Cultivation strategies for prokaryotes from extreme environments

Abstract

The great majority of microorganisms are as‐yet‐uncultivated, mostly found in extreme environments. High‐throughput sequencing provides data‐rich genomes from single‐cell and metagenomic techniques, which has enabled researchers to obtain a glimpse of the unexpected genetic diversity of “microbial dark matter.” However, cultivating microorganisms from extreme environments remains essential for dissecting and utilizing the functions of extremophiles. Here, we provide a straightforward protocol for efficiently isolating prokaryotic microorganisms from different extreme habitats (thermal, xeric, saline, alkaline, acidic, and cryogenic environments), which was established through previous successful work and our long‐term experience in extremophile resource mining. We propose common processes for extremophile isolation at first and then summarize multiple cultivation strategies for recovering prokaryotic microorganisms from extreme environments and meanwhile provide specific isolation tips that are always overlooked but important. Furthermore, we propose the use of multi‐omics‐guided microbial cultivation approaches for culturing these as‐yet‐uncultivated microorganisms and two examples are provided to introduce how these approaches work. In summary, the protocol allows researchers to significantly improve the isolation efficiency of pure cultures and novel taxa, which therefore paves the way for the protection and utilization of microbial resources from extreme environments.

Chelatococcus albus sp. nov., a bacterium isolated from hot spring microbial mat

“…The in situ temperature and pH of the hot spring were 46 °C and pH 6.62, respectively. In order to isolate micro-organisms from the HNT-2 sample, the isolation process was performed by using a standard dilution plate method [15] on 02YE agar: yeast extract 0.5 g, KH 2 PO 4 0.38 g, K 2 HPO 4 0.39 g, (NH 4 ) 2 SO 4 0.5 g, 5 ml trace salt (FeSO 4 ·7H 2 O 1.11 g, MgSO 4 ·7H 2 O 24.65 g, CaCl 2 ·2H 2 O 2.94 g, NaCl 23.4 g, MnSO 4 ·4H 2 O 111 mg, ZnSO 4 ·7H 2 O 28.8 mg, Co(NO 3 ) 2 ·6H 2 O 29.2 mg, CuSO 4 ·5H 2 O 25.2 mg, Na 2 MoO 4 ·2H 2 O 24.2 mg, H 3 BO 3 31.0 mg, trisodium EDTA 4.53 g, ddH 2 O 1000 ml), 1 ml complex vitamins (nicotinic acid 100 mg, thiamine hydrochloride 100 mg, biotin 5 mg, p -aminobenzoic acid 50 mg, cobalamin 1 mg, calcium pantothenate 50 mg, pyridoxine hydrochloride 50 mg, folic acid 0.5 mg, ddH 2 O 100 ml), agar 15 g, ddH 2 O 1000 ml, pH 7.0) [16]. The agar plates were placed in an incubator set at a temperature of 45 °C for 7 days.…”

Flagellimonas halotolerans sp. nov., a novel bacterium isolated from the South China Sea

“…The purified strains were stored as aqueous glycerol suspensions (20 %, v/v) at −80 °C for further use. The isolation steps were carried out according to Yang et al [16]. In this study, 156 and 196 strains were isolated from different samples, respectively, and were identified using their 16S rRNA gene sequences.…”