title: Effects of Gelling Agent and Extracellular Signaling Molecules on the Culturability of Marine Bacteria.
abstract: 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 this study was to investigate if improved cultivation conditions, including the use of an alternative gelling agent and supplementation with signaling molecules, improve the culturability of bacteria from seawater. Replacing agar with gellan gum improved viable counts 3- to 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 acylated homoserine lactones (AHLs) did not have a positive effect on total viable counts or a strong effect on culturable diversity. However, low concentrations of AHLs increased the relative abundance of sphingobacteria. Hence, with alternative growth substrates, it is possible to significantly increase the number and diversity of cultured marine bacteria.IMPORTANCESerious challenges to human health, such as the occurrence and spread of antibiotic resistance and an aging human population in need of bioactive pharmaceuticals, have revitalized the search for natural microbial products. The marine environment, representing the largest ecosystem in the biosphere, harbors an immense and virtually untapped microbial diversity producing unique bioactive compounds. However, we are currently able to cultivate only a minute fraction of this diversity. The lack of cultivated microbes is hampering not only bioprospecting efforts but also our general understanding of marine microbes. In this study, we present a means to increase the number and diversity of cultured bacteria from seawater, showing that relatively simple changes to medium components may facilitate the isolation and growth of hitherto unknown bacterial orders.
authors.affiliation: Department of Biotechnology and Biomedicine, Technical University of Denmark, Kongens Lyngby, Denmark.
authors.forename: Anita Mac
authors.initials: AM
authors.lastname: Rygaard
authors.affiliation: Department of Biotechnology and Biomedicine, Technical University of Denmark, Kongens Lyngby, Denmark.
authors.forename: Mariane Schmidt
authors.initials: MS
authors.lastname: Thøgersen
authors.affiliation: Department of Biotechnology and Biomedicine, Technical University of Denmark, Kongens Lyngby, Denmark.
authors.forename: Kristian Fog
authors.initials: KF
authors.lastname: Nielsen
authors.affiliation: Department of Biotechnology and Biomedicine, Technical University of Denmark, Kongens Lyngby, Denmark.
authors.forename: Lone
authors.initials: L
authors.lastname: Gram
authors.affiliation: Department of Biotechnology and Biomedicine, Technical University of Denmark, Kongens Lyngby, Denmark mibti@bio.dtu.dk.
authors.forename: Mikkel
authors.identifier: http://orcid.org/0000-0002-7888-9845
authors.initials: M
authors.lastname: Bentzon-Tilia
mesh_terms: D059001:Aquatic Organisms / Q000187:drug effects* / Q000254:growth & development*; D001419:Bacteria / Q000187:drug effects* / Q000254:growth & development*; D001431:Bacteriological Techniques / Q000379:methods*; D044822:Biodiversity; D003470:Culture Media / Q000737:chemistry*; D036341:Intercellular Signaling Peptides and Proteins / Q000378:metabolism*; D050296:Microbial Viability; D011135:Polysaccharides, Bacterial / Q000378:metabolism
chemical_list: D003470:Culture Media; D036341:Intercellular Signaling Peptides and Proteins; D011135:Polysaccharides, Bacterial; C048288:gellan gum
keywords: AHLs; cultivation; gellan gum; marine microbiology; microbial communities