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From ecophysiology to cultivation methodology: filling the knowledge gap between uncultured and cultured microbes

  • Corresponding author: Wen-Jun Li, liwenjun3@mail.sysu.edu.cn
  • Received Date: 2020-05-26
    Accepted Date: 2020-07-22
    Published online: 2020-09-08
  • Edited by Chengchao Chen.
  • SPECIAL TOPIC:Cultivation of uncultured microorganisms
  • 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.
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    Zhang Y-G, Chen J-Y, Wang H-F, Xiao M, Yang L-L, Guo J-W, Zhou E-M, Zhang Y-M, Li W-J. (2016a). Egicoccus halophilus gen. nov., sp. nov., a halophilic, alkalitolerant actinobacterium and proposal of Egicoccaceae fam. nov. and Egicoccales ord. nov. Int J Syst Evol Microbiol, 66:530-535 doi: 10.1099/ijsem.0.000749
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From ecophysiology to cultivation methodology: filling the knowledge gap between uncultured and cultured microbes

    Corresponding author: Wen-Jun Li, liwenjun3@mail.sysu.edu.cn
  • 1. State Key Laboratory of Biocontrol, Guangdong Provincial Key Laboratory of Plant Resources and Southern Marine Sciences and Engineering Guangdong Laboratory (Zhuhai), School of Life Science and School of Ecology, Sun Yat-Sen University, Guangzhou, China
  • 2. State Key Laboratory of Desert and Oasis Ecology, Xinjiang Institute of Ecology and Geography, Chinese Academy of Sciences, Urumqi, China

Abstract: 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.

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Introduction
  • Bacterial cultivation was revolutionized with the accidental discovery of agar as a solidifying agent in the preparation of culture media in the late nineteenth century (Hesse 1992). Subsequently, plenty of bacterial and fungal strains have been brought into pure cultures in laboratory conditions for downstream investigation. As of April 2020, 16, 529 bacterial and archaeal species have been described with valid nomenclature (https://lpsn.dsmz.de/text/numbers; accessed on 24 May 2020). These described species largely fall within the bacterial phyla of Proteobacteria, Firmicutes, Actinobacteria, and Bacteroidetes (Rinke et al. 2013).

    Bacterial diversity has been largely estimated on the basis of molecular markers, particularly the 16S rRNA gene sequence (Pace 1997). A systematic comparison between the described bacterial species and the global bacterial diversity indices indicated that most (> 99%) bacteria escaped cultivation, i.e., not-yet-cultured (Bernard et al. 2000; Hofer 2018; Kim and Yu 2012; Lok 2015; Overmann et al. 2017). The culture-elusive bacteria are, therefore, often considered as the "microbial dark matter" (Lok 2015). Their unculturability may be ascribed to various factors, including actual resistance to grow on conventional media and requirement of specific growth factors or conditions (Vartoukian et al. 2010). In addition, many biodiversity hotspots remain underexplored even by the conventional approaches (Mincer et al. 2002; Nimaichand et al. 2012, 2013; Qin et al. 2009).

    Much effort has been devoted to cultivation of the not-yet-cultured microbial majority over the last decades (Asem et al. 2018; Rinke et al. 2014; Zhou et al. 2018). The conventional approach is often tedious, with a strong bias to the isolation of certain dominant bacterial groups. Frequently, this leads to isolation of already known strains (Fig. 1). However, many novel strains were effectively isolated by employing selective isolation procedures through pre-treating samples (Nimaichand et al. 2012) and/or through optimizing media to mimic the natural environment (Kaeberlein et al. 2002). Moreover, certain strains could not cope with the simulated environment during subsequent cultivation, and thus lose their viability. In recent years, development and advancement of sequencing technology have helped devise new and advanced culturomics approaches (Cross et al. 2019; Lagier et al. 2016; Xian et al. 2020). Despite their limitations, these new developments are expected to provide access to many novel previously uncultivated bacteria, and to divulge their metabolic pathways as well as ecological roles.

    Figure 1.  Progress in cultivation methods and promising strategies for future microbial isolation. Newly developed methods include selective isolation procedures (pre-treating samples, mimicking the environmental conditions), culturomics approaches, dilution media, diffusion chambers, co-culture, physical cell sorting using microfluidic streak, and enrichment before isolation. Promising strategies include network-directed isolation, culturomics approach, and the "Reverse-genomics" cultivation approach

Biofilm as a major factor for unculturability
  • Factors causing unculturability for microorganisms have been discussed in several reviews (Gutleben et al. 2018; Overmann et al. 2017; Vartoukian et al. 2010). The most common factor that limits our capacity to culture bacteria is the lack of understanding of suitable growth conditions. Conversely, a considerable number of bacteria exist in the form of biofilms in natural habitats (Nikolaev and Plakunov 2007); and no sufficient methods can effectively segregate a particular species from a complex community without affecting their viability (Fig. 2).

