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.

中文翻译：

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底物浓度对通过高通量稀释灭绝栽培分离的异养土壤微生物的可培养性的影响。

尚未在受控的实验室条件下分离或研究居住在贫营养的浅层地下土壤环境中的绝大多数微生物。在一定程度上，与分离浅层地下微生物有关的挑战可能会继续存在，因为深层土壤中的微生物会适应低养分利用率或低品质。在这里，我们使用高通量的稀释灭绝培养法从美国亚利桑那州的针叶林中分离出浅层地下微生物。我们假设微生物生长培养基中异养底物的浓度会影响从这些土壤中可培养出哪种微生物类群。为了测试这一点，我们将从土壤中提取的细胞稀释到两种定制设计的定义生长培养基之一中，该培养基的氨基酸和有机碳浓度相差100倍。在两种媒体之间，我们总共分离了133种纯培养物，所有这些培养物都被归类为放线菌属或Alphaproteobacteria。底物可用性决定了哪些放线菌属菌种是可培养的，但对Alphaproteobacteria的可培养性没有显着影响。我们分离了代表最丰富菌种的培养物。土壤微生物群落（Bradyrhizobium spp。）以及放线菌细菌最丰富的10种系统型中的5种，包括诺卡氏菌，分枝杆菌和其他几种系统发育的谱系。核酸染色细胞的流式细胞术表明，在低底物培养基上分离的培养物的核酸荧光明显低于在高底物培养基上分离的培养物。这些结果表明，从稀释到灭绝是一种分离大量土壤微生物的有效方法，并且培养基中底物的浓度会影响特定微生物谱系的可培养性。重要事项隔离环境微生物并在受控条件下研究其生理是微生物的重要方面。了解他们的生态。地下生态系统通常是养分贫乏的环境，具有各种微生物群落，其中大多数迄今尚未养殖。在这项研究中，我们使用改良的高通量栽培方法来分离地下土壤微生物。我们表明，是否可以从地下土壤中培养微生物的一个因素是培养基中生长底物的浓度。