Cultivation of previously uncultured sponge-associated bacteria using advanced cultivation techniques: A perspective on possible key mechanisms

Abstract

Most of the microbes from natural habitats cannot be cultivated with standard cultivation in laboratory, and sponge-associated microbes are no exception. We used two advanced methods based on a continuous-flow bioreactor (CF) and in situ cultivation (I-tip) to isolate previously uncultivated marine sponge-associated bacteria. We also characterized the physiological properties of the isolates from each method and attempted to clarify the mechanisms operating in each cultivation method. A greater number of novel bacteria were isolated using CF and in situ cultivation compared to standard direct plating (SDP) cultivation. Most isolates from CF cultivation were poor growers (with lower specific growth rates and saturated cell densities than those of isolates from SDP cultivation), demonstrating that it is effective to carry out pre-enrichment cultivation targeting bacteria that are less competitive on conventional cultivation, especially K-strategists and bacterial types inhibited by their own growth. Isolates from in situ cultivation showed a positive influence on cell recovery stimulated by chemical compounds in the extract of sponge tissue, indicating that some of the bacteria require a “growth initiation factor” that is present in the natural environment. Each advanced cultivation method has its own distinct key mechanisms allowing cultivation of physiologically and phylogenetically different fastidious bacteria for cultivation compared with conventional methods.

A targeted liquid cultivation method for previously uncultured non-colony forming microbes

“…Previous studies have successfully demonstrated that liquid cultivation using a continuous-flow bioreactor led to isolating previously uncultured species, especially bacterial types inhibited by their own growth. Thus, this type formed significantly smaller-sized colonies on agar media than strains from SDP during the sub-cultivation step (Jung et al, 2022). Therefore, the growth of some isolates from NCFLC in this study was also likely inhibited on agar plates because of sensitivity to their own metabolic compounds.…”

“…Eilers et al (2000) and Simu et al (2005) found that non-colony-forming bacteria are abundant, sometimes dominant, in pelagic bacterial communities. In our previous study, we demonstrated that long-term liquid cultivation using a continuous-flow bioreactor led to cultivating previously uncultured bacteria from marine sponges (Jung et al, 2022). Interestingly, physiological analysis of bacterial species isolated using the bioreactor showed that these species had difficulty forming colonies.…”

Short-chain fatty acids (SCFAs) as potential resuscitation factors that promote the isolation and culture of uncultured bacteria in marine sediments

“…Many factors contribute to the discovery of microorganisms that are not yet cultured with dormancy being considered one of the most difficult factors to overcome when attempting to isolate and culture most marine bacteria (Zhang et al 2021 ). Although a “simulated habitat” strategy has successfully resulted in the culture of some uncultured microbial groups (Jung et al 2022 ), it is not effective on dormant bacteria or those found in low abundance.…”

Diversity of prokaryotic communities associated with sponges in Vietnam and their secondary metabolite biosynthetic potential

“…In addition to traditional agar-plating techniques, numerous additional isolation approaches have been used to culture sponge symbionts, including the use of liquid cultures, floating filters (Sipkema et al, 2011), microcapsule-based cultivation (Gerardo Toledo et al, 2012), diffusion growth chambers (Steinert et al, 2014), in situ cultivation devices (Jung et al, 2014;Knobloch et al, 2019;Jung et al, 2021;Jung et al, 2022), the addition of antibiotics (Versluis et al, 2017), sodium pyruvate, catalase (Olson et al, 2000), alpha-butyrolactone (Selvin et al, 2009), sponge extracts (Webster et al, 2001a;Sipkema et al, 2011;Steinert et al, 2014;Esteves et al, 2016), spongin-based sponge skeleton (Kaboré et al, 2019), and arsenic (Keren et al, 2015;Ray et al, 2016) in culture media, as well as the incorporation of sponge-derived physiological and genomic information (Lavy et al, 2014;Gutleben et al, 2020). Such tremendous culture efforts often yield large numbers of isolates, many of which are new species and even produce novel secondary metabolites.…”

“…A large number of studies on sponge-associated microbes has revealed the discrepancy of microbial communities recovered by cultivation-dependent and -independent approaches (Sipkema et al, 2011;Versluis et al, 2017;Knobloch et al, 2019;Gutleben et al, 2020;Anteneh et al, 2022;Jung et al, 2022), and only a few true sponge symbionts have been successfully cultured so far. For instance, members of the alphaproteobacterial genus Pseudovibrio are isolated frequently from various sponges (Muscholl-Silberhorn et al, 2008;Santos et al, 2010;Bondarev et al, 2013;Lavy et al, 2014;Versluis et al, 2017).…”