Current Articles

Editorial
Cultivation of uncultured marine microorganisms
Fengping Wang, Meng Li, Li Huang, Xiao-Hua Zhang
2021, 3(2): 117-120. doi: 10.1007/s42995-021-00093-z
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Marine Bacteria
Strategies for culturing active/dormant marine microbes
Da-Shuai Mu, Yang Ouyang, Guan-Jun Chen, Zong-Jun Du
2021, 3(2): 121-131. doi: 10.1007/s42995-020-00053-z
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Microorganisms are ubiquitous in the ocean environment and they play key roles in marine ecosystem function and service. However,many of their functions and phenotypes remain unknown because indigenous marine bacteria are mostly difficult to culture. Although many novel techniques have brought previously uncultured microbes into laboratory culture,there are still many most-wanted or key players that need to be cultured from marine environments. This review discusses possible reasons for 'unculturable microbes' and categorizes uncultured bacteria into three groups: dominant active bacteria,rare active bacteria,and dormant bacteria. This review also summarizes advances in cultivation techniques for culturing each group of unculturable bacteria. Simulating the natural environment is an effective strategy for isolating dominant active bacteria,whereas culturomics and enrichment culture methods are proposed for isolating rare active bacteria. For dormant bacteria,resuscitation culture is an appropriate strategy. Furthermore,the review provides a list of the most-wanted bacteria and proposes potential strategies for culturing these bacteria in marine environments. The review provides new insight into the development of strategies for the cultivation of specific groups of uncultured bacteria and therefore paves the way for the detection of novel microbes and their functions in marine ecosystems.
From ecophysiology to cultivation methodology: filling the knowledge gap between uncultured and cultured microbes
Nimaichand Salam, Wen-Dong Xian, Mipeshwaree Devi Asem, Min Xiao, Wen-Jun Li
2021, 3(2): 132-147. doi: 10.1007/s42995-020-00064-w
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Earth is dominated by a myriad of microbial communities, but the majority fails to grow under in situ laboratory conditions. The basic cause of unculturability is that bacteria dominantly occur as biofilms in natural environments. Earlier improvements in the culture techniques are mostly done by optimizing media components. However, with technological advancement particularly in the field of genome sequencing and cell imagining techniques, new tools have become available to understand the ecophysiology of microbial communities. Hence, it becomes easier to mimic environmental conditions in the culture plate. Other methods include co-culturing, emendation of growth factors, and cultivation after physical cell sorting. Most recently, techniques have been proposed for bacterial cultivation by employing genomic data to understand either microbial interactions (network-directed targeted bacterial isolation) or ecosystem engineering (reverse genomics). Hopefully, these techniques may be applied to almost all environmental samples, and help fill the gaps between the cultured and uncultured microbial communities.
Application of in situ cultivation in marine microbial resource mining
Dawoon Jung, Liwei Liu, Shan He
2021, 3(2): 148-161. doi: 10.1007/s42995-020-00063-x
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Microbial communities in marine habitats are regarded as underexplored reservoirs for discovering new natural products with potential application. However,only a few microbes in nature can be cultivated in the laboratory. This has led to the development of a variety of isolation and cultivation methods,and in situ cultivation is one of the most popular. Diverse in situ cultivation methods,with the same basic principle,have been applied to a variety of environmental samples. Compared with conventional approaches,these new methods are able to cultivate previously uncultured and phylogenetically novel microbes,many with biotechnological potential. This review introduces the various in situ cultivation methods for the isolation of previously uncultured microbial species and their potential for marine microbial resource mining. Furthermore,studies that investigated the key and previously unidentifed mechanisms of growing uncultivated microorganisms by in situ cultivation,which will shed light on the understanding of microbial uncultivability,were also reviewed.
