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Volume 3 Issue 2
Apr.  2021
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Citation:

Application of in situ cultivation in marine microbial resource mining

  • Corresponding author: Shan He, heshan@nbu.edu.cn
  • Received Date: 2020-04-23
    Accepted Date: 2020-06-28
    Published online: 2020-10-06
  • SPECIAL TOPIC: Cultivation of uncultured microorganisms.
  • Edited by Chengchao Chen.
  • 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.
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Application of in situ cultivation in marine microbial resource mining

    Corresponding author: Shan He, heshan@nbu.edu.cn
  • 1. Li Dak Sum Yip Yio Chin Kenneth Li Marine Biopharmaceutical Research Center, College of Food and Pharmaceutical Sciences, Ningbo University, Ningbo 315832, China

Abstract: 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.

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Introduction
  • Microorganisms from marine environments are regarded as a major source for natural products discovery. These represent a wide diversity of biological activities, including antimicrobial, anti-inflammatory and anti-tumor activities (Blunt et al. 2018; Liu et al. 2010; Srilekha et al. 2017; Xiong et al. 2012, 2013). Pure cultivation of microorganisms is essential to enable efficient discovery of novel natural products with industrial and biotechnological potential, especially for new antibiotics. Despite the availability of culture-independent molecular tools for analysis of microbial communities in natural environments (Wang et al. 2012) and heterologous expression of biosynthetic gene clusters (Gomez-Escribano and Bibb 2014; Nah et al. 2017; Ongley et al. 2013), culture-dependent approaches are still necessary, because a comprehensive physiological characterization and a full assessment of application potential can only be performed through the isolation of individual bacterial strains in pure culture (Vartoukian et al. 2010).

    Most marine microbial species, however, are unculturable and cannot grow under regular laboratory conditions (Rinke et al. 2013). Only a few of these (generally about 1%) can grow into colonies on standard agar media, thus leading to the 'great plate count anomaly', a feature for more than 120 years (Amann 1911; Amann et al. 1995; Staley and Konopka 1985; Winterberg 1898). This oldest unresolved problem in microbiology is still the major bottleneck in bioprospecting of marine microbial resources. A lot of effort has been devoted to developing alternative methods to overcome the limitations of conventional cultivation. Modifying growth conditions by the addition of specific organic or inorganic compounds to the media (Nguyen et al. 2018), lowering nutrient concentrations (Janssen et al. 2002), extending incubation times (Davis et al. 2005), enrichment culturing (Mu et al. 2018), and the use of alternative gelling agents (Tamaki et al. 2005) have all allowed an improvement in microbial recovery, but most of the postulated microbial taxa from natural environments remain uncultured. This suggests that microbial uncultivability is not easily explained by the unfitness of specific species to certain cultivation conditions, such as nutrient of culture media, pH, temperature, and gas composition (Stewart 2012). Therefore, other essential but unknown factors, that could more generally and critically affect the growth of such uncultured microbes, would be absent from those artificial conditions.

    One simple solution to the problem of unidentified growth factors is to cultivate the microbes in their native environments. Applying this concept to microbial cultivation has resulted in the development of in situ cultivation techniques, such as diffusion chambers, that aim to better simulate environment conditions (Kaeberlein et al. 2002). Furthermore, several modified in situ cultivation approaches, that share the same basic rationale, have been proposed and tested in diverse environments, e.g., activated sludge, alkaline soda lakes, hot springs, sediments, sponges and soil environments. These have led to the isolation of many previously uncultured microbes (Aoi et al. 2009; Bollmann et al. 2007; Jung et al. 2013, 2014, 2016, 2018; Steinert et al. 2014). Therefore, in situ incubation can be a powerful alternative to discover novel and valuable marine microbial resources. Furthermore, the mechanisms that were identified through in situ cultivation approaches will further support and foster the innovation of isolation techniques, which may be the turning point for bioprospecting of marine microbial resources.

    The purpose of this paper is to summarize in situ cultivation techniques for the isolation of previously uncultivated bacterial species and to assess the potential of these approaches for marine microbial resource mining. In the first part of the review, the history of in situ cultivation techniques is outlined and their advantages and potential are discussed (Table 1). In the second part the integration of different methods to improve the understanding of microbial uncultivability is discussed.

