Effects Of Microaerobic Condition On Bacterial Isolation And Evaluation Cultivability Of Microorganisms Based On High-throughput Sequencing

A large number of molecular ecology studies directly on environmental samples show that the vast majority of microorganisms in nature have not been isolated and cultured in the laboratory.They are called uncultured microorganisms or microbial dark matter.On the one hand,through literature research and preliminary experiments,we proposed that microaerobic condition(below 1%oxygen concentration)is an important factor that may affect the inability of many microorganisms to grow.On the other hand,we plan to collect all culturable microorganisms by means of scrapping cultural plate and based on 16S rRNA gene high-throughput sequencing technology to effectively detect these microbial populations to evaluate microbial cultivability under different culture conditions.In this paper,microorganisms were isolated from activated sludge samples and cultured under three oxygen concentration conditions(aerobic condition,oxygen concentration of approximately 0.05%as microaerobic condition,and anaerobic condition),in order to study the effect of microaerobic condition on bacterial isolation and evaluate microbial cultivability on cultural plate.The main research results show that:(1)By adding internal control,improving data process,and eliminating cross talking contamination among samples generating by sequencing,it is proved that the research of this paper is methodologically feasible,and the experimental results are reliable.(2)A total of 904 zOTUs were detected in the activated sludge samples(uncultured).464 zOTUs of those were detected on all our culture plates.The number of zOTUs detected in aerobic,microaerobic,and anaerobic condition were 327,409,and 361,respectively.From the perspective of the number of zOTUs,we evaluate microbial cultivability under the tested activated sludge samples and culture conditions as high as 51.3%.Suggesting that there are a large number of culturable microorganisms on the culture plate were ignored,which may be due to their low abundance or difficult to pick in routine separation experiment.We believe that the method of evaluate microbial cultivability based on the number of zOTUs can better reflect the truth whether microorganisms can be isolated and cultured.It has been considered in the past that microbial cultivability from the perspective of cell number is less than 1%,and more than 99%of the microorganisms cannot be culturable is likely to be an overestimate of the seriousness for the problem of uncultured microorganisms.(3)1959 zOTUs were detected among all the 48 scrapping cultural plate samples,of which 809 zOTUs were identified as potential new taxa at different rank of classification.Among these 809 zOTUs,the number of zOTUs were detected in aerobic,microaerobic,anaerobic condition was 434,601,and 487,respectively(and the new species obtained under microaerobic conditions were the most).In addition,any microbial dark matter at Phylum lever was not detected on the cultural plates,but and 1,6,5,45,and 90 new taxa were identified at the rank of Class,Order,Family,Genus,Species classification,respectively.(4)Analyzing Principal Coordinate Analysis(Pcoa)of the original activated sludge and cultural plate samples,it was found that the microbial populations cultured under microaerobic or anaerobic conditions are closer to the microbial community under the real environment of the original activated sludge.At the same time,in situ medium used as the culture medium was similar to the original activated sludge sample,indicating that microaerobic or anaerobic and insitu nutrient conditions are more suitable for the separation of certain specific microorganisms.Microbial populations in activated sludge may be more inclined to microaerobic and anaerobic metabolism.(5)In the separation experiment by Petri dish,12 strains of pure culture with 16S rRNA gene similarity less than 95%were isolated.Under microaerobic conditions,potential rank of new families,new genera,and new species level classification microorganisms were isolated 1,3,and 1,respectively.Based on our research,evaluating cultivability of microorganisms from the perspective of species(zOTU)is a more reasonable method for assessing microbial cultivability.And prompting that the reconsideration of the seriousness for the problem of uncultured microorganisms.Compared with aerobic and anaerobic conditions,many new microbial populations can be cultured under microaerobic conditions,suggesting that a considerable number of environmental microorganisms have a special adaptability to oxygen.Although this study has not yet cultured microbial dark matter at Phylum level,microbial dark matter at Class level and below Class level have been harvested,and many microorganisms have been cultured,which were consider to be uncultured microorganisms in previous study.Therefore,the use of microaerobic conditions for the isolation and culture of bacteria and based on high-throughput sequencing technology to identify the culturable bacteria is expected to be applied to other natural and artificial environment samples,to provide more ideas and confidence in the isolation of microbial dark matter.