Geomicrobiology Of Karst Cave:Bacterial Community And Controls On Calcium Carbonate Formation

Karst speleothems?e.g.stalagmite and stalactite?are unique data sources for climate change research.The stable isotopes of oxygen and carbon in speleothems are very important proxies to trace paleoclimate and paleoenvironment.Especially,oxygen isotope has been used to reveal the paleoclimate changes in many studies.The variations of carbon isotope in cave sediments are complex and little is known about how microbial action on carbon isotope,e.g.its abnormality has been always found according to previous studies.It is necessary to study the biogeochemical processes of carbon isotope evolution in karst cave systems combined with the isotope information of microbial sediments.Because there are some problems using existing criteria for the identification of sediment carbon isotope fractionation as some microbial activities such as extracellular carbonic anhydrase?Extracellular carbonic anhydrase,CAex?may cause large deviation,affecting the accuracy of carbon isotope proxy in the process of the reconstruction of paleoenvironment.It is necessary to collect the isotopic information of the cave sediments under the action of microorganisms,and further to study the geochemical process of the carbon isotope evolution of the karst cave system.In this research,an underground river and dripping water of two caves were studied,called Xueyu Cave and Shuiming Cave,respectively,both located in Chongqing,southwest China.The bacteria that can deposit calcium carbonate were screened and investigated to study the distribution of microorganism calcium carbonate deposition,to clarify the mechanism and isotopic composition of carbonate minerals under the action of microbial carbonic anhydrase,so as to provide a scientific basis for revealing the deviation caused by microbial activity in the paleoclimate and paleoenvironmental changes based on the calcium carbonate carbon isotope information.In this study,samples of microorganisms were collected from the cave sediment surface and dripping water in these two caves.Samples of microorganisms were separated by a B-4 culture medium,a total of 12 strains that can produce solid crystals on the surface of the colony were selected from two caves.The 16 S rDNA gene sequences were obtained by sequencing the bacterial group DNA to determine the classification status on the NCBI website and apply for the login number?Acession No.?.It is identified that most of the bacteria belong to the Bacillus genus.To understand the type,proportion and distribution of bacteria that can deposit calcium carbonate in bacterial community compositions in the underground river and dripping water in two caves,filtered materials from the underground river and dripping water were collected and 16S rDNA gene sequences were obtained for high-throughput sequencing.In the underground river of two caves,Proteobacteria,represented mostly by ?-Proteobacterium and Bacteroidetes dominated in both cave systems,but bacterial community composition were different.In the dripping water of Xueyu Cave,Proteobacteria and Firmicutes were dominant,accouting for 81% and 13%,respectively.The ?-Proteobacterium and Bacillus were the predominant class.Acinetobacter,Pseudomonas spp.are prevalent bacteria,accouting for 65% and 27% in the ?-Proteobacterium,Streptococcus and Lactococcus are prevalent in bacteria,accouting for 55% and 14%,Anoxybacillus and Bacillus accouting for 12% in the Bacillus,respectively.The Lysinibacillus sp.strain LHXY2,which have been screened and identified,and with large amount of deposition,was selected for laboratory simulation.The physicochemical conditions of microbial deposition process from the perspective of microbial extracellular carbonic anhydrase were studied.The cultivation of adding extracellular enzyme of carbonic anhydrase inhibitor?acetazolamide,AZ?has been done in B-4 culture medium to study the deposit quantity,mineral composition and crystal morphology of calcium carbonate deposited by Lysinibacillus sp.strain LHXY2 at different AZ concentrations.The mineral composition and morphology of the microbially induced CaCO₃ crystals were analyzed using an X-ray diffraction analyzer?XRD?and a scanning electron microscope?SEM?,respectively.The results from laboratory show that AZ concentration was positively correlated cell culture medium p H and Ca2+?r=0.55,0.87?,but negatively correlated with deposit quantity?r=-0.81??It means that p H and Ca2+ concentration increased when AZ inhibited CA enzyme of LHXY2,indicating carbonic anhydrase plays an important role in the process of microbial induced calcium carbonate.In addition,there was an “dose effect” between AZ and CA enzyme,which affected mineral species and morphology.It formed rhombohedral calcite when AZ concentration<1 mmol/L but spherical vaterite and the mixture of irregular vaterite and calcite were formed when AZ concentration>1 mmol/L.Under different AZ concentrations,the morphologys of strain LHXY2 were like a funnel or petals and the signs from surface microbial effects were significant to prove that CA enzyme of LHXY2 had an important impact on the precipitation of calcite carbonate.The Lysinibacillus sp.strain LHXY2,which as the main strain,was selected for cave simulation.In order to expound the effects of microbes on CaCO₃ precipitation in the karst caves combining with the monitoring data and geological background,samples of microorganisms collected from the surface of the cave sediment in Xueyu cave.Additionally,the dynamic simulation process of cave dripping water was to performed study microbially induced CaCO₃ precipitation which will influence the primitive cave deposits.The feature and carbon isotope information of the primitive cave deposit crystals were investigated for that need.The simulations in laboratory and caves provided an evidence that microbial induced deposits of calcite carbonate were mixed with calcite and Mg calcite.Moreover,the pristine secondary sediments were mixed with low Mg calcite and high Mg calcite.It shows that the mineral composition of the cave sediment mainly comes from the bedrock,indicating that microbes run through the whole dripping process.The stable carbon isotope analysis showed that the carbonic anhydrase produced by Lysinibacillus sp.strain LHXY2 resulted in approximately-7‰ of carbon isotope deviation in the laboratory simulation,approximately-1.4‰ in the cave simulation.The analysis may be caused by the superposition of the cave environment parameters and the chemical background of the dripping water and the microorganisms in the open cave system.In this research,using Lysinibacillus sp.strain LHXY2 for the laboratory and cave simulation,it is the first time to explore the effect of microorganism on cave sediments carbon isotope fractionation from the perspective of extracellular carbonic anhydrase.The results indicate that microbial carbonic anhydrase can significantly cause the carbon isotope fractionation of calcium carbonate sediments.For example,Isotopic analysis in the laboratory showed that the carbon isotopic composition of crystals induced by CA of LHXY2 strains presented a gradually depleted trend companying with increased AZ concentration.Moreover,with the increase of the concentration of AZ,the CA of LHXY2 strains resulted in approximately-7‰ of the stable carbon isotope deviation in the laboratory simulation,of approximately-1.4‰ in the cave simulation,which could cause a large deviation in the reconstruction of paleoenvironment based on the cave speleothems carbon isotope composition,it provides a new idea for the interpretation of the paleoenvironment by carbon isotope composition of the cave sediments.