    Figure 2.  Biofilm and unculturability. Microorganisms occur as complex multispecies communities in biofilms. Limited knowledge of cell-to-cell interactions caused the unculturability of majority of members therein. These interactions occur either within the species or between different species, and may include diffusion of small molecules, such as amino acids, siderophores, and vitamins

    In the marine ecosystem, bacteria form aggregates on the available particulate matter (Teeling et al. 2012). The bacterial diversity profile is highly diverse in marine biofilms, and consists of several unclassified groups, including members of the classes Solibacteres, Acidobacteria, and Blastocatellia (Zhang et al. 2019). Moreover, > 70% of the bacterial community in hot spring microbial mats belong to not-yet-cultured microbial groups (Xian et al. 2020). Similarly, uncultured microbial communities are dominant in biofilms associated with most of other unique and underexplored environments, such as deserts, acid mine drainages, human guts (Amin et al. 2020; Lear et al. 2009; Lozupone et al. 2012; Madinger et al. 2016), or even in the largely explored soil ecosystem (Lindahl 1996) where they aggregate and adhere to any available surface.

    Within a biofilm, microorganisms occur as complex multispecies communities (Stoodley et al. 2002). The resistance of a biofilm community to external stresses is linked to its complex coordination (Fig. 2), often related to quorum sensing, rather than the physiological activity of individual cells (Ratzke and Gore 2018). This closed coordination involved cross-signalling in chemical language within the communities through cell-to-cell interactions (Zengler and Palsson 2012). The microbial populations therein are, therefore, adapted to a more specialized life form, which is physiologically and evolutionarily different from free-living planktonic forms of the constituent populations (Burmølle et al. 2014). In certain cases, the genomic constituents of the biofilm communities are streamlined, indicating a probable dependency of individual cells on the community interactions (He et al. 2015; Mee et al. 2014; Morris et al. 2012).

    Diffusion of small molecules, for example, amino acids, siderophores, and vitamins, is part of the central metabolism in biofilm communities (Cordero and Datta 2016; D'Onofrio et al. 2010) (Fig. 2). When proximal microbial populations share complementary metabolic stocks, they perform physiological functions better than planktonic free-living cells (Cordero and Datta 2016). The dependency of most biofilm microbial populations on the neighbouring cells for growth factors is one major factor that makes them recalcitrant to culture in its pure form (Mee et al. 2014). Another important factor of unculturability of biofilm components is the adaptive gene loss within the microbial population that drives species co-occurrence (Zelezniak et al. 2015).

Microbial ecophysiology
  • In microbial ecology, the –omics study based on nucleic acids (metagenomics and metatranscriptomics), peptides and small molecules (metaproteomics), and metabolites (metabolomics) has advanced our understanding of the microbial biosphere (Gutleben et al. 2018). Development in sequencing technology, particularly the rRNA sequencing, has helped us to determine microbial community structure. Yet, their ecophysiological roles are largely unknown and cannot be predicted (Narihiro et al. 2009). Additionally, environmental heterogeneity and inter- and intra-species competition of resources influence the spatial distribution pattern of the bacterial communities. Consequently, relationship cannot be determined between the dynamic bacterial community structure and their in situ function in the habitat without an understanding of local microenvironments (Okabe et al. 2011). Besides, the physiological properties predicted from cultured representatives may not also sufficiently define the actual function in the ecology. Advanced techniques, such as fluorescence in situ hybridization, microsensors, and microautoradiography, are now employed to analyse the in situ function of single microbial communities (Okabe et al. 2004). A combined use of the above-mentioned techniques can sufficiently connect specific microbial population or cells to a specific metabolic function within complex microbial communities.

    Alternatively, the physiology of one microorganism may be assessed from its genome (Wagner et al. 2002), but it may not always be generalized. For example, some bacteria are versatile in substrate utilization (Kragelund et al. 2006), whereas others are very specialized (Andreasen and Nielsen 2000). Many bacteria possess an unusual physiology and ecology. Despite the limitations, metagenomes-assembled genomes and single-cell genomics are used often to determine the ecological and physiological aspects of uncultivated microorganisms (Rinke et al. 2013). In most cases, a good quality genome can provide a more accurate prediction about the lifestyle of single organisms. For example, the genome quality of single-cell amplified genomes is usually very low; but an improved method devised by Rinke et al. (2014) based on fluorescence-activated cell sorting-based single-cell genomics generates up to 1 μg of genomic DNA for single microbial cells. Based on the sensitivity of the sequencing protocols, an average of~50% completeness may be recorded.

    Coming to the concept of ecophysiology, let us consider the example of the phylum Chloroflexi. Cultivated members of Chloroflexi are anoxygenic photoautotrophs, aerobic chemoheterotrophs, thermophiles, and anaerobes that obtain their energy by dehalogenation of organic chlorinated compounds (Gupta et al. 2013). Chloroflexi lacks a typical lipid outer cell member, and is considered a monoderm (Sutcliffe 2010, 2011). Members of this phylum have often eluded cultivation, despite representing a larger portion in the community structure of many ecosystems, including freshwater and marine environments (up to 30% (Mehrshad et al. 2018)), extreme environments such as hot springs (Lau et al. 2009), and hypersaline microbial mats (Ley et al. 2006) and activated sludge treatment plants (Kragelund et al. 2007; Speirs et al. 2019). Some common features, such as slow growth and syntrophy, make Chloroflexi (particularly the classes Anaerolineae and Caldilineae) recalcitrant to isolation. Growth of these groups is easily outcompeted by fast-growing heterotrophic anaerobes.

