Methods and Strategies to Uncover Coral-Associated Microbial Dark Matter

Abstract

The vast majority of environmental microbes have not yet been cultured, and most of the knowledge on coral-associated microbes (CAMs) has been generated from amplicon sequencing and metagenomes. However, exploring cultured CAMs is key for a detailed and comprehensive characterization of the roles of these microbes in shaping coral health and, ultimately, for their biotechnological use as, for example, coral probiotics and other natural products. Here, the strategies and technologies that have been used to access cultured CAMs are presented, while advantages and disadvantages associated with each of these strategies are discussed. We highlight the existing gaps and potential improvements in culture-dependent methodologies, indicating several possible alternatives (including culturomics and in situ diffusion devices) that could be applied to retrieve the CAM "dark matter" (i.e., the currently undescribed CAMs). This study provides the most comprehensive synthesis of the methodologies used to recover the cultured coral microbiome to date and draws suggestions for the development of the next generation of CAM culturomics.

Keywords: coral metaorganism; coral microbiome; coral probiotics; coral-associated microbes; culture-dependent techniques; culturing; culturomics; microbial dark matter.

FIG 1

“Coral juice” preparation for cultivation of coral-associated microorganisms. First, a 5-g coral fragment of interest (A) is macerated (B) to obtain a homogeneous mixture. Then, the mixture (C) is then added to an Erlenmeyer flask with 45 mL of saline solution (2.5%) and 5-mm glass beads for overnight incubation. After incubation, the contents are transferred into a centrifuge tube (D), centrifuged at 10,000 g for 5 min (E), and the supernatant is collected (F). Subsequently, successive filtration steps must be performed (G), starting with membranes of 1 μm (for debris retention) and ending with 0.22-μm pore size (for sterilization). The sterile supernatant is the coral juice. Then (H) 5% vol/vol of coral juice is loaded into autoclaved Marine Minimal Medium at 70°C, followed by mixing the blend and plating 20 mL in each Petri dish. Coral juice is the sole carbon source in the medium, favoring the growth of coral-associated microbes. After the seeding of samples of interest, plate dishes can be incubated under different oxygen demand conditions, for different periods of incubation and temperatures. Created using Biorender.com.

FIG 2

Overview of the current knowledge of culture-dependent methods being used in coral microbiology, in addition to novel and alternative strategies that can be applied for culturing coral microbiomes. Created using Biorender.com.