    Technique Sample Properties Advantage Disadvantage References
    Diffusion chamber Marine sediment and soil Leading technique of in situ cultivation Simple manufacturing process Low efficiency for high-throughput cultivation Bollmann et al. (2007); Kaeberlein et al. (2002)
    Trap for in situ cultivation Soil Trap methods for filamentous actinobacteria Selective cultivation of actinobacteria Novelty of isolates is not so high Gavrish et al. (2008)
    Ichip Marine sediment and soil Containing multiple (384) diffusion chambers in one device High-throughput cultivation Requires special equipment Nichols et al. (2010)
    Modified ichip Soil Simpler version of original ichip much easier and economical to make Leading to build-up of anoxic conditions below the ichip Berdy et al. (2017)
    I-tip Sponge Trap method for isolation of sponge-associated microbes More flexible for work with irregular surface such as aquatic invertebrates Complex manufacturing process Jung et al. (2014)
    Double encapsulation technique Coral mucus, wastewater and soil Encapsulation of bacteria in agarose spheres and incubated in situ Isolation of novel bacteria and unicellular eukaryotic organisms Complex manufacturing process Ben-Dov et al. (2009)
    In situ culturing device for flowing borehole Following borehole The device using acetate, formate, methanol, Fe3 citrate and SO4 enriched agar Understanding subsurface lithoautotrophic microbial ecosystems (SLiMEs) Enriched microbes in the in situ cultivation could not be cultured in lab cultivations Silver et al. (2010)
    Diffusion bioreactor Soil Diffusion bioreactor containing liquid media Use of liquid enrichment media for long-term incubations The concept is similar to diffusion chamber Chaudhary et al. (2019)

    Table 1.  List of in situ cultivation techniques applied to various environments, and its properties, advantages and disadvantages of each technique. Techniques listed in the table followed the order described in this paper

Development and modification of in situ cultivation methods

    Diffusion chamber and microbial trap as a leading technique of in situ cultivation

  • In situ cultivation methods began with the following questions; why do most microorganisms that already grow well in their natural environment, not grow under laboratory conditions? Given that they grow well in their natural environment, why not just cultivate them there? To answer these questions, Kaeberlein et al. (2002) first developed a diffusion chamber that aimed to better simulate natural conditions. Diffusion chambers consist of a stainless steel or plastic washer and membranes with a 0.03 µm pore size (Fig. 1a). After the membrane is attached to the bottom of the washer, the inoculum (diluted environmental cells mixed with 40 ℃ agar) is placed inside the chamber, another membrane then seals the chamber. Once assembled, the chamber is placed in the original natural environment and the inoculated microbes are incubated for 1-4 weeks. During in situ cultivation, the membranes allow the exchange of growth factors and nutrients between the natural habitat and the agar. This approach should minimize differences in the chemical environment on either side of the membrane, thus simulating the natural conditions inside the chamber. Diffusion chambers have been used with samples from diverse environments (e.g., sediment, soil, and marine sponges) and have proved to be highly efficient in terms of both microbial recovery and the novelty of isolated species (Bollmann et al. 2007; Jung et al. 2016; Kaeberlein et al. 2002). Starting with this technology, many in situ cultivation methods have been developed to isolate previously uncultured novel microorganisms.

    Figure 1.  Conceptual design and application of the diffusion chamber (a) and the microbial trap (b) (Epstein et al. 2010)

    Gavrish et al. (2008) used diffusion chambers to cultivate filamentous bacteria, such as Actinobacteria, and fungi, known to be rich sources of bioactive compounds. Filamentous fungi and actinomycetes grow as thread-like filaments capable of penetrating substrates (Gavrish et al. 2008). Since actinomycetes hyphae can pass through 0.2 µm pores, a trap was designed to capture these filamentous microorganisms (Fig. 1b). The design of the trap is similar to the diffusion chamber, except that the pore size of the membrane is larger (0.2 µm) and the cells are not inoculated onto the agar at the beginning (Fig. 1a, b). This method has been used with soil samples and resulted in the isolation of unusual and rare actinomycetes. This result suggests that modification of the in situ cultivation method can make it widely applicable in many fields.