    The first genomic insights related to the lifestyle of uncultivated Chloroflexi as being flagellated, aerobic photoheterophic and capacity to demineralize organic matter was predicted from a lake metagenome (Denef et al. 2016). Analysis of the 117 metagenomes of Chloroflexi from lakes, reservoirs, and river samples showed that there is a remarkably high diversity of Chloroflexi among the aquatic ecosystem. However, different lineages have different distribution patterns among the analysed samples. For example, cluster JG30-KF-CM66 sequences are distributed preferentially in rivers rather than in lakes. GOS datasets are basically freshwater dominant lineages, instead of marine groups as originally assigned; or SAR202-related clades are not related to water columns (Mehrshad et al. 2018). Also, these genomes showed the presence of genes necessary for central carbohydrate metabolism and denitrification, and the absence of assimilatory sulphate reduction gene. This suggests a totally heterotrophic lifestyle for Chloroflexi. The absence of genes encoding assimilatory sulphate reduction could be related to the utilization of exogenous reduced sulphur for growth, as shown with the SAR11 group (Tripp et al. 2008). Energy generation in Chloroflexi is generally driven through rhodopsin (prediction of carotenoid biosynthesis in Chloroflexia) or aerobic anoxygenic phototrophy (presence of genes encoding enzymes for PS-Ⅱ, and bacteriochlorophyl and carotenoid biosynthesis; absence of carbon fixing pathway); and is depicted also among different lineages of Chloroflexi (Mehrshad et al. 2018). In another study, it was predicted that there was a massive gene loss for phototrophism in most lineages of Chloroflexi. However, a few lineages acquired phototrophy later via horizontal gene transfer from a different ancestral donor (Ward et al. 2018).

Culturing the not-yet-cultured microbial majority
  • To meet the needs of various analyses, it is necessary to obtain microbes in pure culture. Exploration of new microbial resources was initiated mostly on the basis of searching for new microbial metabolites for the treatment of human diseases. The conventional plating method was largely successful until the mid-1950s. Subsequently, the isolation of new isolates became redundant as the majority of the isolation procedures was focused on soil microbes. Here, there was a biased selection for fastidious isolates on the growth medium. Over the years, many research groups have attempted new techniques, including supplementation of antibiotics to inhibit the growth of fungi and fast-growing bacteria (Williams and Davies 1965), emending media with compounds that reduce growth stress (Martin et al. 1976), or overlaying of porous membranes on isolation plates (Hirsch and Christensen 1983). Dilution culture (Button et al. 1993), use of diffusion chambers (Kaeberlein et al. 2002), co-culture (D'Onofrio et al. 2010), and physical cell sorting using microfluidic streaks (Jiang et al. 2016) are a few of the other techniques that have led to the description of new taxa. However, the overlap is still very low between the number of cultured and not-yet-cultured microorganisms.

    Our laboratory has been focusing on microbial exploration in extreme and unusual habitats, particularly hypersaline environments, hot springs, deserts, caves, alkaline soils, acid mine drainage, marine and estuary, and tissues of medicinal plants, and determining the accurate taxonomy of the cultivated isolates (Fig. 3). To date, we have finished the description of at least 500 novel bacterial and archaeal taxa, most of which were recovered by improving isolation procedures, including pre-treatment conditions, media optimization, and designing new isolation methods (Asem et al. 2018; Qin et al. 2009; Xian et al. 2020). These novel microbial taxa consisted of one new class, 23 new orders/sub-orders, 29 new families, and over 80 new genera.

    Figure 3.  Isolating previously uncultivated microbes through media optimization. Previously uncultured members were continuously cultivated from hot springs (a), deserts (e), and salt lakes (i). Some filamentous Chloroflexi isolates (c, d) were isolated from hot spring microbial mats (a) on an SCM medium (c) in comparison to the conventional medium (b) realized the potential of directed isolation of targeted members in this phylum. Novel species belonging to Micromonospora (g, h) were retrieved from hyper-arid desert soils (e) using a diluted (1/4) R2A medium (f). This isolation procedure provided an effective way of isolating putatively uncultured Micromonospora species from desert soils. Halophilic archaeal Halorubrum spp. (k, l) from salt lake can be cultivated with a media of high salt concentration (j, k), the crystallization of the salt can be seen in the agar plates (k). All the strains were isolated in our laboratory

  • Media optimization

  • Nutrient concentrations in extreme habitats are mostly limited. For example, hypersaline environments have high salt contents; chemical composition of salts may differ depending on the origin of the habitats (Ventosa et al. 2008). Adaptability to the unusually high content of salts limits microbial distribution in such environments (Ventosa et al. 2015). Also, microbial community structures differ greatly along an ecological gradient of hypersaline soil and sediments (Hollister et al. 2010). Based on the physiology of the microorganisms at different concentrations of salts, they are classified as non-halophiles (<  1% NaCl), halotolerant (non-halophiles but tolerate as high as 25% NaCl), slight halophiles (optimal growth with 1–3% NaCl), moderate halophiles (optimal growth with 3–15% NaCl), and extreme halophiles (grow optimally with 15–25% NaCl or with saturated salt concentrations) (Kushner and Kamekura 1988).

