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Volume 3 Issue 2
Apr.  2021
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Citation:

Cultivation of uncultured marine microorganisms

  • Corresponding author: Xiao-Hua Zhang, xhzhang@ouc.edu.cn
  • Received Date: 2020-12-18
    Accepted Date: 2021-01-11
    Published online: 2021-03-15
  • Edited by Chengchao Chen.
  • SPECIAL TOPIC: Cultivation of uncultured microorganisms.
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  • Alongi DM (2014) Carbon cycling and storage in mangrove forests. Ann Rev Mar Sci 6: 195-219 doi: 10.1146/annurev-marine-010213-135020
    Asem MD, Shi L, Jiao JY, Wang D, Han MX, Dong L, Liu F, Salam N, Li WJ (2018) Desertimonas flava gen. nov., sp. nov., isolated from a desert soil, and proposal of Ilumatobacteraceae fam. nov. Int J Syst Evol Microbiol 68: 3593-3599 doi: 10.1099/ijsem.0.003038
    Baker BJ, De Anda V, Seitz KW, Dombrowski N, Santoro AE, Lloyd KG (2020) Diversity, ecology and evolution of Archaea. Nat Microbiol 5: 887-900 doi: 10.1038/s41564-020-0715-z
    Castelle CJ, Banfield JF (2018) Major new microbial groups expand diversity and alter our understanding of the tree of life. Cell 172: 1181-1197 doi: 10.1016/j.cell.2018.02.016
    Cui H-L, Dyall-Smith ML (2021) Cultivation of halophilic archaea (class Halobacteria) from thalassohaline and athalassohaline environments. Mar Life Sci Technol 3: 242-250 doi: 10.1007/s42995-020-00087-3
    Ding L, Xu P, Zhang W, Yuan Y, He X, Su D, Shi Y, Naman CB, Yan X, Wu B, Lazaro JEH, Li S, He S (2020) Three new diketopiperazines from the previously uncultivable marine bacterium Gallaecimonas mangrovi HK-28 cultivated by iChip. Chem Biodiv 17: e2000221 
    Gutleben J, Chaib De Mares M, van Elsas JD, Smidt H, Overmann J, Sipkema D (2018) The multi-omics promise in context: from sequence to microbial isolate. Crit Rev Microbiol 44: 212-229 doi: 10.1080/1040841X.2017.1332003
    Hofer U (2018) The majority is uncultured. Nat Rev Microbiol 16: 716-717 doi: 10.1038/s41579-018-0097-x
    Hu B, Xu B, Yun J, Wang J, Xie B, Li C, Yu Y, Lan Y, Zhu Y, Dai X, Huang Y, Huang L, Pan J, Du W (2020) High-throughput single-cell cultivation reveals the underexplored rare biosphere in deep-sea sediments along the Southwest Indian Ridge. Lab Chip 20: 363-372 doi: 10.1039/C9LC00761J
    Hu B, Xu P, Ma L, Chen D, Wang J, Dai X, Huang L, Du W (2021a) One cell at a time: droplet-based microbial cultivation, screening and sequencing. Mar Life Sci Technol 3: 168-187 doi: 10.1007/s42995-020-00082-8
    Hu H, Natarajan VP, Wang F (2021b) Towards enriching and isolation of uncultivated archaea from marine sediments using a refined combination of conventional microbial cultivation methods. Mar Life Sci Technol 3: 230-241 doi: 10.1007/s42995-021-00092-0
    Hug LA, Baker BJ, Anantharaman K, Brown CT, Probst AJ, Castelle CJ, Butterfield CN, Hernsdorf AW, Amano Y, Ise K, Suzuki Y, Dudek N, Relman DA, Finstad KM, Amundson R, Thomas BC, Banfield JF (2016) A new view of the tree of life. Nat Microbiol 1: 16048 doi: 10.1038/nmicrobiol.2016.48
    Imachi H, Nobu MK, Nakahara N, Morono Y, Ogawara M, Takaki Y, Takano Y, Uematsu K, Ikuta T, Ito M, Matsui Y, Miyazaki M, Murata K, Saito Y, Sakai S, Song C, Tasumi E, Yamanaka Y, Yamaguchi T, Kamagata Y et al (2020) Isolation of an archaeon at the prokaryote-eukaryote interface. Nature 577: 519-525 doi: 10.1038/s41586-019-1916-6
    Jiang C, Dong L, Zhao J, Hu X, Shen C, Qiao Y, Zhang X, Wang Y, Ismagilov RF, Liu S, Du W (2016) High-throughput single-cell cultivation on microluidic streak plates. Appl Environ Microbiol 82: 2210-2218 doi: 10.1128/AEM.03588-15
    Jiao JY, Liu L, Hua ZS, Fang BZ, Zhou EM, Salam N, Hedlund BP, Li WJ (2020) Microbial dark matter coming to light: challenges and opportunities. Natl Sci Rev. https://doi.org/10.1093/nsr/nwaa280 doi: 10.1093/nsr/nwaa280
    Jung D, Liu L, He S (2021) Application of in situ cultivation in marine microbial resource mining. Mar Life Sci Technol 3: 147-160 doi: 10.1007/s42995-020-00063-x
    Liu H, Xue R, Wang Y, Stirling E, Ye S, Xu J, Ma B (2021) FACS-iChip: a high-efficient iChip system for microbial 'dark matter' mining in sediments. Mar Life Sci Technol 3: 161-167
    Lok C (2015) Mining the microbial dark matter. Nature 522: 270-273 doi: 10.1038/522270a
    Lyu L, Wang Q, Wang G (2021) Cultivation and diversity analysis of novel marine thraustochytrids. Mar Life Sci Technol 3: 262-274 doi: 10.1007/s42995-020-00069-5