  • High-throughput in situ cultivation of previously inaccessible microorganisms and discovery of novel antibiotics using ichip

  • The demand for new microbial resources for in situ cultivation has led to the continued development of the method. Diffusion chambers have produced larger and more novel microbial diversities than standard techniques, but they need laborious separation for pure cultivation and so cannot efficiently perform high-throughput cultivation. To streamline this process into a high-throughput system, Nichols et al. (2010) designed a cultivation platform that allowed the cultivation of novel species and simultaneously support pure cultivation with hundreds of individual chambers on a single device. This platform was named 'ichip'. The ichip is an array of miniature diffusion chambers that are loaded with one cell on average per individual culture.

    The original ichip contained 384 holes that become individual small growth chambers. The device consists of a central plate for cultivating bacterial cells, membranes with a 0.03 µm pore size on both the top and bottom of the plate and side panels holding all other parts. When the plate containing the multiple through-holes is dipped into a suspension of microbial cells in warm agar (Fig. 2a), each hole captures a micro volume of this mixture and is then covered by the membranes after solidification (Fig. 2b). The number of cells in each chamber is controlled by adjusting the initial concentration of cells in the warm agar used for inoculum. To control the cell density of the inoculum, cell numbers in the pretreated sample were counted by fluorescence microscopy and the optimal dilution was determined. The membranes were fixed in place by the side panels and all structures were secured with screws (Fig. 2c). The ichip was tested with seawater and soil samples. Compared with the standard direct agar plating method, ichip allows better recovery and greater biodiversity of taxa, including many of previously uncultivated novel bacterial species.

    Figure 2.  Conceptual design of the ichip. A plate including 384 through-holes is dipped into a suspension containing microbial cells in warm agar (a). The dilution of the suspension is such that each hole captures one cell on average (b). Membranes cover through holes on each side; upper and bottom plates with matching holes press the membranes against the center loaded plate. Screws provide sufficient pressure to seal the content of individual through holes, which become a miniature diffusion chamber (c) (Nichols et al. 2010)

    The ichip was opitimized into a much simpler form by Berdy et al. (2017). This used an empty, commercially-produced pipet tip rack. This contained 96 through-holes, and any autoclavable material, such as plastic plate, rubber sheet, silicone sheet and polydimethylsiloxane (PDMS), can be used in construction. After a rectangular membrane is glued to one side of the plate, the diluted cell suspension (one cell on average) with warm agar is inoculated inside by a multichannel pipette; another membrane closes the chambers. This modified ichip shares the same principle as the original but is much easier and more economical to make. Besides, the through-holes of the rack correspond to wells of standard 96-well plates. This allows much greater parallel growth and pretreatment for further pure cultivation and identification of grown isolates.

    The ichip technique has been successfully applied to the high-throughput mining of uncultured microbes for novel antibiotic discovery by Novobiotic Pharmaceuticals in Boston (co-founded by Slava Epstein, the major inventor of the diffusion chamber and ichip). From tens of thousands of uncultured microbes obtained by ichip, a novel compound with potent antibacterial activity and a novel mechanism of action was isolated from Eleftheria terrae (new species of Betaproteobacteria). The molecule, named "teixobactin", showed high efficacy in the treatment of some gram-positive pathogens, such as Mycobacterium and Staphylococcus, without generating any detectable resistance (Ling et al. 2015). Teixobactin is the first novel antibiotic discovered from bacteria in decades (McCarthy 2019; Piddock 2015). It turned out to be a new class of antibiotics with a novel mechanism of action. This result suggests that the new microbial cultivation techniques will lead to further antibiotic discoveries.

  • I-tip, an in situ cultivation method for aquatic invertebrate symbiotic microbes

  • Most aquatic invertebrates are rich sources of secondary metabolites that have great potential for their antitumoral, antiviral, antibacterial and neuroprotective properties (Anjum et al. 2016; Koopmans et al. 2009; Liu et al. 2019). It is known that many of these bioactive compounds are actually from their symbiotic microbes (Flatt et al. 2005; König et al. 2006; Wang 2006). Therefore, the abundant microbial resources in these symbiotic ecosystems have been noted widely for their potential for new natural products (Blunt et al. 2018).