    Halophilic and halotolerant microorganisms were isolated usually by supplementing high concentrations of salt (10–30% NaCl or KCl) in the conventional media (Cui et al. 2001b; Jiang et al. 2006; Li et al. 2004a, c) (Fig. 3j, k). All the halophilic/halotolerant microorganisms isolated in our laboratory are listed in Table 1. Mechanisms for the dependency of these halophiles to different concentrations of salts (Na+, K+, Ca2+, Mg2+) seem to be complex (Jiang et al. 2006). However, halophilic bacteria and archaea respond to the osmotic pressure of the high salt concentration in the environment most likely by subsequent cellular adaptation including morphological changes (Pianetti et al. 2009) and excretion of exopolysaccharide layers (Liu et al. 2019; Xue et al. 2018).

    Name of novel taxa Highest taxonomic rank proposed with the novel taxa Isolation condition* Source/sampling site References
    Actinopolyspora alba Novel species CCMS + 15% NaCl/37 ℃/ 3 weeks Baicheng salt field/Xinjiang, China (Tang et al. 2011a)
    Actinopolyspora erythraea Novel species CCMS + 15% NaCl/37 ℃/3 weeks Baicheng salt field/Xinjiang, China (Tang et al. 2011a)
    Aidingimonas halophila Novel genus CCMS Aiding lake sediment/Xinjiang, China (Wang et al. 2009b)
    Alkalibacillus halophilus Novel species SG + 25% NaCl/37 ℃/2–3 weeks Hypersaline soil/Xinjiang, China (Tian et al. 2007)
    Alteromonas halophila Novel species MA + 20% NaCl/28 ℃/1–4 weeks Sea anemone/Naozhou Island, China (Chen et al. 2009i)
    Amycolatopsis halophila Novel species CCMS / 37 ℃/3 weeks Qijiaojing Lake soil/Xinjiang, China (Tang et al. 2010a)
    Amycolicicoccus subflavus Novel genus ASW + 2.4% NaCl/30 ℃/2 days Oil-polluted saline soil/Shengli Oilfield, China (Wang et al. 2010)
    Arthrobacter halodurans Novel species MA + 20% NaCl/28 ℃/2 weeks Sea water/Naozhou Island, South China Sea (Chen et al. 2009g)
    Brevibacterium album Novel species ISP5 + 15% KCl / 37 ℃/2 weeks Saline soil/ Xinjiang, China (Tang et al. 2008c)
    Corynebacterium halotolerans Novel species ISP5 + 15% KCl + trace elements /28 ℃ / 2–3 days Saline soil/Xinjiang, China (Chen et al. 2004)
    Egibacter rhizosphaerae Novel order R2A + 10% NaCl (pH 10)/ 30 ℃ / 4 weeks Rhizosphere of Tamarix hispida/Xinjiang, China (Zhang et al. 2016e)
    Egicoccus halophilus Novel order MA (pH 10)/30 ℃/4 weeks Saline-alkali soil/Shihezi, Xinjiang, China (Zhang et al. 2016a)
    Georgenia alba Novel species R2A/28 ℃/5 days Desert sand/Saudi Arabia (Li et al. 2019)
    Georgenia deserti Novel species R2A/28 ℃/5 days Desert sand/Saudi Arabia (Hozzein et al. 2018)
    Georgenia halophila Novel species GTY + 10% NaCl/37 ℃/3 weeks Qijiaojing Lake soil/Xinjiang, China (Tang et al. 2010b)
    Gracilibacillus halophilus Novel species MA + 20% NaCl/30 ℃/4 weeks Saline soil/Qinghai, China (Chen et al. 2008c)
    Gracilibacillus quinghaiensis Novel species MA/28 ℃ Xiaochaidamu salt lake sediment/Qinghai, China (Chen et al. 2008d)
    Gracilibacillus saliphilus Novel species ISP5 + 10% NaCl Ebinur Lake sample/Xinjiang, China (Tang et al. 2009c)
    Haladaptatus pallidirubidus Novel species CCMS/30 ℃/4 weeks Saline soil from Lop Nur/Xinjiang, China (Liu et al. 2014)
    Halegenticoccus soli Novel genus GM + 20% NaCl / 37 ℃ / 4 weeks Ebi Lake soil/ Xinjiang, China (Liu et al. 2019)
    Haloactinopolyspora alkaliphila Novel species CCMS / 30 ℃/3 weeks Saline-alkali soil/Xinjiang, China (Zhang et al. 2014)
    Haloactinobacterium album Novel genus GTY + 10% NaCl / 37 ℃/3 weeks Qijiaojing Lake soil/Xinjiang, China (Tang et al. 2010e)
    Haloactinopolyspora alba Novel genus CCMS/37 ℃/3 weeks Qijiaojing lake sample/Xinjiang, China (Tang et al. 2011b)
    Haloactinospora alba Novel genus CCMS + 10% NaCl/37 ℃/3 weeks Salt lake/Xinjiang, China (Tang et al. 2008a)
    Halobacillus hunanensis Novel species MA + 30% NaCl/28 ℃/1–4 weeks Brine sample from Xiangli Salt Mine/Hunan, China (Peng et al. 2009)
    Halobacillus naozhouensis Novel species MA + 5% NaCl / 28 ℃/2 weeks Sea anemone/Naozhou, China (Chen et al. 2009f)
    Halobacillus salsuginis Novel species MA + 5% NaCl / 30 ℃/2 weeks Brine sample from Xiangli Salt Mine/Hunan, China (Chen et al. 2009k)
    Haloechinothrix alba Novel genus CCMS + 10% NaCl/37 ℃/3 weeks Qijiaojing Lake soil/Xinjiang, China (Tang et al. 2010c)
    Haloglycomyces albus Novel genus CCMS + 10% NaCl/ 37 ℃/2–4 weeks Hypersaline soil/Xinjiang, China (Guan et al. 2009)
    Halomonas flava Novel species GTY + 10% NaCl / 37 ℃ / 1 week Qijiaojing Lake sediment/Xinjiang, China (Chen et al. 2011a)
    Halomonas litopenaei Novel species MA / 28 ℃/5 days Larviculture water/ Donghai Island, Guangdong, China (Xue et al. 2018)
    Halomonas lutea Novel species ISP5 + 10% NaCl/37 ℃ Ebinur Lake sample/Xinjiang, China (Wang et al. 2008)
