Abstract
Sponges host complex microbial communities of recognized ecological and biotechnological importance. Extensive cultivation efforts have been made to isolate sponge bacteria, but most still elude cultivation. To identify the bottlenecks of sponge bacterial cultivation, we combined high-throughput 16S rRNA gene sequencing with a variety of cultivation media and incubation conditions. We aimed to determine the extent to which sample processing and cultivation conditions can impact bacterial viability and recovery in culture. We isolated 325 sponge bacteria from six specimens of Cymbastela concentrica and three specimens of Scopalina sp. These isolates were distributed over 37 different genera and 47 operational taxonomic units (defined at 97% 16S rRNA gene sequence identity). The cultivable bacterial community was highly specific to its sponge host and different media compositions yielded distinct microbial isolates. Around 97% of the isolates could be detected in the original sponge and represented a large but highly variable proportion (0.5–92% total abundance, depending on sponge species) of viable bacteria obtained after sample processing, as determined by propidium monoazide selective DNA modification of compromised cells. Our results show that the most abundant viable bacteria are also the most predominant groups found in cultivation, reflecting, to some extent, the relative abundances of the viable bacterial community, rather than the overall community estimated by direct molecular approaches. Cultivation is therefore shaped not only by the growth conditions provided, but also by the different cell viabilities of the bacteria that constitute the cultivation inoculum. These observations highlight the need to perform experiments to assess each method of sample processing for its accurate representation of the actual in situ bacterial community and its yield of viable cells.
Highlights
Culturing is an important tool to study the physiology and ecological function of microorganisms (Stewart, 2012) and it can provide access to bacteria that have eluded detection with molecular approaches (Donachie et al, 2007; Shade et al, 2012)
A higher number of isolates and higher counts of bacteria per sponge specimen was retrieved from Scopalina sp., C. concentrica harbors a slightly richer cultivable community, spanning 25 bacterial genera and 32 iOTUs compared to 19 taxa and 19 iOTUs found in Scopalina sp
At the operational taxonomic units (OTUs) level, Gammaproteobacteria showed higher richness, with 13 iOTUs and 11 different bacterial genera, against 10 iOTUs and eight genera within the Alphaproteobacteria (Table 2). These results differ among sponge species, with Scopalina sp. showing higher richness for Alphaproteobacteria than that found in Gammaproteobacteria
Summary
Culturing is an important tool to study the physiology and ecological function of microorganisms (Stewart, 2012) and it can provide access to bacteria that have eluded detection with molecular approaches (Donachie et al, 2007; Shade et al, 2012). Several innovative culturing approaches have been developed, including complex microfluidic and laser manipulation systems (Fröhlich and König, 2000; Zhang and Liu, 2008; Liu et al, 2009; Yamaguchi et al, 2009), high-throughput cultivation technologies based on dilution-to-extinction and microencapsulation (Connon and Giovannoni, 2002; Zengler et al, 2005; Ben-Dov et al, 2009), diffusion devices that allow the exchange of small molecules with the environment (Kaeberlein et al, 2002; Bollmann et al, 2007; Nichols et al, 2010), filters and membrane systems to simulate the natural environment (de Bruyn et al, 1990; Ferrari et al, 2008), co-culture approaches (Ohno et al, 2000; Nichols et al, 2008), formulation of new media compositions based on metagenomic information (Tyson et al, 2005) and establishment of growth conditions to mimic the natural environment with low-nutrient media and longer incubation times (Song et al, 2009) These approaches have made significant breakthroughs by increasing the diversity and recovery rates of bacteria retrieved in culture and enabling the cultivation of ecologically relevant microorganisms, like the SAR11 clade (Rappé et al, 2002; Bollmann et al, 2007). While in the past decade a comprehensive picture of the phylogenetic diversity and composition of the sponge microbiome has been generated through culture-independent methods (Webster and Taylor, 2012), the in situ activity and function of these microbial symbionts are the major research focus (Thomas et al, 2010; Hentschel et al, 2012; Radax et al, 2012; Ribes et al, 2012; Fan et al, 2013)
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