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

Abstract

Although molecular approaches can identify members of microbial communities in the environment, genomic information does not necessarily correlate with environmental phenotype. Understanding functional roles can be done by cultivating representative species, yet the culturablility of bacteria from caves remains low, at 0.02%, limiting our understanding of microbial community interactions and processes. We have investigated several factors influencing culturability of bacteria from a single sample location in Maxwelton Sink Cave, WV, USA. Extended incubation of inoculated plates showed a significant increase in colony counts from two to four weeks, indicating that extended incubations increase culturability. There were no significant differences in plate counts or diversity measures when the sample was suspended in different buffers prior to cultivation, while samples plated immediately after collection demonstrated higher culturability. Although supplementing the media with antibiotics reduced colony counts and cultured diversity, these plates did appear to contain a higher proportion of slow-growing oligotrophs. Finally, among a selection of culture media used, pyruvate agar showed the highest culturability and bacterial diversity, which may be a result of the oxygen radical scavenging effects of pyruvate. By identifying methods that improve culturable diversity, we hope to further understand the roles played by bacteria in cave communities, and test hypotheses that are best assessed using culture-based methods, such as screening for bioactive compounds or confirming in situ metabolic strategies.

Prokaryotes of renowned Karlovy Vary (Carlsbad) thermal springs: phylogenetic and cultivation analysis

“…4 b), including novel species of genera Thermomonas (S), Paenibacillus and Cellulomonas (P). These findings confirm that such unconventional cultivation approaches are successful in isolating rare taxa [ 79 ] and that prolonged cultivation can enhance culturability of slow-growing and rare microbes [ 80 ]. At the same time, the enriched inoculum cultivation approach led to the enrichment of Bacillus -like genera, which are ubiquitous and often represent the cultivable part of microbes from thermal springs [ 15 , 81 ].…”

Reaching unreachables: Obstacles and successes of microbial cultivation and their reasons

“…10.3389/fmicb. 2023.1089630 Frontiers in Microbiology 06 frontiersin.org Adam et al, 2018;Bender et al, 2020). In a study by Davis et al (2005), autochthonous soil cells, as well as non-native cells from constructed consortia, were counted on six different media at 7-day intervals.…”

“…Since many unreachables are slow-growers, prolonged incubation times can lead to their successful cultivation. Prolonged cultivations, usually coupled with culturing diluted cell suspensions, have proved to be useful in many studies ( Eilers et al, 2001 ; Connon and Giovannoni, 2002 ; Rappé et al, 2002 ; Kakumanu and Williams, 2012 ; Adam et al, 2018 ; Bender et al, 2020 ). In a study by Davis et al (2005) , autochthonous soil cells, as well as non-native cells from constructed consortia, were counted on six different media at 7-day intervals.…”

Shedding light on the composition of extreme microbial dark matter: alternative approaches for culturing extremophiles

“…The first step to successfully culture extremophiles, as with any other microbe (Bellali et al, 2019;Schultz et al, 2022a), is to effectively detach the microbial cells from the sample substrate. Using three different resuspension buffers (Ringer's solution, PBS, and sterile cave water, pH 8.2) for rocks from cave samples, Bender et al (2020) identified the largest number of colonies and diverse phenotypes in water. This suggests that suspension solutions buffered to physiological conditions (i.e., pH, sodium, and potassium) should be effective in increasing microbial culturability (Padan et al, 2005).…”

“…In order to obtain pure cultures and fully understand the inadequately exploited cultured extremophiles, developing innovative culturing methods is crucial (Vartoukian et al, 2010;Bull and Goodfellow, 2019). Several old and recently developed techniques and strategies have been applied to isolate previously uncultured microbes from extreme habitats, such as prolonging incubation times (Davis et al, 2005;de Jesus et al, 2015;Pulschen et al, 2017), using different concentrations of oxygen and other gases (Lopez et al, 2019;Volpiano et al, 2021), using low-nutrient culture media (Do Carmo et al, 2011;Peixoto et al, 2011;Grzesiak et al, 2015;Pulschen et al, 2017), adding antibiotics to inhibit fast-growing microorganisms and prevent contamination with unwanted microbial groups (Bender et al, 2020), changing the gelling agent, e.g., gellan gum (Das et al, 2015), or using a cellulose plate (Tsudome et al, 2009) instead of agar, in situ diffusion devices (Nichols et al, 2010;Palma Esposito et al, 2018), and celltargeting methods (Huber et al, 2000;Antunes et al, 2008a). However, although these alternative strategies may yield new microbes, most of the microbes presumed to occur in extreme environments remain uncultured.…”