    Halomonas nanhaiensis Novel species ISP5 + 3.5% sea salt /8 ℃/30 days Sediment sample/South China Sea (Long et al. 2013)
    Halomonas qijiaojingensis Novel species GTY + 10% NaCl / 37 ℃/1 week Qijiaojing Lake sediment/Xinjiang, China (Chen et al. 2011a)
    Halomonas taeanensis Novel species MA + 8% NaCl / 35 ℃ / 2 days Solar saltern sample/Taean, Korea (Lee et al. 2005)
    Halomonas xianhensis Novel species SSDM Saline soil/ Xianhe, Shangdong, China (Zhao et al. 2012)
    Halomonas xiaochaidanensis Novel species R2A + ASW/15 ℃/3 days (after enrichment for 2 days in the same liquid media) Xiaochaidan Lake sediment/Tibet, China (Liu et al. 2016)
    Halomonas zhanjiangensis Novel species MA / 28 ℃ / 1 week Sea urchin/Naozhou Island, China (Chen et al. 2009j)
    Halopelagius fulvigenes Novel species GM + 10% NaCl/37 ℃/4 weeks Qijiaojing lake soil/Xinjiang, China (Liu et al. 2013)
    Isoptericola halotolerans Novel species HM + 20% NaCl/28 ℃/1 week Saline soil/Qinghai, China (Zhang et al. 2005)
    Jeotgalicoccus huakuii Novel species LB / 30 ℃ Seaside soil/Shandong, China (Guo et al. 2010)
    Jeotgalicoccus marinus Novel species MA + 20% NaCl/28 ℃/4 weeks Sea urchin/Leizhou Bay, China (Chen et al. 2009l)
    Kocuria aeqyptia Novel species HM / 28 ℃/1 week Saline, alkaline desert soil/Egypt (Li et al. 2006a)
    Kocuria halotolerans Novel species ISP5 + 10% NaCl/37 ℃/3 weeks Saline soil from Ganjiahu Suosuo Forest/Xinjiang, China (Tang et al. 2009d)
    Lentibacillus salis Novel species MA + 10% NaCl/35 ℃/3 days Ayakekum salt lake soil/Xinjiang, China (Lee et al. 2008)
    Lipingzhangella halophila Novel genus MA + 2% NaCl (pH 10)/30 ℃/4 weeks Gurbangtϋnggϋt desert soil/Xinjiang, China (Zhang et al. 2016c)
    Marinococcus halotolerans Novel species SG + 25% MgCl2/28 ℃/2 weeks Hypersaline soil/Qinghai, China (Li et al. 2005b)
    Marinococcus luteus Novel species ISP5 + 10% NaCl/28 ℃/1 week Barkol Lake sediment/Xinjiang, China (Wang et al. 2009a)
    Microbacterium album Novel species R2A/28 ℃/5 days Desert sample/Saudi Arabia (Yang et al. 2018b)
    Microbacterium deserti Novel species R2A/28 ℃/5 days Desert sample/Saudi Arabia (Yang et al. 2018b)
    Microbacterium halotolerans Novel species ISP5 + 15% KCl/28 ℃/2 weeks Hypersaline soil/Qinghai, China (Li et al. 2005a)
    Microbulbifer halophilus Novel species ISP5 + 10% MgCl2/37 ℃ Saline soil/Xinjiang, China (Tang et al. 2008b)
    Myceligenerans halotolerans Novel species GTY + 5% KCl/37 ℃/2 weeks Qijiaojing salt lake soil/Xinjiang, China (Wang et al. 2011)
    Nesterenkonia halophila Novel species SG + 25% KCl/28 ℃/2 weeks Saline soil/ Xinjiang, China (Li et al. 2008)
    Nesterenkonia halotolerans Novel species ISP5 + 15% MgCl2/28 ℃/2 weeks Hypersaline soil/ Xinjiang, China (Li et al. 2004b)
    Nesterenkonia natronophila Novel species PCA + 2% NaCl (pH 10)/30 ℃/10 days Lake Magadi sediment/Arusha, Tanzania (Machin et al. 2019)
    Nesterenkonia rhizosphaerae Novel species ISP5 (pH 10)/30 ℃/4 weeks Desert rhizospheric soil of Reaumuria soongorica/Fukang, Xinjiang, China (Wang et al. 2014)
    Nesterenkonia xinjiangensis Novel species ISP5 + 15% KCl/28 ℃/2 weeks Hypersaline soil/Xinjiang, China (Li et al. 2004b)
    Nocardiopsis ansamitocini Novel species ISP2 (pH 10)/30 ℃/4 weeks Saline-alkali soil/Xinjiang, China (Zhang et al. 2016b)
    Nocardiopsis litoralis Novel species MA + 10% NaCl / 25 ℃ / 2 weeks Sea anemone/Naozhou Island, China (Chen et al. 2009h)
    Nocardiopsis salina Novel species ISP5 + 20% NaCl/28 ℃/1 week Hypersaline soil/Xinjiang, China (Li et al. 2004c)
    Nocardiopsis terrae Novel species MA / 30 ℃/2 weeks Saline soil from Qaidam Basin/Qinghai, China (Chen et al. 2010c)
    Nocardiopsis xinjiangensis Novel species ISP5 + 10% NaCl/28 ℃/4 weeks Hypersaline soil/Xinjiang, China (Li et al. 2003a)
    Ornithinicoccus halotolerans Novel species R2A (pH 10)/30 ℃/3 weeks Karamayi desert sample/Xinjiang, China (Zhang et al. 2016d)
    Paracoccus saliphilus Novel species ISP5 + 10% NaCl/28 ℃/2 weeks Saline soil/Xinjiang, China (Wang et al. 2009c)
    Paraliobacillus quinghaiensis Novel species MA + 10% NaCl/28 ℃/4 weeks Sediment sample of Dabuxun salt lake/Qinghai, China (Chen et al. 2009d)
    Phytoactinopolyspora endophytica Novel genus R2A / 28 ℃ / 4 weeks Root tissue of Glycyrrhiza uralensis/Yili county, Xinjiang, China (Li et al. 2015)
    Pontibacillus halophilus Novel species MA + 20% NaCl / 28 ℃ / 4 weeks Sea urchin/Leizhou Bay, China (Chen et al. 2009m)
    Pontibacillus litoralis Novel species MA/28 ℃/2 weeks Sea anemone/Naozhou Island, China (Chen et al. 2010d)
    Prauserella aidingensis Novel species CCMS/37 ℃/3 weeks Brine sample of Aiding Lake/Xinjiang, China (Li et al. 2009)
    Prauserella alba Novel species SCA + 20% NaCl/28 ℃/4 weeks Hypersaline soil/Xinjiang, China (Li et al. 2003c)