    Previous in situ cultivation devices work well in environments with flat surfaces, such as aquatic sediment and soil, but are difficult to use in aquatic invertebrates which have an irregular surface. Therefore, Jung et al. (2014) developed an in situ cultivation device that was more flexible for work on the isolation of symbiotic microbes of aquatic invertebrates. This equipment was named 'in situ cultivation by tip', or 'I-tip' for short. It is so named because the main element of the device was a commercial micropipette tip. The I-tip consists of microbeads and agar layers inside the tip. The pointed end of the tip makes it possible to position it on the surface of the target organism, such as sponge (Fig. 3a). The microbead layer can prevent the invasion of larger organisms from the target sample and agar layer, including the medium, and create space for microbial cultivation. The upper part of the I-tip is covered by a waterproof adhesive to prevent contamination. After the I-tip is installed to target host organisms, microbes are expected to grow in the agar layer with chemicals diffusing from the natural habitat, while the bead layer prevents the entry of too many microbes and larger organisms from the environment (Fig. 3b). The I-tip was tested with endemic sponges in Lake Baikal and showed that it was highly efficient in isolating formerly uncultivated sponge-associated bacteria (Jung et al. 2014).

    Figure 3.  The photo showing installation of the I-tips device into the sponges (a) and schematic images showing structure and principle of I-tip (b) (Jung et al. 2014)

    Since the I-tip shares the same basic rationale with the trap method (Gavrish et al. 2008; Fig. 1b), it is able to target filamentous bacteria such as Actinobacteria. When the I-tip is packed with glass beads, 50-100 μm in diameter, it produces 0.2-0.3 μm interstitial channels (Jung et al. 2014). Since actinomycetes hyphae can pass through 0.2 µm pores and that size is too small for the passage of eukaryotes and other bacterial cells, I-tip with a smaller sized bead-layer could provide better conditions for the selective isolation of Actinobacteria, known to be a rich source of bioactive compounds (Gavrish et al. 2008). The results suggested that most actinomycetes were obtained using I-tips with a smaller size of beads (50-100 μm in diameter) whereas the larger beads (150-212 μm) were better able to isolate other groups of bacteria and fungi.

  • Other in situ cultivation methods that have led to the discovery of novel microbial species

  • Other than the in situ cultivation methods mentioned above, there are many other innovative in situ cultivation techniques that have led to the isolation of novel microbial species. Ben-Dov et al. (2009) encapsulated bacteria from coral mucus, wastewater and soil in agarose spheres encased in polysulfone, and incubated them in either simulated or natural environments. This double encapsulation technique stimulates the growth of the entrapped bacteria by allowing them access to essential growth factors from their natural environments. The environmental microbe containing agar spheres were incubated in situ and allowed the isolation of previously uncultured bacteria and unicellular eukaryotic organisms.

    Silver et al. (2010) applied an in situ cultivation device to a flowing borehole in a mafic sill at a depth of 1474 m in the Evander Au mine, South Africa and found subsurface lithoautotrophic microbial ecosystems (SLiMEs). Even though enriched microbes in the in situ cultivation device could not be cultured in lab cultivations, 16S rDNA sequences indicated that a diverse community, including methanogenic, Fe reducing and SO4 reducing microbes, was incubated in situ and that the subsets of this community could be enriched according to which combination of electron acceptors and donors in the environment were utilized.

    Recently, Chaudhary et al. (2019) developed a 'diffusion bioreactor' for the isolation of uncultured soil bacteria. This new cultivation technique shared a similar basic principle to the diffusion chamber but is distinct from former with regard to the size of the bioreactor, the membrane pore size, and the use of liquid enrichment media. The diffusion bioreactor used soil samples and successfully enriched bacterial species diversity during an in situ incubation and facilitates their cultivation on agar plates in a further step. In summary, a number of in situ culture methods have been developed or modified, and these have significantly contributed to the isolation of new microbes from a wide range of environments.

