Influence of Substrate Concentration on the Culturability of Heterotrophic Soil Microbes Isolated by High-Throughput Dilution-to-Extinction Cultivation
ABSTRACT The vast majority of microbes inhabiting oligotrophic shallow subsurface soil environments have not been isolated or studied under controlled laboratory conditions. In part, the challenges associated with isolating shallow subsurface microbes may persist because microbes in deeper soils are...
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| Format: | Article |
| Language: | English |
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American Society for Microbiology
2020-02-01
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| Series: | mSphere |
| Subjects: | |
| Online Access: | https://journals.asm.org/doi/10.1128/mSphere.00024-20 |
| _version_ | 1818394927172681728 |
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| author | Ryan P. Bartelme Joy M. Custer Christopher L. Dupont Josh L. Espinoza Manolito Torralba Banafshe Khalili Paul Carini |
| author_facet | Ryan P. Bartelme Joy M. Custer Christopher L. Dupont Josh L. Espinoza Manolito Torralba Banafshe Khalili Paul Carini |
| author_sort | Ryan P. Bartelme |
| collection | DOAJ |
| description | ABSTRACT The vast majority of microbes inhabiting oligotrophic shallow subsurface soil environments have not been isolated or studied under controlled laboratory conditions. In part, the challenges associated with isolating shallow subsurface microbes may persist because microbes in deeper soils are adapted to low nutrient availability or quality. Here, we use high-throughput dilution-to-extinction culturing to isolate shallow subsurface microbes from a conifer forest in Arizona, USA. We hypothesized that the concentration of heterotrophic substrates in microbiological growth medium would affect which microbial taxa were culturable from these soils. To test this, we diluted cells extracted from soil into one of two custom-designed defined growth media that differed by 100-fold in the concentration of amino acids and organic carbon. Across the two media, we isolated a total of 133 pure cultures, all of which were classified as Actinobacteria or Alphaproteobacteria. The substrate availability dictated which actinobacterial phylotypes were culturable but had no significant effect on the culturability of Alphaproteobacteria. We isolated cultures that were representative of the most abundant phylotype in the soil microbial community (Bradyrhizobium spp.) and representatives of five of the top 10 most abundant Actinobacteria phylotypes, including Nocardioides spp., Mycobacterium spp., and several other phylogenetically divergent lineages. Flow cytometry of nucleic acid-stained cells showed that cultures isolated on low-substrate medium had significantly lower nucleic acid fluorescence than those isolated on high-substrate medium. These results show that dilution-to-extinction is an effective method to isolate abundant soil microbes and that the concentration of substrates in culture medium influences the culturability of specific microbial lineages. IMPORTANCE Isolating environmental microbes and studying their physiology under controlled conditions are essential aspects of understanding their ecology. Subsurface ecosystems are typically nutrient-poor environments that harbor diverse microbial communities—the majority of which are thus far uncultured. In this study, we use modified high-throughput cultivation methods to isolate subsurface soil microbes. We show that a component of whether a microbe is culturable from subsurface soils is the concentration of growth substrates in the culture medium. Our results offer new insight into technical approaches and growth medium design that can be used to access the uncultured diversity of soil microbes. |
| first_indexed | 2024-12-14T06:08:59Z |
| format | Article |
| id | doaj.art-801e4ab8f441446f941be4752124f2af |
| institution | Directory Open Access Journal |
| issn | 2379-5042 |
| language | English |
| last_indexed | 2024-12-14T06:08:59Z |
| publishDate | 2020-02-01 |
| publisher | American Society for Microbiology |
| record_format | Article |
| series | mSphere |
