不同二氧化碳浓度岩溶洞穴可培养细菌群落特征--以重庆雪玉洞和水鸣洞为例

Characteristics of culturable bacterial communities in karst caves with different CO2 concentrations:An example of the Xueyu cave and Shuiming cave in Chongqing

以重庆雪玉洞和水鸣洞为例,在测试地球化学参数的基础上,在不同CO2浓度条件下利用4种分离培养基,富集培养不同类型(洞穴沉积物及洞壁)样品可培养细菌并进行系统进化分析。结果表明:(1)水鸣洞可培养细菌丰度明显高于雪玉洞;(2)分离纯化得到的244株细菌基于16S rRNA基因序列比对分析发现,细菌主要由变形菌门(Proteobacteria)、放线菌门(Actinobacteria)、厚壁菌门(Firmicutes)、拟杆菌门(Bacteroidetes)与异常球菌-栖热菌门(Deinococcus-Thermus)构成。Bacteroidetes只存在于高CO2浓度的雪玉洞,推测Bacteroidetes能耐受高CO2浓度;(3)CO2浓度是影响岩溶洞穴细菌丰度、群落与活动的重要因素之一,推测雪玉洞和水鸣洞的δ13C-CO2值偏负可能是微生物与水文地球化学作用共同影响的结果。

Despite many reports on the excavation of bacterial resources in caves.There is still no detailed report on how the culturable bacteria in caves respond to different concentrations of environmental factors(especially CO2 concentration).The purpose of this work was to examine the community characteristics of culturable bacteria in karst caves with different CO2 concentrations and reveal the response of culturable bacteria to relevant geochemical indicators.In 2018,sediment and rock wall samples of karst caves were collected from the Xueyu cave and Shuiming cave with different CO2 concentrations in Chongqing.The gas collection bag was used to collect gas in the caves to measure CO2 concentration.Water samples from the surface of the sediments in the two holes were collected and packed in polyethylene plastic bottles for the determination of HCO3-.The rock wall and sediments of the two caves and water bodies were collected and packed in sterile bags for culture and geochemical index determination.Four kinds of isolation media were used to enrich and culture different types of bacteria,and phylogenetic analysis was carried out.The 16S rRNA gene sequence was amplified by PCR using 27F/1492r as the forward and reverse primers(27F:AGAGTTTGATCCTGGCT;1492r:GGTTACCTTGTTACGACTT).The amplification conditions were pre denaturation at 95℃ for 5 min,denaturation at 94℃ for 30 s,annealing at 52℃ for 30 s,extension at 72℃ for 1.5 min,35 cycles,extension at 72℃ for 10 min,and preservation at 12℃,respectively.Cluster x software was used for multiple sequence alignment,and the phylogenetic tree was constructed by MEGA5.0,and SPSS 25 software and Pearson correlation coefficient method were used to analyze the correlation between geochemical parameters and dominant OTUs.The results are as follows,(1)The abundance of culturable bacteria in the Shuiming cave is significantly higher than that in Xueyu cave.(2)Based on 16S rRNA gene sequence analysis,244 strains of bacteria are isolated and purified,which belong to 87 different OTUs.Culturable bacteria are mainly composed of Proteobacteria,Actinobacteria,Firmicutes,Bacteroidetes and Deinococcus-Thermus.Bacteroidetes only exist in the Xueyu cave with high CO2 concentration,then it is speculated that Bacteroidetes can tolerate high CO2 concentration.(3)CO2 concentration is one of the important factors that affect the bacterial abundance,community and activity in karst caves.It is speculated that the negative δ13C-CO2 values of the Xueyu cave and Shuiming cave may be the result of the interaction between microorganisms and hydrogeochemistry.These results suggest that high CO2 concentration inhibit most microbial abundance and diversity.The high concentration of CO2 may be attributed to reduction of the pH inside and outside the cells affecting the metabolic activity of bacteria and the bacterial enzyme system.However,some special cases exist,for example,the relative abundance of Bacteroides increases from 3.09% to 21.20%with the increase of CO2 leakage.The respiration and hydrogeochemistry of overlying soil can lead to the negative value of δ13C-CO2 in caves.