FACS-iChip: a high-efficiency iChip system for microbial 'dark matter' mining
Haoze Liu, Ran Xue, Yiling Wang, Erinne Stirling, Shudi Ye, Jianming Xu, Bin Ma
2021, 3(2): 162-168. doi: 10.1007/s42995-020-00067-7
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The isolation chip method (iChip) provides a novel approach for culturing previously uncultivable microorganisms; this method is currently limited by the user being unable to ensure single-cell loading within individual wells. To address this limitation, we integrated flow cytometry-based fluorescence-activated cell sorting with a modified iChip (FACS-iChip) to effectively mine microbial dark matter in soils. This method was used for paddy soils with the aim of mining uncultivable microorganisms and making preliminary comparisons between the cultured microorganisms and the bulk soil via 16S rRNA gene sequencing. Results showed that the FACS-iChip achieved a culture recovery rate of almost 40% and a culture retrieval rate of 25%. Although nearly 500 strains were cultured from 19 genera with 8 FACS-iChip plates, only six genera could be identified via 16S rRNA gene amplification. This result suggests that the FACS-iChip is capable of detecting strains in the currently dead spaces of PCR-based sequencing technology. We, therefore, conclude that the FACS-iChip system provides a highly efficient and readily available approach for microbial 'dark matter' mining.
One cell at a time:droplet-based microbial cultivation, screening and sequencing
Beiyu Hu, Peng Xu, Liang Ma, Dongwei Chen, Jian Wang, Xin Dai, Li Huang, Wenbin Du
2021, 3(2): 169-188. doi: 10.1007/s42995-020-00082-8
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Microbes thrive and, in turn, influence the earth's environment, but most are poorly understood because of our limited capacity to reveal their natural diversity and function. Developing novel tools and effective strategies are critical to ease this dilemma and will help to understand their roles in ecology and human health. Recently, droplet microfluidics is emerging as a promising technology for microbial studies with value in microbial cultivating, screening, and sequencing. This review aims to provide an overview of droplet microfluidics techniques for microbial research. First, some critical points or steps in the microfluidic system are introduced, such as droplet stabilization, manipulation, and detection. We then highlight the recent progress of droplet-based methods for microbiological applications, from high-throughput single-cell cultivation, screening to the targeted or whole-genome sequencing of single cells.
Viable but nonculturable bacteria and their resuscitation: implications for cultivating uncultured marine microorganisms
Xiao-Hua Zhang, Waqar Ahmad, Xiao-Yu Zhu, Jixiang Chen, Brian Austin
2021, 3(2): 189-203. doi: 10.1007/s42995-020-00041-3
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Culturing has been the cornerstone of microbiology since Robert Koch first successfully cultured bacteria in the late nineteenth century. However, even today, the majority of microorganisms in the marine environment remain uncultivated. There are various explanations for the inability to culture bacteria in the laboratory, including lack of essential nutrients, osmotic support or incubation conditions, low growth rate, development of micro-colonies, and the presence of senescent or viable but nonculturable (VBNC) cells. In the marine environment, many bacteria have been associated with dormancy, as typified by the VBNC state. VBNC refers to a state where bacteria are metabolically active, but are no longer culturable on routine growth media. It is apparently a unique survival strategy that has been adopted by many microorganisms in response to harsh environmental conditions and the bacterial cells in the VBNC state may regain culturability under favorable conditions. The resuscitation of VBNC cells may well be an important way to cultivate the otherwise uncultured microorganisms in marine environments. Many resuscitation stimuli that promote the restoration of culturability have so far been identified; these include sodium pyruvate, quorum sensing autoinducers, resuscitation-promoting factors Rpfs and YeaZ, and catalase. In this review, we focus on the issues associated with bacterial culturability, the diversity of bacteria entering the VBNC state, mechanisms of induction into the VBNC state, resuscitation factors of VBNC cells and implications of VBNC resuscitation stimuli for cultivating these otherwise uncultured microorganisms. Bringing important microorganisms into culture is still important in the era of high-throughput sequencing as their ecological functions in the marine environment can often only be known through isolation and cultivation.
