In the classical microbial isolation technique,the isolation process inevitably destroys all microbial interactions and thus makes it difficult to culture the many microorganisms that rely on these interactions for su...In the classical microbial isolation technique,the isolation process inevitably destroys all microbial interactions and thus makes it difficult to culture the many microorganisms that rely on these interactions for survival.In this study,we designed a simple coculture technique named the“sandwich agar plate method,”which maintains microbial interactions throughout the isolation and pure culture processes.The total yield of uncultured species in sandwich agar plates based on eight helper strains was almost 10-fold that of the control group.Many uncultured species displayed commensal lifestyles.Further study found that heme was the growth-promoting factor of some marine commensal bacteria.Subsequent genomic analysis revealed that heme auxotrophies were common in various biotopes and prevalent in many uncultured microbial taxa.Moreover,our study supported that the survival strategies of heme auxotrophy in different habitats varied considerably.These findings highlight that cocultivation based on the“sandwich agar plate method”could be developed and used to isolate more uncultured bacteria.展开更多
Many marine bacteria are difcult to culture because they are dormant,rare or found in low-abundances.Enrichment culturing has been widely tested as an important strategy to isolate rare or dormant microbes.However,man...Many marine bacteria are difcult to culture because they are dormant,rare or found in low-abundances.Enrichment culturing has been widely tested as an important strategy to isolate rare or dormant microbes.However,many more mechanisms remain uncertain.Here,based on 16S rRNA gene high-throughput sequencing and metabolomics technology,it was found that the short-chain fatty acids(SCFAs)in metabolites were signifcantly correlated with uncultured bacterial groups during enrichment cultures.A pure culture analysis showed that the addition of SCFAs to media also resulted in high efciency for the isolation of uncultured strains from marine sediments.As a result,238 strains belonging to 10 phyla,26 families and 82 species were successfully isolated.Some uncultured rare taxa within Chlorobi and Kiritimatiellaeota were successfully cultured.Amongst the newly isolated uncultured microbes,most genomes,e.g.bacteria,possess SCFA oxidative degradation genes,and these features might aid these microbes in better adapting to the culture media.A further resuscitation analysis of a viable but non-culturable(VBNC)Marinilabiliales strain verifed that the addition of SCFAs could break the dormancy of Marinilabiliales in 5 days,and the growth curve test showed that the SCFAs could shorten the lag phase and increase the growth rate.Overall,this study provides new insights into SCFAs,which were frst studied as resuscitation factors in uncultured marine bacteria.Thus,this study can help improve the utilisation and excavation of marine microbial resources,especially for the most-wanted or key players.展开更多
Microorganisms are ubiquitous in the ocean environment and they play key roles in marine ecosystem function and service.However,many of their functions and phenotypes remain unknown because indigenous marine bacteria ...Microorganisms are ubiquitous in the ocean environment and they play key roles in marine ecosystem function and service.However,many of their functions and phenotypes remain unknown because indigenous marine bacteria are mostly difficult to culture.Although many novel techniques have brought previously uncultured microbes into laboratory culture,there are still many most-wanted or key players that need to be cultured from marine environments.This review discusses possible reasons for‘unculturable microbes’and categorizes uncultured bacteria into three groups:dominant active bacteria,rare active bacteria,and dormant bacteria.This review also summarizes advances in cultivation techniques for culturing each group of unculturable bacteria.Simulating the natural environment is an effective strategy for isolating dominant active bacteria,whereas culturomics and enrichment culture methods are proposed for isolating rare active bacteria.For dormant bacteria,resuscitation culture is an appropriate strategy.Furthermore,the review provides a list of the most-wanted bacteria and proposes potential strategies for culturing these bacteria in marine environments.The review provides new insight into the development of strategies for the cultivation of specific groups of uncultured bacteria and therefore paves the way for the detection of novel microbes and their functions in marine ecosystems.展开更多
基金supported by the National Natural Science Foundation of China(Nos.32070002 and 41876166)Science&Technology Fundamental Resources Investigation Program(Grant Nos.2022FY101100 and 2019FY100700)+1 种基金China Postdoctoral Science Foundation(2022M721923)Natural Science Foundation of Shandong Province,China(ZR2023QD187).