Application of in situ cultivations to marine environmental samples and aquatic invertebrates

    Results of diffusion chamber and ichip from marine sediment samples (growth recovery and novelty of isolates)

  • The diffusion chamber was applied to marine sediment in Massachusetts Bay, USA. After 1-3 weeks of in situ cultivation, the chambers were retrieved and the bacterial colonies that had grown in agar were examined, counted and subcultured by transfer into new chambers. Growth recovery of the inoculated cells was significantly different between the in situ cultivation (diffusion chamber) and standard agar plate cultivation. A recovery rate of ~ 22% (up to 40%) was observed in the diffusion chambers, exceeding by many hundred times what could be achieved using standard agar media (Kaeberlein et al. 2002). This suggests that the in situ cultivation led to the isolation of uncultured microbes that were fastidious or impossible to be cultivated using standard cultivation methods.

    Nichols et al. (2010) applied the ichip to seawater in Nahant, USA, examining the colony count and bacterial diversity of isolates from the in situ cultivation and compared the results with those from standard direct plating methods. After two weeks of in situ cultivation, about 50 agar samples with growing bacteria were retrieved from the ichip so the colonies could be examined by microscope. The remaining plugs were used to isolate DNA for identification by 16S rRNA gene analysis. The results led to three clear conclusions. Firstly, the ichip allowed a higher cell recovery during in situ cultivation, which exceeded the the number of colonies recovered by the standard direct plating cultivation by about fivefold. Secondly, only one isolated species was common between isolates from the ichip and the standard cultivation method, even the same inoculum used for each. Thirdly, the novelty of isolates from ichip was much higher than that of isolates from the standard cultivation method (Fig. 4). These results suggest that ichip cultivation allowed the isolation of microbial species from diverse groups, which produced different isolates from conventional methods. In addition, this approach led to the isolation of rarely cultivated classes and phyla, such as Deltaproteobacteria, Epsilonproteobacteria, Verrucomicrobia and Spirochaetes that could not be cultivated on standard agar plates (Nichols et al. 2010). Thus, the ichip technique allows in situ cultivation in a high-throughput manner; this can produce a greater number and more unique microbial species with a minimal amount of effort and time from the marine environment.

    Figure 4.  Novelty of seawater and soil microbial strains grown in ichip and standard direct plating cultivation. Sequence novelty, in percentage of diversion from the known species, and its equation to taxonomic rank (genus, family level, etc.) is very approximate (Nichols et al. 2010)

  • Application of in situ cultivation methods, I-tip and modified diffusion chambers for studying microbial diversity in sponges and isolation of previously uncultivated bacteria

  • Aquatic invertebrates, such as sponges and corals, contain diverse microbial symbionts that have great potential for valuable secondary metabolites (Anjum et al. 2016; Wang 2006), however, pure cultivation of such species is a challenge due to their host dependency. To overcome this, a new in situ cultivation device, I-tip and a modified version of the diffusion chamber, has been applied to several sponge samples.

    I-tips were embedded in two species of endemic sponges in Lake Baikal, Baicalospngia sp. and Lubomirskia baicalensis and then incubated for 4 weeks (Jung et al. 2014). After the devices were retrieved from the sponges, subculturing was performed on agar media using the I-tip agar layers (Fig. 3b). This resulted in the isolation of 34 species belonging to five taxonomic groups, Actinobacteria, Alphaproteobacteria, Betaproteobacteria, Firmicutes, and Gammaproteobacteria, whereas standard direct plating method yielded a much lower diversity of 16 species in the taxonomic groups, Betaproteobacteria, Firmicutes, and Gammaproteobacteria (Fig. 5). Shannon-Weaver diversity index confirmed that bacterial species from I-tip were much more diverse than those from standard cultivation (I-tip: 26.8, standard cultivation: 13.1). In addition, microbial species from I-tip cultivation showed a higher novelty compared to the results from standard direct plating, checked statistically using the Chi-Square statistic. These results indicate that the newly developed I-tip can isolate more diverse and novel species which better represents the diversity in the environment. They also work well in more challenging environmental samples, such as aquatic invertebrates.