Microorganisms from deep-sea hydrothermal vents
Xiang Zeng, Karine Alain, Zongze Shao
2021, 3(2): 204-230. doi: 10.1007/s42995-020-00086-4
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With a rich variety of chemical energy sources and steep physical and chemical gradients, hydrothermal vent systems offer a range of habitats to support microbial life. Cultivation-dependent and independent studies have led to an emerging view that diverse microorganisms in deep-sea hydrothermal vents live their chemolithoautotrophic, heterotrophic, or mixotrophic life with versatile metabolic strategies. Biogeochemical processes are mediated by microorganisms, and notably, processes involving or coupling the carbon, sulfur, hydrogen, nitrogen, and metal cycles in these unique ecosystems. Here, we review the taxonomic and physiological diversity of microbial prokaryotic life from cosmopolitan to endemic taxa and emphasize their significant roles in the biogeochemical processes in deep-sea hydrothermal vents. According to the physiology of the targeted taxa and their needs inferred from meta-omics data, the media for selective cultivation can be designed with a wide range of physicochemical conditions such as temperature, pH, hydrostatic pressure, electron donors and acceptors, carbon sources, nitrogen sources, and growth factors. The application of novel cultivation techniques with real-time monitoring of microbial diversity and metabolic substrates and products are also recommended.
Marine Archaea
Towards enriching and isolation of uncultivated archaea from marine sediments using a refined combination of conventional microbial cultivation methods
Haining Hu, Vengadesh Perumal Natarajan, Fengping Wang
2021, 3(2): 231-242. doi: 10.1007/s42995-021-00092-0
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The archaea that can be readily cultivated in the laboratory are only a small fraction of the total diversity that exists in nature. Although molecular ecology methods, such as metagenomic sequencing, can provide valuable information independent of cell cultivation, it is only through cultivation-based experiments that they may be fully characterized, both for their physiological and ecological properties. Here, we report our efforts towards enriching and isolation of uncultivated archaea from marine sediments using a refined combination of conventional microbial cultivation methods. Initially, cells were retrieved from the sediment samples through a cell extraction procedure and the sediment-free mixed cells were then divided into different size-range fractions by successive filtration through 0.8 njm, 0.6 njm and 0.2 njm membranes. Archaeal 16S rRNA gene analyses indicated noticeable retention of different archaeal groups in different fractions. For each fraction, supplementation with a variety of defined substrates (e.g., methane, sulfate, and lignin) and stepwise dilutions led to highly active enrichment cultures of several archaeal groups with Bathyarchaeota most prominently enriched. Finally, using a roll-bottle technique, three co-cultures consisting of Bathyarchaeota (subgroup-8) and a bacterial species affiliated with either Pseudomonas or Glutamicibacter were obtained. Our results demonstrate that a combination of cell extraction, size fractionation, and roll-bottle isolation methods could be a useful protocol for the successful enrichment and isolation of numerous slow-growing archaeal groups from marine sediments.