    Prauserella flava Novel species CCMS / 37 ℃/3 weeks Brine sample of Aiding Lake/Xinjiang, China (Li et al. 2009)
    Prauserella halophila Novel species SCA + 20% NaCl/28 ℃/4 weeks Hypersaline soil/Xinjiang, China (Li et al. 2003c)
    Prauserella salsuginis Novel species CCMS/37 ℃/3 weeks Brine sample of Aiding Lake/Xinjiang, China (Li et al. 2009)
    Prauserella sediminis Novel species CCMS/37 ℃/3 weeks Brine sample of Aiding Lake/Xinjiang, China (Li et al. 2009)
    Psychroflexus sediminis Novel species MA/28 ℃/2 weeks Dachaidamu salt lake sediment/Qinghai, China (Chen et al. 2009a)
    Saccharomonospora paurometabolica Novel species ISP5 + 20% NaCl/28 ℃/4 weeks Soil sample/Xinjiang, China (Li et al. 2003b)
    Saccharomonospora saliphila Novel species ISP5 + 20% NaCl/28 ℃/4 weeks Muddy soil/Gulbarga, India (Syed et al. 2008)
    Saccharopolyspora deserti Novel species R2A + 5% NaCl/37 ℃/1 week Desert sand/Saudi Arabia (Yang et al. 2018a)
    Saccharopolyspora halophila Novel species CCMS + 15% NaCl/28 ℃/3 weeks Hypersaline soil/Xinjiang, China (Tang et al. 2009a)
    Saccharopolyspora qijiaojingensis Novel species CCMS / 28 ℃/3 weeks Soil sample of Qijiaojing salt lake/Xinjiang, China (Tang et al. 2009e)
    Salinicoccus albus Novel species MA + 30% NaCl/28 ℃/4 weeks Brine sample of Yipinlang salt mine/Yunnan, China (Chen et al. 2009b)
    Salinicoccus luteus Novel species MA + 15% NaCl/28 ℃/2 weeks Desert soil, Wadi Sannur, Egypt (Zhang et al. 2007b)
    Salinicoccus salitudinis Novel species MA + 10% NaCl/28 ℃/2 weeks Saline soil/Qaidam, China (Chen et al. 2008a)
    Salinimicrobium terrae Novel species MA/28 ℃/2 weeks Saline soil from Chaka salt lake/Qinghai, China (Chen et al. 2008b)
    Salinisphaera halophila Novel species CCMS/28 ℃/3 weeks Brine sample/Shiyang salt well, Yunnan, China (Zhang et al. 2012a)
    Saliphagus infecundisoli Novel genus GM + 13% NaCl/37 ℃/4 weeks Saline soil/Loulan, Xinjiang, China (Yin et al. 2017)
    Sinococcus qinghaiensis Novel genus SG + 25% KCl/28 ℃ / 2 weeks Hypersaline soil/Qinghai, China (Li et al. 2006b)
    Sphingomonas hunanensis Novel species MA + 5% NaCl/28 ℃ / 2 weeks Forest soil/Hunan, China (Chen et al. 2011b)
    Streptomonospora alba Novel species SCA + 20% NaCl/28 ℃/2 weeks Hypersaline soil/Xinjiang, China (Li et al. 2003d)
    Streptomonospora amylolytica Novel species SCA + 20% NaCl/28 ℃/2 weeks Hypersaline soil/Xinjiang, China (Cai et al. 2009)
    Streptomonospora flavalba Novel species SCA + 20% NaCl/28 ℃/2 weeks Hypersaline soil/Xinjiang, China (Cai et al. 2009)
    Streptomonospora halophila Novel species ISP5 + 10% NaCl/28 ℃/2 weeks Hypersaline soil/Xinjiang, China (Cai et al. 2008)
    Streptomonospora salina Novel genus ISP5 + 15% NaCl/28 ℃/2 weeks Hypersaline soil/Xinjiang, China (Cui et al. 2001)
    Streptomyces fukangensis Novel species CCMS (pH10)/30 ℃/4 weeks Saline-alkaline soil/Fukang, Xinjiang, China (Zhang et al. 2013)
    Streptomyces tritolerans Novel species SCA/28 ℃/2 weeks Alkaline soil/Gulbarga, India (Syed et al. 2007)
    Tenuibacillus halotolerans Novel species CCMS/37 ℃/1 week Qijiojing Lake sediment/ Xinjiang, China (Gao et al. 2013)
    Virgibacillus albus Novel species CCMS/28 ℃/2 weeks Lop Nur salt lake/Xinjiang, China (Zhang et al. 2012b)
    Virgibacillus litoralis Novel species MA + 5% NaCl/30 ℃/2 weeks Saline soil/Naozhou Island, South China Sea (Chen et al. 2009e)
    Virgibacillus sediminis Novel species MA + 5% NaCl/30 ℃/2 weeks Keke salt lake/Qinghai, China (Chen et al. 2009c)
    Yania halotolerans Novel genus ISP5 + 15% KCl / 28 ℃/2 weeks Saline soil/Xinjiang, China (Li et al. 2004a)
    Yaniella soli Novel species NA + 10% NaCl / 30 ℃/2 weeks Forest soil/Hunan, China (Chen et al. 2010a)
    Yimella lutea Novel genus ISP5 + 5% NaCl / 37 ℃/3 weeks Contaminated plate (Tang et al. 2010d)
    Zhihengliuella alba Novel species ISP5 + 10% NaCl/37 ℃/3 weeks Saline sample/Xinjiang, China (Tang et al. 2009b)
    Zhihengliuella halotolerans Novel genus MA + 15% NaCl/28 ℃/2 weeks Saline soil/Qinghai, China (Zhang et al. 2007a)
    Zhihengliuella salsuginis Novel species MA + 5% NaCl/30 ℃/2 weeks Xiangli Salt Mine/Hunan, China (Chen et al. 2010b)
    * ASW Artifcial sea water agar (Eguchi et al.1996), CCMS Cellulose-casein multi salt agar (Tang et al.2008a), GM Gauze medium (Atlas 1993), GTY Glucose-tryptone-yeast medium (Tang et al.2010e), HM Horikoshi medium (Horikoshi 1990), ISP5 International Streptomyces Project medium 5(Shirling and Gottlieb 1966), LB Lutia Berteni medium (Atlas 1993), MA Marine Agar (Difco), NA Nutrient Agar (Atlas 1993), R2A Reasoner's 2A agar (Reasoner and Geldreich 1985), SCA Starch Casein Agar (Kϋster and Williams 1964), SG Sehgal and Gibbon medium (Sehgal and Gibbons 1960), SSDM Sea-salt defned medium (Quesada et al.1987)

