Effects of gelling agent and extracellular signaling molecules on the culturability of marine bacteria

Only 1 % of marine bacteria are currently culturable using standard laboratory procedures and this is a major obstacle for our understanding of the biology of marine microorganisms and for the discovery of novel microbial natural products. Therefore, the purpose of the present study was to investigate if improved cultivation conditions, including the use of an alternative gelling agent, and supplementation with signaling molecules, could improve the culturability of bacteria from seawater. Substituting agar with gellan gum improved viable counts 3 – 40-fold, depending on medium composition and incubation conditions, with a maximum of 6.6 % culturability relative to direct cell counts. Through V4 amplicon sequencing we found that culturable diversity was also affected by a change in gelling agent, facilitating the growth of orders not culturable on agar-based substrates. Community analyses showed that communities grown on gellan gum substrates were significantly different from communities grown on agar, and that they covered a larger fraction of the seawater community. Other factors, such as incubation temperature and time, had less obvious effects on viable counts and culturable diversity. Supplementation with acyl homoserine lactones (AHLs) did not have a positive effect on total viable counts and no strong effect on culturable diversity. However, low concentrations of AHLs did increase the relative abundance of Sphingobacteria. Hence, with alternative growth substrates it is possible to significantly increase the number and diversity of cultured marine bacteria.