Culturomics of the plant prokaryotic microbiome and the dawn of plantbased culture media – A review

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Culturomics of the plant prokaryotic microbiome and the dawn of plantbased culture media – A review. Improving cultivability of a wider range of bacterial and archaeal community members, living natively in natural environments and within plants, is a prerequisite to better understanding plant-microbiota interactions and their functions in such very complex systems. Sequencing, assembling, and annotation of pure microbial strain genomes provide higher quality data compared to environmental metagenome analyses, and can substantially improve gene and protein database information. Despite the comprehensive knowledge which already was gained using metagenomic and metatranscriptomic methods, there still exists a big gap in understanding in vivo microbial gene functioning in planta, since many differentially expressed genes or gene families are not yet annotated. Here, the progress in culturing procedures for plant microbiota depending on plant-based culture media, and their proficiency in obtaining single prokaryotic isolates of novel and rapidly increasing candidate phyla are reviewed. As well, the great success of culturomics of the human microbiota is considered with the main objective of encouraging microbiologists to continue minimizing the gap between the microbial richness in nature and the number of species in culture, for the benefit of both basic and applied microbiology.
Contents lists available at ScienceDirect
Journal of Advanced Research
Review
Culturomics of the plant prokaryotic microbiome and the dawn of plant-
based culture media – A review
Mohamed S. Sarhana, Mervat A. Hamzaa, Hanan H. Youssefa, Sascha Patzb, Matthias Beckerc,
Hend ElSaweya, Rahma Nemra, Hassan-Sibroe A. Daanaad, Elhussein F. Mourada, Ahmed T. Morsia,
Mohamed R. Abdelfadeela, Mohamed T. Abbase, Mohamed Fayeza, Silke Ruppelf, Nabil A. Hegazia,
a Environmental Studies and Research Unit (ESRU), Department of Microbiology, Faculty of Agriculture, Cairo University, Giza 12613, Egypt
b Algorithms in Bioinformatics, Center for Bioinformatics, University of Tübingen, Tübingen 72076, Germany
c Institute for National and International Plant Health, Julius Kühn-Institute – Federal Research Centre for Cultivated Plants, 38104 Braunschweig, Germany
d Department of Genetics, School of Life Science, The Graduate University for Advanced Studies (SOKENDAI), Shizuoka 411-8540, Japan
e Department of Microbiology, Faculty of Agriculture & Natural Resources, Aswan University, Aswan, Egypt
f Leibniz Institute of Vegetable and Ornamental Crops (IGZ), Großbeeren, 14979, Germany
h i g h l i g h t s
g r a p h i c a l
a b s t r a c t
 The plant microbiome culturomics is
substantially lagging behind the
human microbiome.
 Conventional chemically-synthetic
culture media recover < 10% of plant-
Hello fellow “Endo”!
We have an invitation on dinner tonight
Warm Petri dish of
meat extract + pepton,
they call it “Nutrient Agar”
Oh my God!
Those people!!!
They don‘t realize that
we are vegetarians!
associated microbiota.
 Plant-based culture media (PCM) are
introduced as a novel tool for plant
microbiome culturomics.
 PCM extended the microbiota
culturability to recover unculturable
bacterial taxa.
 Streamlined- and large-genomes
NA LB BAP TSA
conspicuously contribute to the
dilemma of unculturability.
a r t i c l e
i n f o
a b s t r a c t
Article history:
Improving cultivability of a wider range of bacterial and archaeal community members, living natively in
Received 18 January 2019
Revised 11 April 2019
Accepted 12 April 2019
Available online 19 April 2019
natural environments and within plants, is a prerequisite to better understanding plant-microbiota inter-
actions and their functions in such very complex systems. Sequencing, assembling, and annotation of
pure microbial strain genomes provide higher quality data compared to environmental metagenome
analyses, and can substantially improve gene and protein database information. Despite the comprehen-
Keywords:
Plant microbiome
Metagenomics
Plant-based culture media
Culturomics
sive knowledge which already was gained using metagenomic and metatranscriptomic methods, there
still exists a big gap in understanding in vivo microbial gene functioning in planta, since many differen-
tially expressed genes or gene families are not yet annotated. Here, the progress in culturing procedures
for plant microbiota depending on plant-based culture media, and their proficiency in obtaining single
prokaryotic isolates of novel and rapidly increasing candidate phyla are reviewed. As well, the great suc-
Unculturable bacteria
cess of culturomics of the human microbiota is considered with the main objective of encouraging micro-
Candidate Phyla Radiation (CPR)
biologists to continue minimizing the gap between the microbial richness in nature and the number of
species in culture, for the benefit of both basic and applied microbiology. The clear message to fellow
Peer review under responsibility of Cairo University.