    Figure 5.  Phylogenetic affiliations of bacterial isolates from Baikalian sponges by standard cultivation (a) and I-tip (b) on the basis of 16S rRNA gene sequences. Left part of each cultivation represent the result from Baicalospongia sp., and right part from Lubomirskia baicalensis. In situ cultivation technology, I-tip could isolate a richer collection of more novel microbial strains that is better representation of the natural diversity (Jung et al. 2014)

    Modified diffusion chambers were applied to marine sponges to isolate previously uncultured bacterial symbionts. The isolation efficiency was compared with those using the standard cultivation method (Jung et al. under review). A rectangular silicone rubber frame (w = 3 cm, l = 1.2 cm, h = 0.3 cm) was used instead of the stainless steel washer that was previously used in the original diffusion chamber. It was thus small enough to be installed into the target sponges with minimum damage to the organisms. After the small diffusion chambers were assembled with the inoculum, the same size of the groove as the diffusion chamber were made on the surface of the marine sponges by cutting with a surgical knife and the devices were then installed. After in situ cultivation for one week, chambers were opened, and subculturing was performed on agar media using the grown microbes. The diffusion chambers enabled the isolation of 37 species belonging to six phyla, whereas the conventional method isolated only 13 species belonging to three phyla (Fig. 6a). Shannon-Weaver diversity index of isolated bacterial species from diffusion chamber was much higher than that obtained by standard cultivation (diffusion chamber: 25.0, standard cultivation: 9.7). The ratios of novel species also differed between the two approachs, only one isolate (1.7%) from standard cultivation was a novel species, while 24 isolates (40%) from the diffusion chambers were novel species (Fig. 6b).

    Figure 6.  Diversity and novelty of isolates from in situ (diffusion chamber) and standard cultivation method. Venn diagram consisting of phylogenetic trees based on the 16S rRNA gene of isolates from each cultivation method (a). The numbers in parentheses represent the numbers of novel species. Similarity of isolates from each cultivation method based on 16S rRNA gene to the closest known relative in GenBank databases (b). Numbers in the bar graphs represent OTUs numbers in each similarity level and numbers in parentheses represent the ratio of novel species (Jung et al. under review)

  • Application of in situ cultivation for marine microbial resource mining

  • Previous studies have shown that in situ cultivation methods are guaranteed to isolate previously uncultivated microbial diversity to discover new natural products (Berdy et al. 2017). The diffusion chamber and ichip techniques are being used by a microbial cultivation lab at Marine Biopharmaceutical Research Center (Ningbo University, China) to isolate previously uncultivated microorganisms as a source of new marine natural products (Jung et al. under review). Using marine environmental samples (seawater and marine sediment) in the intertidal zone, numerous previously uncultured microorganisms were isolated (Fig. 7). In addition, the ichip cultivation method was also applied to the mangrove sediment environment of Hainan Island, China by the same research group. Several novel species were isolated and some of those were reported after phylogenetic and physiological characterization (Zhang et al. 2018a, b ). The complete genomic sequencing of one novel species was also performed, Saccharospirillum mangrovi HK-33T, to understand its ecological status in the mangrove environment (Zhang et al. 2019).

    Figure 7.  Phylogenetic tree based on 16S rRNA gene sequences of novel isolates from marine and mangrove environments. The sequences of isolates derived from in situ cultivation methods are shown in blue (diffusion chamber) or red (ichip) with related the closest strain in databases of EZBIOCLOUD (https://www.ezbiocloud.net). Circle (sea water), square (sea sediment) and triangle (mangrove sediment) indicate environmental sources of the isolates. The trees were constructed by the Neighbor-Joining method with the MEGA package using partial 16S rRNA gene sequences. The bar represents five substitutions per 100 nucleotide positions. Bootstrap probabilities are indicated at branch nodes. The NCBI/GenBank accession numbers for isolates are shown in parentheses

  • Principle of in situ cultivation to access previously uncultured microorganisms

  • In situ cultivation methods have certainly produced new microbial species but the reasons why these approaches are able to cultivate previously uncultured species are not well understood. Recently, Jung et al. (under review) investigated the key and unidentified mechanisms of growing uncultivated microorganisms by focusing on growth triggering via resuscitation factors in natural environments. Previous studies have noted that the in situ growth of otherwise uncultivated microbes is adapted for ex situ growth (Berdy et al. 2017; Bollmann et al. 2007; Jung et al. 2013, 2014). Therefore, that study examined what mechanism of in situ cultivation could explain this observation. It was assumed that some in situ factor is present that induces nongrowing microbes and those that were activated to maintain their growth, even on standard agar plate.

