Importance of isolation and culture of difficult-to-cultivation microorganisms in deep sea

Tenericutes are a group of bacteria without cell wall and with small genome, which have special research value in environmental adaptation mechanism and evolution, but most of them have not obtained pure culture.

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Deep-sea microorganisms (bacteria, archaea and eukaryotic organisms, etc.) have abundant genetic and metabolic diversities, but due to the limitations of sampling and culture conditions, many deep-sea microorganisms cannot be isolated and cultured, the fundamental role of deep-sea microorganisms (especially difficult-to-cultivation microorganisms) in the cycle of geochemical elements is still poorly understood. Thus, for different deep-sea environments (such as abyss, hydrothermal, cold spring, seamounts, etc.) of microbial pure culture and reveals their metabolic pathway mediated substances drive element circulation mechanism, help to clarify for deep-sea microbes in the ocean and the whole earth scope element circulation contribution, and achieve the depth of deep-sea microbial resources mining.

The deep-sea cold seep is a chemosynthetic ecosystem and contain an extensive diversity of bacteria and archaea which play important roles in element metabolisms, such as carbon cycling, nitrogen cycling, sulfur cycling and phosphorus cycling (Jaekel et al., 2013; Zhang et al., 2020). We have tried to culture these difficult-to-culture microorganisms from deep-sea sediments using different isolation strategies. The deep-sea samples were collected by RV KEXUE from a typical cold seep in the South China Sea (E119º17'07.322'', N22º06'58.598'') (Fig.1 and Fig.2).

Fig.1. Deep-sea sampling devices.

Fig.2. The scientific expedition vessel-Kexue.

Fortunately, we have obtained the first isolation of a Candidatus Izemoplasma representative from the deep-sea methane seep, strain zrk13, using a DNA degradation-driven method given Izemoplasma’s prominent DNA-degradation potentials. Candidatus Izemoplasma, an intermediate in the reductive evolution from Firmicutes to Mollicutes, was proposed to represent a novel class of free-living wall-less bacteria within the phylum Tenericutes. Strain zrk13 could degrade and utilize the extracellular DNA for growth in both laboratorial and deep-sea conditions (Fig.3). Moreover, the predicted genes determining DNA-degradation broadly distribute in the genomes of other Izemoplasma members. Given that extracellular DNA is a particularly crucial phosphorus as well as nitrogen and carbon source for microorganisms in the seafloor, Izemoplasma bacteria are thought to be important contributors to the biogeochemical cycling in the deep ocean.

Fig.3. Views of the in situ experimental apparatus in the deep-sea cold seep where distributed many mussels and shrimps.

Obtaining pure cultures of these difficult-to-culture microorganisms through special isolation strategies is a key step to extensively understand and utilize the microbial resources. However, to truly understand the metabolic characteristics of microorganisms, we cannot rely solely on genomic analysis and physiological characteristics under laboratorial conditions. We need to cultivate these microorganisms in the in situ environment to explore and verify their real metabolic characteristics.

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1. Jaekel U, Musat N, Adam B, Kuypers M, Grundmann O, Musat F. (2013). Anaerobic Degradation of Propane and Butane by Sulfate-Reducing Bacteria Enriched from Marine Hydrocarbon Cold Seeps. Isme J 7: 885-895.

2. Zhang J, Liu R, Xi SC, Cai RN, Zhang X, Sun CM. (2020). A Novel Bacterial Thiosulfate Oxidation Pathway Provides a New Clue About the Formation of Zero-Valent Sulfur in Deep Sea. Isme J 14: 2261-2274.

Rikuan Zheng

Doctor, Institute of oceanology, University of Chinese Academy of Sciences