Cultivation of halophilic archaea (class Halobacteria) from thalassohaline and athalassohaline environments
Heng-Lin Cui, Mike L. Dyall-Smith
2021, 3(2): 243-251. doi: 10.1007/s42995-020-00087-3
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As a group, the halophilic archaea (class Halobacteria) are the most salt-requiring and salt-resistant microorganisms within the domain Archaea. Halophilic archaea flourish in thalassohaline and athalassohaline environments and require over 100Ƀ150 g/L NaCl for growth and structural stability. Natural hypersaline environments vary in salt concentration, chemical composition and pH, and occur in climates ranging from tropical to polar and even under-sea. Accordingly, their resident haloarchaeal species vary enormously, as do their individual population compositions and community structures. These diverse halophilic archaeal strains are precious resources for theoretical and applied research but assessing their taxonomic and metabolic novelty and diversity in natural environments has been technically difficult up until recently. Environmental DNA-based high-throughput sequencing technology has now matured sufficiently to allow inexpensive recovery of massive amounts of sequence data, revealing the distribution and community composition of halophilic archaea in different hypersaline environments. While cultivation of haloarchaea is slow and tedious, and only recovers a fraction of the natural diversity, it is the conventional means of describing new species, and provides strains for detailed study. As of the end of May 2020, the class Halobacteria contains 71 genera and 275 species, 49.8% of which were first isolated from the marine salt environment and 50.2% from the inland salt environment, indicating that both thalassohaline and athalassohaline environments contain diverse halophilic archaea. However, there remain taxa that have not yet been isolated in pure culture, such as the nanohaloarchaea, which are widespread in the salt environment and may be one of the hot spots in the field of halophilic archaea research in the future. In this review, we focus on the cultivation strategies that have been used to isolate extremely halophilic archaea and point out some of the pitfalls and challenges.
Diversity, metabolism and cultivation of archaea in mangrove ecosystems
Cui-Jing Zhang, Yu-Lian Chen, Yi-Hua Sun, Jie Pan, Ming-Wei Cai, Meng Li
2021, 3(2): 252-262. doi: 10.1007/s42995-020-00081-9
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Mangroves comprise a globally significant intertidal ecosystem that contains a high diversity of microorganisms, including fungi, bacteria and archaea. Archaea is a major domain of life that plays important roles in biogeochemical cycles in these ecosystems. In this review, the potential roles of archaea in mangroves are briefly highlighted. Then, the diversity and metabolism of archaeal community of mangrove ecosystems across the world are summarized and Bathyarchaeota, Euryarchaeota, Thaumarchaeota, Woesearchaeota, and Lokiarchaeota are confirmed as the most abundant and ubiquitous archaeal groups. The metabolic potential of these archaeal groups indicates their important ecological function in carbon, nitrogen and sulfur cycling. Finally, some cultivation strategies that could be applied to uncultivated archaeal lineages from mangrove wetlands are suggested, including refinements to traditional cultivation methods based on genomic and transcriptomic information, and numerous innovative cultivation techniques such as single-cell isolation and high-throughput culturing (HTC). These cultivation strategies provide more opportunities to obtain previously uncultured archaea.
Marine Eukaryotic Microorganisms
Cultivation and diversity analysis of novel marine thraustochytrids
Lu Lyu, Qiuzhen Wang, Guangyi Wang
2021, 3(2): 263-275. doi: 10.1007/s42995-020-00069-5
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Thraustochytrids are a group of unicellular marine heterotrophic protists, and have long been known for their biotechnological potentials in producing squalene, polyunsaturated fatty acids (PUFAs) and other bioactive products. There are less than a hundred known strains from diverse marine habitats. Therefore, the discovery of new strains from natural environments is still one of the major limitations for fully exploring this interesting group of marine protists. At present, numerous attempts have been made to study thraustochytrids, mainly focusing on isolating new strains, analyzing the diversity in specific marine habitats, and increasing the yield of bioactive substances. There is a lack of a systematic study of the culturable diversity, and cultivation strategies. This paper reviews the distribution and diversity of culturable thraustochytrids from a range of marine environments, and describes in detail the most commonly used isolation methods and the control of culture parameters. Furthermore, the perspective approaches of isolation and cultivation for the discovery of new strains are discussed. Finally, the future directions of novel marine thraustochytrid research are proposed. The ultimate goal is to promote the awareness of biotechnological potentials of culturable thraustochytrid strains in industrial and biomedical applications.
Correction
Correction to: FACS-iChip: a high-efficiency iChip system for microbial ndark mattero mining
Haoze Liu, Ran Xue, Yiling Wang, Erinne Stirling, Shudi Ye, Jianming Xu, Bin Ma
2021, 3(2): 276-277. doi: 10.1007/s42995-020-00079-3
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