Cultivating marine bacteria under laboratory conditions: Overcoming the “unculturable” dogma

Abstract

Underexplored seawater environments may contain biological resources with potential for new biotechnological applications. Metagenomic techniques revolutionized the study of bacterial communities but culture dependent methods will still be important to help the biodiscovery of new products and enzymes from marine bacteria. In this context, we promoted the growth of bacteria from a marine rock pond by culture dependent techniques and compared the results with culture independent methods. The total number of bacteria and diversity were studied in different agar plate media during 6 weeks. Agar plate counting was of the same order of magnitude of direct microscopy counts. The highest efficiency of cultivation was 45% attained in marine agar medium. Molecular analysis revealed 10 different phyla of which only four were isolated by the culture dependent method. On the other hand, four taxonomic orders were detected by cultivation but not by the molecular technique. These include bacteria from the phyla Bacillota and Actinomycetota. Our study shows that it is possible to grow more than the traditionally considered 1% of bacteria from a seawater sample using standard agar plate techniques and laboratorial conditions. The results also demonstrate the importance of culture methods to grow bacteria not detected by molecular approaches for future biotechnological applications.

Bioactivity Profiling and Untargeted Metabolomics of Microbiota Associated with Mesopelagic Jellyfish Periphylla Periphylla

“…Marine agar was the most suitable isolation medium used herein and is designed to support growth of mostly oligotrophic marine bacteria [ 55 ]. Similar to Marine agar, Hastings and modified Wickerham media contain yeast extract, but differ in their protein source (WSP: peptone, Hastings: tryptone), hence, were selected for this study.…”

Marine Bioprospecting, Biocatalysis and Process Development

“…The dogma coined originally in 1985 by Staley and Konopka [ 8 ] was eventually generalized to all kinds of environmental samples. It is currently well accepted by the scientific community that only 1% of bacteria from the environment grow under laboratory conditions, although the ‘great plate count anomaly’ has been proven not to be true [ 24 , 94 ]. The dogma started because only a small part of the population visible under the microscope provided colonies on agar plates.…”

“…Several works have shown that significantly more than 1% of microbial species could be grown and that cultivation techniques (including the use of multiple culture media, refinement of culture media composition, and co-culture of multiple species) could detect isolates not detectable by biased metagenomic techniques [ 24 , 94 , 103 ]. We have recently shown that we could grow ca.…”

“…We have recently shown that we could grow ca. 45% of the cells in a sample from a marine rock pond using marine agar medium after spread-plating the cells on different agar media compositions and concentrations, and by waiting for 6 weeks [ 24 ]. Although metataxonomy showed members of ten different phyla of which only four were isolated by the culture-dependent method, the latter detected four taxonomic orders not detected by the cultivation-independent technique.…”

“…The difficulty to mimic natural conditions in vitro and the lack of time to test numerous media formulations necessary for the growth of “unculturable” bacteria are some of the reasons for the perpetuation of the dogma that the majority of environmental bacteria do not grow in the laboratory [ 22 , 23 ]. However, recent studies have showed that a much larger percentage of marine bacteria may be isolated [ 24 , 25 ], including one study from our laboratory comparing cultivation-dependent and -independent techniques [ 24 ], and a study using a continuous-flow down-flow hanging sponge bioreactor to favor growth of deep sediment microorganisms [ 26 ].…”