    Table 1.  List of halophilic and halotolerant microorganisms isolated in our laboratory

    Often, desert and other arid environments are marked by high spatial heterogeneity, particularly involving variation in the nutrient content (Schlesinger et al. 1996). Considering the tremendous abiotic stresses, particularly high temperature, high radiation, low nutrient, and low water, vegetation is sparse in arid environments (Schade and Hobbie 2005). As a result, soil function in arid environments is dependent largely on the function of soil microbial communities (Belnap et al. 2005), as evidenced by high occurrence of the genus Frankia (Connon et al. 2007). However, in most cases, microbial communities in arid environments enter a state of anhydrobiosis (Billi and Potts 2002). Dominant cultured bacteria from deserts belong to the phyla Actinobacteria, Proteobacteria, and Deinococcus-Thermus (Hozzein et al. 2018; Hussain et al. 2016; Yang et al. 2017, 2018a, 2019). They are readily cultivable using conventional nutrient rich media. However few recent studies showed that diluted conventional media achieves the growth of normally slow-growing rare taxa of the phyla Actinobacteria, Bacterioidetes and Proteobacteria (Asem et al. 2018; Dong et al. 2019; Han et al. 2019). Interestingly, these bacteria fail to grow in most commercial rich nutrient media, but exhibit optimum growth in nutrient-poor media, and in the absence of salt. To date, the exact mechanisms for this oligotrophism have not been studied. However, genome analyses predict that some of these bacteria are involved in quorum sensing activities, which are necessary for maintenance and cell-to-cell communications among desert microbial communities (data unpublished).

