Expanding Actinomycetota Diversity in the TBRC Culture Collection through Metabarcoding and Simulated In Situ Cultivation of Thailand’s Mekong River Microbiota

Abstract

Culture-independent and culture-dependent approaches were employed to investigate the taxonomic diversity and biosynthetic gene cluster potential of Actinomycetota in the Mekong River. Through 16S rRNA gene metabarcoding, 21,103 OTUs were revealed to represent 190 genera and at least 595 species of Actinomycetota, including putatively novel taxa. Conventional and in situ cultivation (IC) methods provided 75 Actinomycetota isolates representing 72 species from 21 genera. Of these, 45 species in 4 genera were new to the Thailand Bioresource Research Center (TBRC), a collection of 20,079 Actinomycetota strains from 660 species. Applying both culture-independent and culture-dependent approaches to the same sample revealed greater diversity among the Actinomycetota in the Mekong River than one approach alone.

Rhodoferax potami sp. nov. and Rhodoferax mekongensis sp. nov., isolated from the Mekong River in Thailand

“…The culture plates were then kept at 30 ±2 °C for 72 h. The IC method was employed as described in detail by Kitikhun et al . [18]. In brief, a 180 µl cell suspension (10 −4 dilution) was diluted with sterile water to a final volume of 960 µl before being used as an IC inoculum.…”

“…For the dilution plate method, water samples from each sampling location were serially diluted with normal saline solution (0.85 % (w/v) NaCl) and inoculated into soil extracted medium supplemented with 50 mg l −1 cycloheximide [17] to inhibit fungal growth. The culture plates were then kept at 30 ±2 °C for 72 h. The IC method was employed as described in detail by Kitikhun et al [18]. In brief, a 180 µl cell suspension (10 −4 dilution) was diluted with sterile water to a final volume of 960 µl before being used as an IC inoculum.…”