Department of Environmental Science, University of Arizona, Tucson, Arizona, USA.
Department of Environment and Sustainability, J. Craig Venter Institute, La Jolla, California, USA.
mSphere. 2020 Jan 29;5(1):e00024-20. doi: 10.1128/mSphere.00024-20.
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 or The substrate availability dictated which actinobacterial phylotypes were culturable but had no significant effect on the culturability of We isolated cultures that were representative of the most abundant phylotype in the soil microbial community ( spp.) and representatives of five of the top 10 most abundant phylotypes, including spp., 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. 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.
绝大多数栖息在贫营养浅地下土壤环境中的微生物尚未在受控实验室条件下被分离或研究。部分原因是，从深层土壤中分离浅层地下微生物的挑战仍然存在，因为深层土壤中的微生物适应于低营养可用性或质量。在这里，我们使用高通量稀释灭绝培养法从美国亚利桑那州的一片针叶林分离浅层地下微生物。我们假设微生物生长培养基中异养底物的浓度会影响可从这些土壤中培养的微生物分类群。为了验证这一点，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中的一种，这两种培养基中氨基酸和有机碳的浓度相差 100 倍。在这两种培养基中，我们总共分离出 133 株纯培养物，均被分类为 或 底物可用性决定了哪些放线菌分类群可培养，但对 的可培养性没有显著影响。我们分离出的培养物代表了土壤微生物群落中最丰富的分类群（ spp.）和前 10 个最丰富的分类群中的 5 个代表，包括 spp.， spp.和其他几个系统发育上不同的谱系。用核酸染色细胞的流式细胞术显示，在低底物培养基上分离的培养物的核酸荧光显著低于在高底物培养基上分离的培养物。这些结果表明，稀释灭绝是一种有效的方法，可以分离丰富的土壤微生物，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。在受控条件下分离环境微生物并研究其生理学是理解其生态学的重要方面。地下生态系统通常是营养贫乏的环境，其中栖息着多样化的微生物群落——其中大多数迄今尚未培养。在这项研究中，我们使用改良的高通量培养方法来分离地下土壤微生物。我们表明，微生物是否可从地下土壤中培养出来的一个因素是培养基中生长底物的浓度。我们的研究结果为可以用来获取土壤微生物未培养多样性的技术方法和培养基设计提供了新的见解。
