Effective Soil Extraction Method for Cultivating Previously Uncultured Soil Bacteria

Abstract

Here, a new medium, named intensive soil extract medium (ISEM), based on new soil extract (NSE) using 80% methanol, was used to efficiently isolate previously uncultured bacteria and new taxonomic candidates, which accounted for 49% and 55% of the total isolates examined (n = 258), respectively. The new isolates were affiliated with seven phyla (Proteobacteria, Acidobacteria, Firmicutes, Actinobacteria, Verrucomicrobia, Planctomycetes, and Bacteroidetes). The result of chemical analysis showed that NSE included more diverse components of low-molecular-weight organic substances than two conventional soil extracts made using distilled water. Cultivation of previously uncultured bacteria is expected to extend knowledge through the discovery of new phenotypic, physiological, and functional properties and even roles of unknown genes. IMPORTANCE Both metagenomics and single-cell sequencing can detect unknown genes from uncultured microbial strains in environments, and either method may find the significant potential metabolites and roles of these strains. However, such gene/genome-based techniques do not allow detailed investigations that are possible with cultures. To solve this problem, various approaches for cultivation of uncultured bacteria have been developed, but there are still difficulties in maintaining pure cultures by subculture.

The impact of sample processing and media chemistry on the culturable diversity of bacteria isolated from a cave

“…The role of antibiotics was evaluated by plating identical media with and without the addition of chloramphenicol and nalidixic acid (lack of antibiotics is indicated by nAB). Alternate carbon sources in media included pyruvate agar (see Barton et al, 2005), soil agar (see Nguyen et al, 2018), and ISP media 2, 4, 5, and 7 from the International Streptomyces project (see Shirling & Gottlieb, 1966). All crushed rock portions were resuspended in 10 mL of the appropriate buffer for each set of sampling conditions (Table 1) and a 0.1 mL volume was inoculated onto plates using a sterile swab.…”

Metagenomics-Guided Survey, Isolation, and Characterization of Uranium Resistant Microbiota from the Savannah River Site, USA

“…To improve microbial growth and isolation from environmental samples, common approaches, such as varying media composition and growth conditions, have been developed [4]. Others approaches significantly deviate from traditional culturing, such as high throughput extinction culturing [5], high throughput single-cell encapsulation [6], diffusion chambers [7], using different gelling agents, antioxidants, or signaling molecules [8,9], and even increasing incubation times along with reducing nutrient concentrations or simple alterations in the media preparation [10,11,12]. Another interesting technique recently shown to retrieve previously uncultivable microbiota from environmental sample entails soil printing, called biological laser printing (BioLP), which provided a deeper understanding on the nature of microbiota colonizing a miniscule soil niche of 100 μm in size [13].…”

A Rapid Method for the Selection of Amidohydrolases from Metagenomic Libraries by Applying Synthetic Nucleosides and a Uridine Auxotrophic Host

“…The hydrolytic activity of the recombinant enzymes was assayed qualitatively by testing 29 different substrates ( Figure 4A): N 4 -acylated (2'-deoxy)cytidines and cytosines (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16); N 2 -acetylisocytosines (17); chromogenic substrates such as p-nitroacetanilide (18) and p-nitrobenzanilide (19); various p-nitrophenyl (pNP) esters (20)(21)(22)(23)(24); terephthalate derivatives bis(2-hydroxyethyl) terephthalate (26), dimethyl terephthalate (27), and monomethyl terephthalate (28); and the beta-lactam nitrocefin (29). Hydrolysis of chromogenic substrates (18)(19)(20)(21)(22)(23)(24)(25) was analysed spectrophotometrically at 405 nm, and that of other substrates was analysed spectrophotometrically at 240-320 nm and using TLC or HPLC-MS ( Figures S4-S10).…”

“…It is estimated that the Earth is inhabited by 10 11 -10 12 microbial species, of which only~10 4 have been cultured in the laboratory [1]. Nonetheless, the unculturable microbiome has provided science with new genes, biocatalysts, natural compounds, and bioproducts [2][3][4][5]. New enzymes that could allow provide novel approaches for designing enzymatic processes are in constant demand, and so are the screening methods for them.…”