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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Autores principales:	Ryan P. Bartelme, Joy M. Custer, Christopher L. Dupont, Josh L. Espinoza, Manolito Torralba, Banafshe Khalili, Paul Carini
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2020
Materias:	genome streamlining microbial cultivation oligotrophy soil microbial ecology Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/801e4ab8f441446f941be4752124f2af
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spelling	oai:doaj.org-article:801e4ab8f441446f941be4752124f2af2021-11-15T15:27:53ZInfluence of Substrate Concentration on the Culturability of Heterotrophic Soil Microbes Isolated by High-Throughput Dilution-to-Extinction Cultivation10.1128/mSphere.00024-202379-5042https://doaj.org/article/801e4ab8f441446f941be4752124f2af2020-02-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mSphere.00024-20https://doaj.org/toc/2379-5042ABSTRACT 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.Ryan P. BartelmeJoy M. CusterChristopher L. DupontJosh L. EspinozaManolito TorralbaBanafshe KhaliliPaul CariniAmerican Society for Microbiologyarticlegenome streamliningmicrobial cultivationoligotrophysoil microbial ecologyMicrobiologyQR1-502ENmSphere, Vol 5, Iss 1 (2020)
institution	DOAJ
collection	DOAJ
language	EN
topic	genome streamlining microbial cultivation oligotrophy soil microbial ecology Microbiology QR1-502
spellingShingle	genome streamlining microbial cultivation oligotrophy soil microbial ecology Microbiology QR1-502 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
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.
format	article
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
title	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_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_sort	influence of substrate concentration on the culturability of heterotrophic soil microbes isolated by high-throughput dilution-to-extinction cultivation
publisher	American Society for Microbiology
publishDate	2020
url	https://doaj.org/article/801e4ab8f441446f941be4752124f2af
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Influence of Substrate Concentration on the Culturability of Heterotrophic Soil Microbes Isolated by High-Throughput Dilution-to-Extinction Cultivation

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