| spelling | doaj.art-801e4ab8f441446f941be4752124f2af2022-12-21T23:14:12ZengAmerican Society for MicrobiologymSphere2379-50422020-02-015110.1128/mSphere.00024-20Influence of Substrate Concentration on the Culturability of Heterotrophic Soil Microbes Isolated by High-Throughput Dilution-to-Extinction CultivationRyan P. Bartelme0Joy M. Custer1Christopher L. Dupont2Josh L. Espinoza3Manolito Torralba4Banafshe Khalili5Paul Carini6Department of Environmental Science, University of Arizona, Tucson, Arizona, USADepartment of Environmental Science, University of Arizona, Tucson, Arizona, USADepartment of Environment and Sustainability, J. Craig Venter Institute, La Jolla, California, USADepartment of Environment and Sustainability, J. Craig Venter Institute, La Jolla, California, USADepartment of Environment and Sustainability, J. Craig Venter Institute, La Jolla, California, USADepartment of Ecology and Evolutionary Biology, University of California, Irvine, California, USADepartment of Environmental Science, University of Arizona, Tucson, Arizona, USAABSTRACT The vast majority of microbes inhabiting oligotrophic shallow subsurface soil environments have not been isolated or studied under controlled laboratory conditions. In part, the challenges associated with isolating shallow subsurface microbes may persist because microbes in deeper soils are adapted to low nutrient availability or quality. Here, we use high-throughput dilution-to-extinction culturing to isolate shallow subsurface microbes from a conifer forest in Arizona, USA. We hypothesized that the concentration of heterotrophic substrates in microbiological growth medium would affect which microbial taxa were culturable from these soils. To test this, we diluted cells extracted from soil into one of two custom-designed defined growth media that differed by 100-fold in the concentration of amino acids and organic carbon. Across the two media, we isolated a total of 133 pure cultures, all of which were classified as Actinobacteria or Alphaproteobacteria. The substrate availability dictated which actinobacterial phylotypes were culturable but had no significant effect on the culturability of Alphaproteobacteria. We isolated cultures that were representative of the most abundant phylotype in the soil microbial community (Bradyrhizobium spp.) and representatives of five of the top 10 most abundant Actinobacteria phylotypes, including Nocardioides spp., Mycobacterium spp., and several other phylogenetically divergent lineages. Flow cytometry of nucleic acid-stained cells showed that cultures isolated on low-substrate medium had significantly lower nucleic acid fluorescence than those isolated on high-substrate medium. These results show that dilution-to-extinction is an effective method to isolate abundant soil microbes and that the concentration of substrates in culture medium influences the culturability of specific microbial lineages. IMPORTANCE Isolating environmental microbes and studying their physiology under controlled conditions are essential aspects of understanding their ecology. Subsurface ecosystems are typically nutrient-poor environments that harbor diverse microbial communities—the majority of which are thus far uncultured. In this study, we use modified high-throughput cultivation methods to isolate subsurface soil microbes. We show that a component of whether a microbe is culturable from subsurface soils is the concentration of growth substrates in the culture medium. Our results offer new insight into technical approaches and growth medium design that can be used to access the uncultured diversity of soil microbes.https://journals.asm.org/doi/10.1128/mSphere.00024-20genome streamliningmicrobial cultivationoligotrophysoil microbial ecology |
| spellingShingle | Ryan P. Bartelme Joy M. Custer Christopher L. Dupont Josh L. Espinoza Manolito Torralba Banafshe Khalili Paul Carini Influence of Substrate Concentration on the Culturability of Heterotrophic Soil Microbes Isolated by High-Throughput Dilution-to-Extinction Cultivation mSphere genome streamlining microbial cultivation oligotrophy soil microbial ecology |
| title | Influence of Substrate Concentration on the Culturability of Heterotrophic Soil Microbes Isolated by High-Throughput Dilution-to-Extinction Cultivation |
| title_full | Influence of Substrate Concentration on the Culturability of Heterotrophic Soil Microbes Isolated by High-Throughput Dilution-to-Extinction Cultivation |
| title_fullStr | Influence of Substrate Concentration on the Culturability of Heterotrophic Soil Microbes Isolated by High-Throughput Dilution-to-Extinction Cultivation |
| title_full_unstemmed | Influence of Substrate Concentration on the Culturability of Heterotrophic Soil Microbes Isolated by High-Throughput Dilution-to-Extinction Cultivation |
| title_short | Influence of Substrate Concentration on the Culturability of Heterotrophic Soil Microbes Isolated by High-Throughput Dilution-to-Extinction Cultivation |
| title_sort | influence of substrate concentration on the culturability of heterotrophic soil microbes isolated by high throughput dilution to extinction cultivation |
| topic | genome streamlining microbial cultivation oligotrophy soil microbial ecology |
| url | https://journals.asm.org/doi/10.1128/mSphere.00024-20 |
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