Corresponding author.
2090-1232/ 2019 The Authors. Published by Elsevier B.V. on behalf of Cairo University.
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
16
M.S. Sarhan et al./Journal of Advanced Research 19 (2019) 15–27
plant microbiologists is to apply plant-tailored culturomic techniques that might open up novel proce-
dures to obtain not-yet-cultured organisms and extend the known plant microbiota repertoire to
unprecedented levels.
 2019 The Authors. Published by Elsevier B.V. on behalf of Cairo University. This is an open access article
The birth and development of in vitro cultivation and pure
the 16S rRNA gene-based high throughput sequencing of PCR
culture studies
amplicon libraries and the PhyloChip microarray technology of
16S rRNA amplicons to oligonucleotide probes hybridization [20],
Since the discovery of microorganisms, in vitro cultivation and
is the PCR-biased amplification efficiency. This is affected by sam-
isolation of bacteria in pure cultures has represented one of the
ple origin, DNA extraction method, primer specificity, and the pro-
major pillars in developing the science of microbiology. Introduc-
portion of target genes within the sample background, which
ing their pioneer work on the germ-disease theory, both Louis Pas-
usually favor highly abundant targets [21]. Nevertheless, data
teur and Robert Koch, and their associates, were able to present
obtained by these methods revealed that members of the ‘‘rare”
their nutrient broth ‘‘Bouillon, Nährflüssigkeit” and solid culture
biosphere are actively attracted by specific environments, and
media, together with single colony isolation and pure cultures
may play an important role despite their low abundance [22].
studies [1]. The well-known solid culture media consisting of meat
Newer next generation sequencing techniques (NGS) did enable
extract, peptones and agar, were developed by the 1890s. With
and simplify metagenomic and metatranscriptomic approaches
extensive progress in selectivity profiles, diagnostic properties,
that partially alleviate the PCR-related problems for just a single
chromogenic reactions, pre- and selective enrichment power, cul-
or a combination of taxonomic/phylogenetic marker genes by
ture media were the main tools to estimate viable counts, enrich,
sequencing all genomic variants within an environmental sample
select and differentiate groups of bacteria. In addition, individuals
[23]. This results in a highly comprehensive dataset of sequenced
were isolated in pure cultures to identify, study properties, test for
microbialreadsrepresenting genomicfragmentsor transcripts,that
secondary metabolites, and determine the genetic composition
aimed to be assigned to operational taxonomic units (OTUs) and/or
(britannica.com/science/pure-culture) [2,3]. Further environmen-
specific genes, to describe microbial taxonomic diversity and to
tal adaptation techniques are discussed in the section ‘‘From syn-
estimate functional variety or activity of a certain taxonomic level,
thetic to environmental cultivation of microbiomes”.
optimally of single strains. Although progresses have been achieved
in extracting DNA/RNA from environmental samples to reduce con-
From plate count anomaly to candidate phyla
tamination and increase purity, there are still limiting factors: (i)
restrictions in sequencing methods (e.g. error rate); (ii) direct
Nutrient agar and many other derived culture media, with their
major components of meat extract and peptone developed for the
isolationofpureisolatesofhumanpathogens,havebeencontinually
used for culturing various types of microbiomes irrespective of the
nature of their environments, whether humans, animals or plants
[4–6]. Additionally, many of the earlier methods continued to be
used, whilediscoveringthe majordifferencesbetween the numbers
of cellsfrom naturalenvironmentsthat form viable colonies on agar
media and the numbers observed by microscopy. This observation
noted at the dawn of microbiology [7] was called ‘‘the great plate
count anomaly” by Staley and Konopka [8], and continued to be
researched by microbiologists over the years [9–12]. The phe-
nomenon was brought sharply into focus, leading to the realization
just how diverse and unexplored microorganisms are, as a result of
analyzingmicrobialsmallsubunitribosomalRNA(SSUor16SrRNA)
gene sequences directly from environmental samples [13].