    Following this hypothesis, the effect of chemical compounds from the environment (sponge extract in that study) on the efficiency of colony formation on agar medium was compared between isolates from in situ and standard cultivation. To evaluate that, the ratio of colony formation between the two culture methods (with and without the sponge extract) were measured for each tested strain. When the sponge extracts (0.1% per total volume of medium) were added to the medium, the colony formation efficiency of the isolates from in situ cultivation was significantly higher, while it did not positively affect isolates from the standard cultivation. Conversely, the maximum growth (carrying capacity on growth curve) and growth rate of isolates from both in situ and standard cultivation were not affected by adding sponge extracts. This outcome suggests several causes to explain the mechanism of in situ cultivation. First, some chemical substances in the environment can improve cell recovery and these signaling molecules selectively work on the isolates from in situ cultivation. Second, such signaling-like molecules do not promote the growth (both growth rate and maximum growth) of microbes in the natural environment but initiate their growth. This is probably causes resuscitation from the nongrowing state, such as dormancy and viable but nonculturable cells (VBNC).

    Many previous studies have suggested that most microbes extracted from the environment are not ready to grow in laboratory conditions; these are referred to as being in a non-growing state such as near-zero growth (NZG), dormancy and VBNC (Lennon and Jones 2011; Panikov et al. 2015; Pinto et al. 2015). This would be one of the major reasons for microbial uncultivability (Fig. 8a). During in situ incubation, some resuscitation factors (signaling molecules) are supplied from the environment (i.e., marine sediments, marine sponge, and seawater) and this trigger the regrowth of some microbial species into a nongrowing state (Fig. 8b). Finally, these cells are sufficiently enriched inside the in situ cultivation device to maintain their growth even after their subcultivation step on a standard agar plate (Fig. 8c). Conversely, such resuscitation factors do not exist in standard media, so most of the cells from the environment do not recover from a non-growing state and thus do not form colonies (Fig. 8c). Since some isolates have been cultivated from conventional cultivation methods and do not need the signaling molecules to grow on standard agar media, they frequently grow in the laboratory but must have been isolated previously. Therefore, microbial species obtained by in situ cultivation are unique and relatively novel compared to those isolated by standard cultivation.

    Figure 8.  Hypothesis explaining how in situ cultivation cultivates different culture collections from microbial recovery in a natural environment. Certain microbial types are not ready to grow (non-growing state) to survive unfavorable conditions such as nutrient limited condition (a). During in situ incubation, dormant microbes are induced from a non-growing state by some resuscitation factors from the outside environment. They then regrow and become enriched, supported by nutrients in the chamber (b). Most microbes from environment do not recover from a non-growing state without resuscitation factors and thus do not form colonies. When they were sub-cultured after the in situ cultivation, they maintain their growth an agar plate, and resulting visible colony appearance (c) (Jung et al. under review)

    Microbes in their natural environments produce a variety of chemical compounds, such as signaling-like molecules (resuscitation factors) but these could be produced by several microbes. A few attempts have been made to discover such microbial interactions via signaling molecules that promote the growth of specific microbial types. Short peptides (Nichols et al. 2008), siderophores (D'Onofrio et al. 2010) and quinones (Fenn et al. 2017) have been identified as signaling molecules that can promote bacterial growth. However, these signaling molecules only work for specific strains, so more effort will be needed to identify additional growth factors that can be widely used widely in microbial cultivation for isolation of unculturable microbes. Therefore, the next challenge will be to identify the growth-initiating factors and the donors and acceptors of such signaling molecules (ex, who produces what and to whom). This will allow a new insight into previously uncultured marine microbes.