    Mu D-S, Liang Q-Y, Wang X-M, Lu D-C, Shi M-J, Chen G-J, Du Z-J (2018) Metatranscriptomic and comparative genomic insights into resuscitation mechanisms during enrichment culturing. Microbiome 6: 230 doi: 10.1186/s40168-018-0613-2
    Mu D-S, Zhou L-Y, Liang Q-Y, Chen G-J, Du Z-J (2020a) Tichowtungia aerotolerans gen. nov., sp. nov., a novel representative of the phylum Kiritimatiellaeota and proposal of Tichowtungiaceae fam. nov., Tichowtungiales ord. nov. and Tichowtungiia class. nov. Int J Syst Evol Microbiol 70: 5001-5011
    Mu D-S, Wang S, Liang Q-Y, Du Z-Z, Tian R, Ouyang Y, Wang X-P, Zhou A, Gong Y, Chen G-J, Nostrand JV, Yang Y, Zhou J, Du Z-J (2020b) Bradymonabacteria, a novel bacterial predator group with versatile survival strategies in saline environments. Microbiome 8: 126 doi: 10.1186/s40168-020-00902-0
    Mu D-S, Ouyang Y, Chen G-J, Du Z-J (2021) Strategies for culturing active/dormant marine microbes. Mar Life Sci Technol 3: 120-130 doi: 10.1007/s42995-020-00053-z
    Nayfach S, Roux S, Seshadri R, Udwary D, Varghese N, Schulz F, Wu D, Paez-Espino D, Chen I-M, Huntemann M, Palaniappan K, Ladau J, Mukherjee S, Reddy TBK, Nielsen T, Kirton E, Faria JP, Edirisinghe JN, Henry CS, Jungbluth SP et al (2020) A genomic catalog of Earth's microbiomes. Nat Biotechnol. https://doi.org/10.1038/s41587-020-0718-6 doi: 10.1038/s41587-020-0718-6
    Oliver JD (2016) The viable but nonculturable state for bacteria: status update. Microbe 11: 159-164 
    Oren A (2014) The family Halobacteriaceae. In: Rosenberg E, DeLong EF, Lory S, Stackebrandt E, Thompson F (eds) The prokaryotes: other major lineages of bacteria and the archaea. Springer, Berlin, pp 41-121
    Overmann J, Lepleux C (2016) Marine bacteria and archaea: diversity, adaptations, and culturability. In: Stal LJ, Cretoiu MS (eds) The marine microbiome: an untapped source of biodiversity and biotechnological potential. Springer International Publishing, Cham, pp 21-56
    Pinto D, Santos MA, Chambel L (2015) Thirty years of viable but nonculturable state research: unsolved molecular mechanisms. Crit Rev Microbiol 41: 61-76 doi: 10.3109/1040841X.2013.794127
    Qin S, Li J, Chen HH, Zhao GZ, Zhu WY, Jiang CL, Xu LH, Li WJ (2009) Isolation, diversity and antimicrobial activity of rare actinobacteria from medicinal plants of tropical rainforests, Xishuangbanna. Appl Environ Microbiol 75: 6176-6186 doi: 10.1128/AEM.01034-09
    Rappé MS, Connon SA, Vergin KL, Giovannoni SJ (2002) Cultivation of the ubiquitous SAR11 marine bacterioplankton clade. Nature 418: 630-633 doi: 10.1038/nature00917
    Salam N, Xian W-D, Asem MD, Xiao M, Li W-J (2021) From ecophysiology to cultivation methodology: filling the knowledge gap between uncultured and cultured microbes. Mar Life Sci Technol 3: 131-146 doi: 10.1007/s42995-020-00064-w
    Spang A, Caceres EF, Ettema TJG (2017) Genomic exploration of the diversity, ecology, and evolution of the archaeal domain of life. Science 357: eaaf3883 doi: 10.1126/science.aaf3883
    Wang W, Li Z, Zeng L, Shao Z (2020) The oxidation of hydrocarbons by diverse heterotrophic and mixotrophic bacteria that inhabit deep-sea hydrothermal ecosystems. ISME J 14: 1994-2006 doi: 10.1038/s41396-020-0662-y
    Xian W-D, Salam N, Li M-M, Zhou E-M, Yin Y-R, Liu Z-T, Ming Y-Z, Zhang X-T, Wu G, Liu L, Xiao M, Jiang H-C, Li W-J (2020) Network-directed efficient isolation of previously uncultivated Chloroflexi and related bacteria in hot spring microbial mats. NPJ Biofilms Microb 6: 20 doi: 10.1038/s41522-020-0131-4
    Xu H-S, Roberts N, Singleton FL, Attwell RW, Grimes DJ, Colwell RR (1982) Survival and viability of nonculturable Escherichia coli and Vibrio cholerae in the estuarine and marine environment. Microb Ecol 8: 313-323 doi: 10.1007/BF02010671
    Zeng X, Alain K, Shao Z (2021) Microorganisms from deep-sea hydrothermal vents. Mar Life Sci Technol 3: 203-229 doi: 10.1007/s42995-020-00086-4
    Zhang C-J, Pan J, Duan C-H, Wang Y-M, Liu Y, Sun J, Zhou H-C, Song X, Li M (2019) Prokaryotic diversity in mangrove sediments across Southeastern China fundamentally differs from that in other biomes. mSystems 4: e00442-e519 
    Zhang C-J, Chen Y-L, Sun Y-H, Pan J, Cai M-W, Li M (2021a) Diversity, metabolism and cultivation of archaea in mangrove ecosystems. Mar Life Sci Technol 3: 251-261 doi: 10.1007/s42995-020-00081-9?utm_medium=cpc
    Zhang X-H, Ahmad W, Zhu X-Y, Chen J, Austin B (2021b) Viable but nonculturable bacteria and their resuscitation: implications for cultivating uncultured marine microorganisms. Mar Life Sci Technol 3: 188-202 doi: 10.1007/s42995-020-00041-3
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Cultivation of uncultured marine microorganisms