文摘In the classical microbial isolation technique,the isolation process inevitably destroys all microbial interactions and thus makes it difficult to culture the many microorganisms that rely on these interactions for survival.In this study,we designed a simple coculture technique named the“sandwich agar plate method,”which maintains microbial interactions throughout the isolation and pure culture processes.The total yield of uncultured species in sandwich agar plates based on eight helper strains was almost 10-fold that of the control group.Many uncultured species displayed commensal lifestyles.Further study found that heme was the growth-promoting factor of some marine commensal bacteria.Subsequent genomic analysis revealed that heme auxotrophies were common in various biotopes and prevalent in many uncultured microbial taxa.Moreover,our study supported that the survival strategies of heme auxotrophy in different habitats varied considerably.These findings highlight that cocultivation based on the“sandwich agar plate method”could be developed and used to isolate more uncultured bacteria.
基金This work was supported by the National Natural Science Foundation of China(41876166)Natural Science Foundation of Shandong Province,China(Grant No.ZR2022QD126)Science and Technology Fundamental Resources Investigation Program(Grant No.2022FY101100,2019FY100700).
文摘Many marine bacteria are difcult to culture because they are dormant,rare or found in low-abundances.Enrichment culturing has been widely tested as an important strategy to isolate rare or dormant microbes.However,many more mechanisms remain uncertain.Here,based on 16S rRNA gene high-throughput sequencing and metabolomics technology,it was found that the short-chain fatty acids(SCFAs)in metabolites were signifcantly correlated with uncultured bacterial groups during enrichment cultures.A pure culture analysis showed that the addition of SCFAs to media also resulted in high efciency for the isolation of uncultured strains from marine sediments.As a result,238 strains belonging to 10 phyla,26 families and 82 species were successfully isolated.Some uncultured rare taxa within Chlorobi and Kiritimatiellaeota were successfully cultured.Amongst the newly isolated uncultured microbes,most genomes,e.g.bacteria,possess SCFA oxidative degradation genes,and these features might aid these microbes in better adapting to the culture media.A further resuscitation analysis of a viable but non-culturable(VBNC)Marinilabiliales strain verifed that the addition of SCFAs could break the dormancy of Marinilabiliales in 5 days,and the growth curve test showed that the SCFAs could shorten the lag phase and increase the growth rate.Overall,this study provides new insights into SCFAs,which were frst studied as resuscitation factors in uncultured marine bacteria.Thus,this study can help improve the utilisation and excavation of marine microbial resources,especially for the most-wanted or key players.
基金This work was supported by the National Natural Science Foundation of China(41876166,and 31770002)Science and Technology Basic Resources Investigation Program of China(2017FY100302).
文摘Microorganisms are ubiquitous in the ocean environment and they play key roles in marine ecosystem function and service.However,many of their functions and phenotypes remain unknown because indigenous marine bacteria are mostly difficult to culture.Although many novel techniques have brought previously uncultured microbes into laboratory culture,there are still many most-wanted or key players that need to be cultured from marine environments.This review discusses possible reasons for‘unculturable microbes’and categorizes uncultured bacteria into three groups:dominant active bacteria,rare active bacteria,and dormant bacteria.This review also summarizes advances in cultivation techniques for culturing each group of unculturable bacteria.Simulating the natural environment is an effective strategy for isolating dominant active bacteria,whereas culturomics and enrichment culture methods are proposed for isolating rare active bacteria.For dormant bacteria,resuscitation culture is an appropriate strategy.Furthermore,the review provides a list of the most-wanted bacteria and proposes potential strategies for culturing these bacteria in marine environments.The review provides new insight into the development of strategies for the cultivation of specific groups of uncultured bacteria and therefore paves the way for the detection of novel microbes and their functions in marine ecosystems.