Microbial Diversity In Major Oil-Shale Mines In China

China has abundant oil-shale resources.Bacteria play a crucial role in the long-term bio-transformations of oil-shale.Nevertheless,very little is known about the true profile of oil-shale microbial resources in China.This study mainly aims to investigate the microbial dominant phyla and genera in major oil-shale mines in China by clone library,polymerase chain reaction-denaturing gradient gel electrophoresis(PCR-DGGE),fluorescence in situ hybridization(FISH),diluted plate culture and Biolog technique,and discuss the influence of environmental factors on the microbial diversity.Three oil-shale mines in China were chosen for the microbial diversity investigation:Fushun Mine in Liaoning Province,Huadian Mine in Jilin Province and Maoming Mine in Guangdong Province.One bacterial clone library and 1 fungal clone library for each mine were respectively constructed by clone library method.Three types of samples were collected from each mine for PCR-DGGE and FISH analysis,including sandy soil samples,freshly mined oil-shale samples and weathered oil-shale samples or underground sandstone samples.Clone library and PCR-DGGE analysis indicated that the main phyla of bacterial or fungal communities were similar but a distinct difference of proportions in different communities.The genus composition had a significant difference between any two mines.Bacterial communities of the three mines were composed of seven phyla:Proteobacteria(Alpha-,Beta-,Gamma-),Firmicutes,Actinobacteria,Acidobacteria,Bacteroidetes,Deinococcus-Thermus and Chloroflexi except unidentified bacteria.Most identified genera were closely affiliated with hydrocarbon-degrading bacteria.Bacterial diversity in the three mines was generally high,but relatively lower in Maoming mine.Phyla Proteobacteria,Firmicutes and genus Pseudomona were common and dominant in three mines.Relatively,the compositions of fungal communities were uncomplicated.Ascomycota was common and dominant in the three mines,but a small number of Basidiomycota only appeared in Fushun and Huadian libraries.The results also indicated that microbial adaptation in oil-shale mines were most possibly driven by a combination of oil-shale characteristics and environmental factors.The relative abundances of bacteria and archaea of Huadian,Maoming and Fushun mine were analyzed by FISH method.It turned out that,bacteria were a dominant group in each sample due to their relative abundance of more than 50%,while the relative abundance of archaea was below 5%far lower than that of bacteria.In each mine,bacterial or archaeal relative abundances of freshly mined oil-shale samples were the highest among all type of samples.There was a difference in the relative abundances of bacteria or archaea between any two mines.Bacteria in Fushun Mine samples were far lower than in Huadian Mine samples,while archaea were just the reverse.In consideration of organic matter characteristics of oil-shale,the relative abundances of bacteria or archaea were independent of the types of kerogen or ratio of O to C.Culturable microorganisms in oil-shale samples from Huadian,Maoming and Fushun mine were separated and purified respectively by dilution plate culture method.Five,5 and 9 bacterial strains were affiliated with Pantoea,Brevibacillus,Paenibacillus,Microbacterium,Arthrobacter,Pseudomonas,Bacillus,Sinomonas,Staphylococcus and Acinetobacter.Eleven,5 and 6 fungal strains were affiliated with Debaryomyces,Coprinopsis,Rhodotorula,Eucasphaeria,Aspergillus,Sporidiobolus,Nectria,Bionectria,Candida,Penicillium,Eupenicillium,Cladosporium' Alternaria,Cryptococcus as well as 3 unidentified fungi.In the beginning of this research,extraction of microbial total DNA,primers of PCR-DGGE and experimental conditions of FISH for oil-shale samples were optimized.First,5 methods(SDS-high-salt extraction,SDS-grinding-in-liquid-nitrogen extraction,SDS repeated freeze-thaw extraction,SDS guanidinium isothiocyanate extraction and kit extraction)were chosen to extract total DNA,showing that the improved SDS-high-salt extraction with simple operation and meeting the following molecular experiments was suitable for samples.Next,four-set primers(968F/1401R,338F/518R,341F/907R and 1055F/1406R)of 16S rDNA high variable target regions(V6-V8,V3,V8 and V9)were used for PCR-DGGE,showing that 968F/1401R and 338F/518R were more suitable due to higher richness index and richer kinds of genera.Finally,optimum conditions for FISH were determined by comparison experiments,showing that the hybridization rate increased obviously through the TRIzol/lysozyme co-processing in pretreatment with hybridization temperature of 42?,hybridization time of 2.5 h and deionized formamide concentration of 20%.At late stage of the study,functional diversity of carbon-source metabolism of oil-shale microorganisms in Fushun Basin Western Open Group was investigated by Biolog-GN2 and Biolog-GP2 microplates method.The results showed that the metabolic level and metabolic rate of microbial communities utilizing different carbon sources were obviously different.For the Gram-negative or Gram-positive bacteria communities,the utilization of carbon sources with amino group was the highest,followed by carboxylic acid,sugar and its derivatives,and sugar phosphates was the lowest.The analyses of Shannon-Wiener,Simpson and McIntosh indexes through multi-point calculation indicated that the carbon metabolic diversity of microbial communities in oil-shale was generally high,but the diversity slightly decreased with incubation time.