    Corresponding author: Xiao-Hua Zhang, xhzhang@ouc.edu.cn
  • 1. State Key Laboratory of Microbial Metabolism, School of Oceanography, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China
  • 2. Shenzhen Key Laboratory of Marine Microbiome Engineering, Institute for Advanced Study, Shenzhen University, Shenzhen 518060, China
  • 3. Key Laboratory of Microbial Resources, Institute of Microbiology Chinese Academy of Sciences, Beijing 100101, China
  • 4. College of Marine Life Sciences and Institute of Evolution and Marine Biodiversity, Ocean University of China, Qingdao 266003, China

Abstract: 

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  • Microorganisms (both pro- and eukaryotes) occur in all marine environments with an estimated abundance of 103-1010 cells/cm3 in sediments and 104-107 cells/ml in seawater; in these habitats they play key roles in marine biogeochemical cycles (Overmann and Lepleux 2016). Surveys employing molecular marker-based high-throughput sequencing and sophisticated metagenomic approaches have revealed the vast diversity of microorganisms in marine environments and have substantially expanded the tree of life (Castelle and Banfield 2018; Hug et al. 2016; Nayfach et al. 2020). However, many of the novel lineages have no cultured representatives. In fact, most (> 99%) of the marine microorganisms have not been cultured under laboratory conditions (Hofer 2018; Jiao et al. 2020) and are seen as "microbial dark matter" (Lok 2015). Although culture-independent approaches have enabled elucidation of the marine microbial assemblies and their metabolic potentials, pure cultures remain needed for microbiological studies. For example, pure cultures are critical for an understanding of the morphology, ecophysiology, and biochemistry of these organisms, which are key to recognizing their functions in complex environmental processes. Likewise, cultures are needed for discovering new bioactive substances, such as anti-tumor bioactive substances, new antibiotics and other secondary metabolites (Ding et al. 2020).

