DataSheet_8_Cultivation of previously uncultured sponge-associated bacteria using advanced cultivation techniques: A perspective on possible key mechanisms.pdf

Most of the microbes from natural habitats cannot be cultivated with standard cultivation in laboratory, and sponge-associated microbes are no exception. We used two advanced methods based on a continuous-flow bioreactor (CF) and in situ cultivation (I-tip) to isolate previously uncultivated marine sponge-associated bacteria. We also characterized the physiological properties of the isolates from each method and attempted to clarify the mechanisms operating in each cultivation method. A greater number of novel bacteria were isolated using CF and in situ cultivation compared to standard direct plating (SDP) cultivation. Most isolates from CF cultivation were poor growers (with lower specific growth rates and saturated cell densities than those of isolates from SDP cultivation), demonstrating that it is effective to carry out pre-enrichment cultivation targeting bacteria that are less competitive on conventional cultivation, especially K-strategists and bacterial types inhibited by their own growth. Isolates from in situ cultivation showed a positive influence on cell recovery stimulated by chemical compounds in the extract of sponge tissue, indicating that some of the bacteria require a “growth initiation factor” that is present in the natural environment. Each advanced cultivation method has its own distinct key mechanisms allowing cultivation of physiologically and phylogenetically different fastidious bacteria for cultivation compared with conventional methods.</p

A Long-Term Cultivation of an Anaerobic Methane-Oxidizing Microbial Community from Deep-Sea Methane-Seep Sediment Using a Continuous-Flow Bioreactor

<div><p>Anaerobic oxidation of methane (AOM) in marine sediments is an important global methane sink, but the physiological characteristics of AOM-associated microorganisms remain poorly understood. Here we report the cultivation of an AOM microbial community from deep-sea methane-seep sediment using a continuous-flow bioreactor with polyurethane sponges, called the down-flow hanging sponge (DHS) bioreactor. We anaerobically incubated deep-sea methane-seep sediment collected from the Nankai Trough, Japan, for 2,013 days in the bioreactor at 10°C. Following incubation, an active AOM activity was confirmed by a tracer experiment using ¹³C-labeled methane. Phylogenetic analyses demonstrated that phylogenetically diverse <i>Archaea</i> and <i>Bacteria</i> grew in the bioreactor. After 2,013 days of incubation, the predominant archaeal components were <u>an</u>aerobic <u>me</u>thanotroph (ANME)-2a, Deep-Sea Archaeal Group, and Marine Benthic Group-D, and <i>Gammaproteobacteria</i> was the dominant bacterial lineage. Fluorescence <i>in situ</i> hybridization analysis showed that ANME-1 and -2a, and most ANME-2c cells occurred without close physical interaction with potential bacterial partners. Our data demonstrate that the DHS bioreactor system is a useful system for cultivating fastidious methane-seep-associated sedimentary microorganisms.</p></div

Comparison of AOM enrichments in different types of continuous-flow bioreactors.

<p>NR: not reported.</p>a<p>Data from long-term incubations with short columns described in Wegener and Boetius <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105356#pone.0105356-Wegener1" target="_blank">[22]</a>.</p>b<p>Data from high-flow experiments with NON-SEEP sediments described in Girguis <i>et al</i>. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105356#pone.0105356-Girguis2" target="_blank">[25]</a>.</p>c<p>Data from new high flow core experiments described in Steeb <i>et al</i>. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105356#pone.0105356-Steeb1" target="_blank">[23]</a>.</p>d<p>Dominant ANME type is shown in bold.</p

(A and G–I) FISH and (B–F) CARD-FISH images of microbial cells cultivated in the DHS bioreactor.

<p>Photomicrographs of DAPI-stained cells (left) and epifluorescence (right) showing identical fields. (A) Chain-forming ANME-1-350-stained cells in the 2,013-day sample (Alexa Fluor 488, green). (B) An ANME-2a-647-stained coccoid-shaped cell in the 2,013-day sample (Fluorescein, green). (C and D) Color overlay of ANME-2c-760- (Fluorescein, green) and EUB338-stained cells (Alexa Fluor 594, red) in the 903-day sample. (E) A MBGB-380-stained coccoid-shaped cell in the 2,013-day sample (Fluorescein, green). (F) A MBGD-318-stained rod-shaped cell in the 2,013-day sample (Fluorescein, green). (G) A MCOCID442-stained coccoid-shaped cell in the 2,013-day sample. (H) Mγ669-stained coccoid-shaped cells in the 2,013-day sample (Alexa Fluor 488, green). (I) UncGAM731-stained irregular rod-shaped cells in the 2,013-day sample (Alexa Fluor 488, green). Bars represent 10 µm.</p

Statistical analysis of clone libraries.

a<p>A phylotype was defined as ≥97% sequence identity.</p>b<p>Numbers in parentheses indicate the 95% confidence interval.</p

Archaeal and bacterial 16S rRNA gene copy numbers in the DHS bioreactor as determined by quantitative real-time PCR.

<p>Values are mean ± SD (n = 3).</p

Microbial community structures based on 16S rRNA gene sequence-based clone libraries.

<p>Phylogenetic affiliations based on (A) archaeal and (B) bacterial 16S rRNA genes and 16S rRNA. The 16S rRNA clone libraries were constructed from the 903-day and 2,013-day samples only. The numbers in parentheses are the total number of sequenced clones.</p

The DHS bioreactor system.

<p>(A) Schematic diagram and photographs of the DHS bioreactor used in this study. (B) The concept of cultivation of an AOM microbial community using the DHS bioreactor. Sponge carriers hang freely, suspended with string, in gaseous methane. Thus, the gaseous methane effectively diffuses not only to the surface but also inside the sponge carriers as the seawater medium flows down into the sponge carriers. The pore space in the sponge carriers serves as the habitat for microbial life.</p