬 发光细菌 灵敏度 毒性测定 

分 类 号：X171.5[环境科学与工程—环境科学]