  • Other strategies and future prospects of in situ cultivation methods

  • Other approaches need to be taken into consideration when attempting to isolate uncultured microorganisms. For example, 'dilution-to-extinction' cultivation is an effective method for cultivating marine bacteria. This approach led to the isolation of the most abundant, but the previously uncultured, group in the marine environment, the SAR11 clade (Connon and Giovannoni 2002; Rappé et al. 2002). This method shares a similar concept with ichip, that uses the dilution of bacteria of up to one to ten cells per well in microplates, but its incubation is performed in liquid media. The exact nature of the adaptations in microbes that foster colony formation on the solid medium is still unknown and this is one of the major reasons for the uncultivability of microbes in the laboratory (Schut et al. 1993). Therefore, liquid medium mixed with sea salt potentially provides easier conditions for marine bacteria to adapt and promotes greater reproduction and growth compared to the solid medium containing the same nutrients.

    Another approach is a simple modification of the agar media to reduce phosphate-catalyzed hydrogen peroxide formation (H2O2). Studies have reported that interaction between phosphate and agar during media preparation produces H2O2, which detrimentally affects the cultivability of bacteria on media (Tanaka et al. 2014). Therefore, H2O2 scavenging agents such as catalase have been used for the preparation of agar media to improve the efficiency of colony formation and isolation (Imazaki and Kobori 2010; Mendis et al. 2018). Recent research has demonstrated that this modification of the agar media can improve the cultivability of bacteria by reducing oxidative stress, compared with the standard agar medium (Kato et al. 2018; Kawasaki and Kamagata 2017). Therefore, it may be necessary to consider applying such strategies when developing new situational cultivation methods. For example, the recently developed diffusion bioreactor used liquid media for in situ cultivation and allowed for the growth of both diverse and previously uncultured soil bacteria (Chaudhary et al. 2019).

    Although many in situ cultivation methods have been demonstrated to be highly capable of microbial cultivation, several limitations need to be considered to further develop this technique. First, previous studies have shown that many interesting microbial groups enriched by in situ cultivation could not be cultivated in subcultivation steps in the laboratory (Silver et al. 2010). In other cases, successfully subcultivated bacterial strains from in situ cultivation have lost their growth activity after several steps of subcultivation on glycerol stock, and some isolates were completely lost from the culture collection (Jung et al. under review). Bollman et al. (2007) attempted to determine whether several rounds of in situ cultivation with diffusion chambers would enable the domestication of enriched strains from the first stage of in situ cultivation. The results indicated that there was a positive correlation between the number of repeated in situ culturing attempts and the probability of obtaining ex situ grown strains. However, since such steps are complicated and time consuming, simpler methods should be developed for the isolation of such missing strains, such as identifying key growth factors and supplying those for subcultivation. Second, some of the in situ cultivation techniques require special materials and complicated processes, so they are difficult to use more generally in laboratories or commercially. To solve such limitations, manufacturing and experimental process of the techniques have to be simplified and their efficiency confirmed.

    In addition, more effort should be put into the identification of the key factors in the mechanism of in situ cultivation. The high diversity of microbial species obtained by in situ cultivation techniques will contribute to the understanding of microbial uncultivability through the further study of such previously uncultured microbes.

    In summary, the application of in situ cultivation methods and discovering the mechanisms are a promising approach to understanding microbial uncultivability, however, it would be not the only way to access uncultured microbial species. Together with other strategies, the in situ cultivation method needs to be modified and continuously developed. It has the potential to be a gamechanger for bioprospecting, not only in marine microbial communities but in a broad range of other environmental habitats.

Data availability
  • Newly determined sequence data have been deposited in GenBank (https://www.ncbi.nlm.nih.gov) under accession numbers MT254895, MT254896, MT254909-MT254915, MT254933-MT254945 and MT260804-MT260837.

  • Acknowledgements

  • This work was funded by the National Natural Science Foundation of China (41776168, 31600016), the National 111 Project of China (D16013), and Li Dak Sum Yip Yio Chin Kenneth Li Marine Biopharmaceutical Development Fund.

  • Author contributions

  • DJ wrote the manuscript. LL and SH revised the manuscript. DJ and SH developed the concept and designed the outline.

Compliance with ethical standards

    Conflict of interest

  • All the authors declare that there are no conficts of interest.

  • Animal and human rights statement

  • This article does not contain any studies with human participants or animals performed by the authors.

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