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 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.

Keywords: genome streamlining; microbial cultivation; oligotrophy; soil microbial ecology.

FIG 1

Dilution-to-extinction workflow. Soils were collected and brought to the lab, where they were homogenized in cell extraction buffer, layered over a Nycodenz solution, and centrifuged (A). The cell layer was extracted from the Nycodenz solution and counted with flow cytometry (B). Counted cells were diluted into growth medium in 96-well microtiter plates to an average density of 5 cells well⁻¹ (C). After incubation, the 96-well microtiter plates were screened for growth with flow cytometry, and wells displaying growth were subcultured into larger volumes (D). After incubating the subcultures, flasks displaying growth were identified by 16S rRNA gene sequencing and molecular phylogeny (E). Aliquots of these identified subcultures were cryopreserved at −80°C (F).

FIG 2

Microbial culturability (v) was greater on ASM-low than on ASM-high. Bar heights are the mean percent culturability ± standard deviation in 96-well microtiter plates (n = 3) as calculated from the initial cell inoculum and the proportion of wells positive for growth (40). Double asterisks indicate Wilcoxon rank sum test P values of ≤0.05.

FIG 3

ASM-low and ASM-high cultured distinct Alphaproteobacteria (a) and Actinobacteria (b). Bar heights are the number of cultures obtained for each taxon and are colored by the medium type on which they were isolated. The genera assigned to bacteria isolated on ASM-low were distinct from those isolated on ASM-high (Kruskal-Wallis rank sum, χ² = 19.05, P = 1.28 × 10⁻⁵). These differences were driven by differences in culturability across medium types for actinobacterial genera but not for alphaproteobacterial genera (Dunn test, P ≤ 0.000 for Actinobacteria and P = 0.128 for Alphaproteobacteria).

FIG 4

The cultures isolated in this study were representative of several abundant soil lineages that show dynamic depth distributions in Oracle Ridge soils. Points are the mean relative abundances ± standard deviation (n = 3) of 16S rRNA gene sequence phylotypes that matched the 16S rRNA gene sequences obtained from cultured isolates at ≥97% identity. Error bars that are not visible are located behind the symbol. Assigned genus names and the average (n = 3) relative rank of each phylotype at 55 cm are shown. Cultures classified at the genus level as Mycobacterium and Arthrobacter cultures matched more than one phylotype in the cultivation-independent surveys. The best-matching OTU number is shown in parentheses. Triangles are Alphaproteobacteria. Circles are Actinobacteria.

FIG 5

The mean nucleic acid fluorescence of taxa isolated on ASM-low was significantly lower than for those microbes isolated on ASM-high. Points are the mean natural logarithm (ln) of the quantified nucleic acid fluorescence (in arbitrary units [AU]) of fixed and SYBR green I stained stationary-phase cultures. The mean fluorescence value was obtained from manually gated histogram plots of fluorescence within the Guava EasyCyte software. Only those cultures that were defined as pure cultures are plotted. The horizontal line in each plot is the mean fluorescence value, and the box surrounding the mean is a 95% confidence interval. Shading illustrates the relative distribution of fluorescence values within each medium type.