    In a recent publication, Xian et al. (2020) report the utilization of co-occurrence network mapping to understand the possible interactions among different OTUs retrieved from 16S rRNA-based Illumina sequencing of hot spring microbial mat samples (Figs. 3, 4). Utilizing this network-modularity data supported by preliminary experiments of growth-promotion assay between cultured representatives of selected OTUs and metabolome analyses, a spent culture isolation medium was designed to specifically target the isolation of Chloroflexi. The study recovered 57 Chloroflexi isolates in the new ameliorated medium in comparison with 12 isolates in the standard isolation media; two of these isolates have 16S rRNA gene sequence identity values of less than 90% with known culture representatives and present distinct lineages in the phylogenetic dendrogram.

    Figure 4.  Network-directed isolation procedure. a. Co-occurrence network analysis of OTUs from a hot spring microbial mat. b Efficacy of ameliorated spent culture media against the conventional media for isolation of Chloroflexi(diagram adapted from Xian et al. 2020)

  • Culturomics approach

  • The concept of culturomics involves using multiple isolation media involving a range of culture conditions with different and prolonged incubation. Technically, similar approaches with limited culture conditions have been attempted for understanding the cultivable microbial diversity. Qin et al. (2009) utilized 11 isolation media and three sample-treatment procedures to study the endophytic actinobacterial diversity among medicinal plant samples of a tropical rain forest. Thus, there was recovery of 32 genera of rare actinobacteria from a total of 2, 174 isolates, including 17 isolates representing novel taxa. Li et al. (2012) isolated 228 actinobacteria from tissues of Artemisia annua. L. by application of six isolation media and four sample-treatment methods. Despite high retrieval of cultivable actinobacterial diversity, the recovery rate with limited expansion in the number of media or culture conditions is unsatisfactory against the existing diverse uncultured populations (Qin et al. 2012).

    High-throughput isolation methods (culturomics) provide an ideal solution for many environments, and have been successful in cultivating the majority of the uncultured microbiome from human gut (Lagier et al. 2018). The method adopted by Lagier et al. (2016) involved a preliminary isolation with a maximum number of probable culture conditions (212 conditions were used in Lagier et al. 2012) and pre-incubation in blood cultures under both aerobic and anaerobic settings. All colonies were rapidly identified with MALDI-TOF MS (or 16S rRNA gene sequencing), followed by optimization of the culture conditions that increase the probability of cultivation of previously uncultured bacteria, whose diversity was already depicted from the metagenomes.

  • "Reverse-genomics" cultivation approach

  • This strategy was developed by Cross et al. (2019), and was applied successfully for targeted isolation of Saccharibacteria, a phylum among the candidate phyla radiation in the tree of life, from one oral cavity sample. The method provided an advantage over the previously described methods in that slow-growing bacteria could be specifically targeted without any lengthy enrichment step.

    A special requirement about this technique is that the genomes (metagenome-assembled genome or single-amplified genomes) of targeted bacteria should contain membrane proteins-encoding genes that are absent in other bacteria. Moreover, these proteins should feature extracellular regions, which may function as surface epitopes. These protein fragments could then be injected into rabbits (or any other suitable animal host) to make antibodies, which are then extracted, purified, and fluorescently labelled. The labelled antibodies are mixed with the microbiome samples to selectively label the targeted organisms. Then, the labelled organisms are sorted from the samples using fluorescence-activated cell sorting, enriched, and cultivated by plating onto several isolation media.

    However, this technique still needs to address major problems related to structural modelling of the surface protein of bacteria without cultured representatives. Besides, the technical issue about the selection of growth media/conditions will be a major hindrance if fluorophore-sorted cells lose viability and/or culturability during subsequent enrichment and isolation.

Conclusions
  • New approaches are frequently being proposed for isolation of previously uncultivated bacteria, and are highly successful for targeted based isolation. However, these approaches are still needed to cover the massive section of the tree of life that is hitherto redundant to cultivation. Besides, all these high-throughput isolation methods are still limited. New improvements have to be brought about to further fill the gap between the cultured and uncultured majority.

Acknowledgements
  • This work is supported by the National Natural Science Foundation of China (Nos. 91951205 and 31850410475).

Author contributions
  • W-JL designed the workflow; NS and W-DX wrote the manuscript. NS and MDA helped in data collection and making figure and tables. MX helped in writing and critical review of the manuscript.

Compliance with ethical standards

    Conflicts of interest

  • The authors declare there is no confict of interest between them.

  • Human and animal rights statement

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

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