Factors affecting the isolation and diversity of marine sponge-associated bacteria.

Marine sponges are an ideal source for isolating as yet undiscovered microorganisms with some sponges having about 50% of their biomass composed of microbial symbionts. This study used a variety of approaches to investigate the culturable diversity of the sponge-associated bacterial community from samples collected from the South Australian marine environment. Twelve sponge samples were selected from two sites and their bacterial population cultivated using seven different agar media at two temperatures and three oxygen levels over 3 months. These isolates were identified using microscopic, macroscopic, and 16S rRNA gene analysis. A total of 1234 bacterial colonies were isolated which consisted of four phyla: Actinobacteria, Firmicutes, Proteobacteria, and Bacteroidetes, containing 21 genera. The diversity of the bacterial population was demonstrated to be influenced by the type of isolation medium, length of the incubation period and temperature, sponge type, and oxygen level. The findings of this study showed that marine sponges of South Australia can yield considerable bacterial culturable diversity if a comprehensive isolation strategy is implemented. Two sponges, with the highest and the lowest diversity of culturable isolates, were examined using next-generation sequencing to better profile the bacterial population. A marked difference in terms of phyla and genera was observed using culture-based and culture-independent approaches. This observed variation displays the importance of utilizing both methods to reflect a more complete picture of the microbial population of marine sponges. KEY POINTS: Improved bacterial diversity due to long incubations, 2 temperatures, and 3 oxygen levels. Isolates identified by morphology, restriction digests, and 16S rRNA gene sequencing. At least 70% of culturable genera were not revealed by NGS methods.