    There are a number of reasons for our inability to cultivate marine microorganisms in the laboratory, including a lack of adequate growth conditions, low growth rates, poor development of colonies, requirements for metabolites generated by other microbes, and the presence of dormant cells (Gutleben et al. 2018; Overmann and Lepleux 2016; Xu et al. 1982). Increasing efforts have been made to culture the uncultured microorganisms. Rapid progress in the development of cultivation techniques has resulted in the successful isolation of a number of novel microorganisms of pivotal importance from marine environments, e.g., the ubiquitous SAR11 marine bacterioplankton clade (Candidatus Pelagibacter ubique; Rappé et al. 2002) and an Asgard archaeon at the prokaryote-eukaryote interface (Candidatus Prometheoarchaeum syntrophicum; Imachi et al. 2020). Cultivation of a microbe is no longer viewed as an outdated craft in recent years, but a skill that requires not only imagination and perseverance but also the latest technologies. However, establishing a pure culture for an important (e.g., the dominant bacterial and archaeal phylotypes in marine environments) and yet uncultured microbe will still be a major challenge as well as a path to potential benefit. This special issue brings together nine review articles and two research papers that highlight various cultivation strategies for marine bacteria, archaea, and eukaryotic microorganisms. Below, we introduce these papers.

    In the ever-challenging marine environment, bacterial taxa may have different abundance and physiological states. In our first paper, based on the bacterial abundance and physiological states, Mu et al. (2021) categorize previously uncultured bacteria into three groups: dominant active bacteria, rare or low-abundance active bacteria, and dormant bacteria. They then propose techniques for isolating each group of uncultured bacteria, i.e., simulating the natural environment for cultivating dominant active bacteria, culturomics and enrichment culture methods for cultivating rare active bacteria, and resuscitation culture for isolating dormant bacteria. By using a modified enrichment culture method (Mu et al. 2018), the authors have isolated and described one novel class (Mu et al. 2020a), three novel orders, five novel families, and over 30 genera of Bacteria. They also found that the isolated bacteria within the order Bradymonadales represented a novel group of predatory bacteria that are widely distributed in the saline environments (Mu et al. 2020b).

    Microorganisms often occur as complicated multispecies communities in biofilms. Currently, we have limited knowledge of cell-to-cell interactions, within or between species; these interactions may alter the growth of species within the community. In our second paper, to culture biofilm microorganisms, Salam et al. (2021) summarize the progress in cultivation techniques and propose some promising strategies for microbial cultivation in future, including network-directed isolation, culturomics approach, and "Reverse-genomics". By improving isolation procedures and designing new cultivation techniques (Asem et al. 2018; Qin et al. 2009; Xian et al. 2020), the authors have isolated and described at least 500 novel bacterial and archaeal taxa, consisting of one new class, 23 new orders/sub-orders, 29 new families, and more than 80 new genera.

    In situ cultivation technique is one of the most popular techniques for cultivating uncultured microorganisms from marine environments (Overmann and Lepleux 2016). In our third paper, Jung et al. (2021) introduce various in situ cultivation methods, including diffusion chamber, microbial trap, iChip (isolation chip), iTip (in situ cultivation by tip), and double encapsulation techniques. They then propose a hypothesis explaining how in situ cultivation can access previously uncultured microorganisms. By using iChip, the authors cultivated a previously uncultivable marine bacterium that can produce three new diketopiperazines (Ding et al. 2020). In our fourth paper, Liu et al. (2021) further develop a FACS-iChip system by integrating iChip with flow cytometry-based fluorescence-activated cell sorting technique. The results of this study offer a highly efficient and readily available approach to mine uncultivable microorganisms in a nature environment.

    Microfluidic droplet-based technique is emerging as a promising new cultivation and screening strategy, with advantages of high-throughput, microscale, single-cell resolution, automation potential, and low cost (Hu et al. 2020; Jiang et al. 2016). In our fifth paper, Hu et al. (2021a) introduce key aspects of droplet microfluidics, including droplet manipulation, operation and detection. They then highlight the applications of microfluidic droplet-based techniques in microbiology, including high-throughput single-cell cultivation of uncultured microorganisms, droplets-based microbial co-cultivation, enzyme screening, as well as single-cell whole-genome sequencing.

    The viable but nonculturable (VBNC) state, first described by Xu et al. (1982), refers to a "dormant" state where bacteria are physiologically or metabolically active, but no longer capable of forming colonies on conventional culture media. This is likely a survival strategy to cope with unfavorable environmental stresses, and the bacterial cells in the VBNC state may restore culturability under appropriate conditions (Oliver 2016; Pinto et al. 2015). In our sixth paper, Zhang et al. (2021b) propose that restoration of VBNC cells into culture may be employed as an important strategy to cultivate the not yet-cultured marine microorganisms. They summarize a number of resuscitation stimuli that could be used to culture the previously uncultured marine bacteria, including resuscitation-promoting factors Rpfs and YeaZ, sodium pyruvate, quorum sensing molecules, catalase, and siderophore.

    Deep-sea hydrothermal vents (DSHV) host a rich diversity of microorganisms including chemolithoautotrophs, heterotrophs, and/or mixotrophs with versatile metabolic strategies. While large efforts have been made to isolate microbes from this unique environment, limited microbial taxa have been incubated in the laboratory. In our seventh paper, Zeng et al. (2021) review and compile available data and provide a list of isolates from DSHV with their taxonomic and physiological properties. Furthermore, they indicate the potential roles of these microbes in biogeochemical processes. It is noteworthy that the first culture representative of SAR324 was isolated from DSHV and confirmed as a mixotroph capable of sulfur oxidation and alkane oxidation (Wang et al. 2020). The authors further proposed putative novel approaches to bring more DSHV microbes into cultivation, especially by designing new cultivation approaches, real-time monitoring, and utilization of meta-omics data.

    Marine sediments host a variety of important, yet uncultivated archaeal groups including the superphylum Asgard, the Bathyarchaeota, the Woesearchaeota, and the ANaerobic MEthanotrophic (ANME) groups within the Euryarchaeota. In our eighth paper, Hu et al. (2021b) report on how they have explored the diversity of such uncultivated archaea using a combination of improved conventional methods, including cell extraction, cell fractionation, enrichment, and roll-bottle isolation. The authors demonstrate these methods by successfully obtaining three co-cultures composed of Bathyarchaeota (subgroup-8) and a bacterial species. This study, therefore, demonstrates that combining and improving conventional cultivation methods can offer a promising means to enrich and isolate slow-growing archaeal groups from marine sediments.

    Haloarchaea, affiliated to class Halobacteria and phylum Euryarchaeota, are a group of halophilic archaea inhabiting high salt environments and generally requiring over 100-150 g/L NaCl for optimum growth (Oren 2014). While haloarchaea are useful resources for theoretical and applied research, there remain widespread taxa (e.g., Nanohaloarchaea) that have not been isolated in pure culture. In our ninth paper, Cui et al. (2021) summarize the strategies (e.g., using washed agar, detergent-free culture vessels, long incubation times, and reduced nutrient concentration) for isolating halophilic archaea from thalassohaline and athalassohaline environments. They then propose new strategies to cultivate uncultured halophilic archaea.

    Mangrove ecosystems are widely distributed in tropical and subtropical coastal areas. They provide many ecological functions, such as protecting coastlines, carbon storage, and maintaining marco- and micro-diversity (Alongi 2014; Zhang et al. 2019). Archaea are becoming an important area of research interest in microbial fields, with many novel archaeal lineages discovered (Baker et al. 2020; Spang et al. 2017). In our tenth paper, Zhang et al. (2021a) summarize the diversity and metabolism of the archaeal community in mangrove ecosystems across the world and find that Bathyarchaeota, Euryarchaeota, Thaumarchaeota, Woesearchaeota, and Lokiarchaeota are the most abundant lineages with potential ecological roles in carbon, nitrogen, and sulfur cycles. In addition, the authors propose some strategies to culture the not yet-cultured archaeal lineages from mangrove ecosystems.

    Thraustochytrids are heterotrophic, unicellular marine protists, and have great potentials in accumulating large amounts of bioactive metabolites, including polyunsaturated fatty acids (PUFAs), carotenoids, and squalene. The isolation of novel thraustochytrid strains from marine environments is a major bottleneck for exploring their applications. In our eleventh paper, Lyu et al. (2021) summarize the commonly used methods (e.g., baiting and direct plating methods) for isolation of strains and propose some promising strategies, including dilution plating, co-cultivation, in situ isolation, diffusion chambers, and sponge-explant-based methods, adopted from other microorganisms (i.e., bacteria, archaea, and fungi).

    The 11 articles compiled in this special issue offer an extensive, but in no way exhaustive, review on strategies for the cultivation of uncultured marine microorganisms and their application in the isolation of various microbes. We hope that these papers will stimulate further research in this growing field.

Acknowledgements
  • The work was supported by the National Key Research and Development Program of China (2018YFE0124100), COMRA project (DY135-B2-12), and National Natural Science Foundation of China (41730530 and 91751202).

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