Historically, until the mid-1980s, most of the available micro-
bial ecology knowledge was based on cultivation techniques and
assignment of reads to their corresponding genes; (iii) gene assem-
bly with the risk of chimaera production among other problems,
and (iv) the quality and availability of annotated genes and gene
families in the databases; which often lead to genes of unknown
functions and consequently to unknown taxa [24].
To overcome the issues above, a huge variety of bioinformatic
tools have been developed to prioritize read quality control and
processing (e.g. FastQC, FastX, PRINSEQ, Cutadapt), contamination
filtering (e.g. BMTagger), and chimaera detection (e.g. Uchime2).
Further tools are applied to assign a specific read to its correspond-
ing gene or protein, function or taxon, that can be alignment-based
(e.g. BLASTn/x, DIAMOND, LAST, RAPSearch2) or alignment-free
(e.g. KRAKEN); the latter mostly uses k-mers to minimize database
inadequacies. Currently, comprehensive tools for taxonomic and/or
functional classification of reads are exemplified by MEGAN6, MG-
RAST, MetaPhlAn2 and Qiita. Notably, some of these metagenomic
tools (e.g. MEGAN-LR) deal with the output of long-read sequenc-
ing techniques, such as of Pacific Biosciences (PacBio) or Oxford
microscopy or enzyme activities measured in laboratories after
Nanopore Technologies (ONT) [25]. Those gains of interest in
substrate induction [14]. Then, Muyzer et al. [15] introduced the
metagenomic research are due to the fact that taxonomic and its
denaturing
gradient
gel
electrophoresis
(DGGE)
technique,
functional annotation do not rely anymore on single genes covered
designed to separate specific PCR-amplified gene fragments, to
by multiple short reads (approx. 50–300 bp) and their gene copy
analyze microbial communities without the need of culturing
number issues (e.g. 16S rRNA) but on multiple genes covered by
microorganisms. As a procedure, DNA samples extracted directly
long reads, with an average read length of 5 to 50 kb, whereof
from the environment were targeted to amplify gene regions such
approx. 50% of the reads are larger than 14 kb [26].
as 16S rRNA for bacterial or ITS regions for fungal communities.
Continuous advances in high throughput genomic sequencing
Concomitantly, terminal restriction fragment length polymor-
technologies, metagenomics and single cell genomics, have con-
phism (T-RFLP) was introduced to produce fingerprints of micro-
tributed new insights into uncultivated lineages. Several of the
bial communities [16]. The emergence of improved sequencing
known microbial phyla, 120 bacterial and 20 archaeal phyla, con-
techniques, and the entailed increase of database-stored sequence
tain few cultivated representatives (ncbi.nlm.nih.gov/Taxonomy/
information in combination with the development of in situ
Browser/wwwtax.cgi). Moreover, phyla composed exclusively of
hybridization probes provided new methods for microbial commu-
uncultured representatives are referred to as Candidate Phyla
nity profiling, especially in the 90s, like the full-cycle or cyclic rRNA
(CP) [27,28]. Such uncultivated majority, approx. 90 bacterial can-
approach [17–19].The major limitation of these methods, including
didate phyla, defined as microbial dark matter and exist in various
In situ & high-
deve opment
-Low-nutrient media
M.S. Sarhan et al./Journal of Advanced Research 19 (2019) 15–27
17
environmental
microbiomes
[6,29–31].
Remarkably,
metage-
nomics and microbiome analyses have detected so many candidate
phyla, and phylogenetic analyses have revealed such a close rela-
tionship among many of them that the term ‘‘Candidate Phyla
throughput cultivation
-Diffusion chamber
-Isolation chip (Ichip)
Radiation” (CPR) was coined for a group of uncultured bacteria that
-Microfluidic Streak Plate (MSP)
share evolutionary history [32–34].
The number of newly discovered candidate phyla is increasing
due to further developments in metagenomic techniques and con-
-Double encapsulation technique
-Soil Substrate Membrane System (SSMS)
-Hollow-Fiber Membrane Chamber (HFMC)
tinual updating of genomic databases, and representing a striking
challenge to the scientific community [27,35]. With increased
metagenomic sampling and analysis, taxonomic boundaries and
nomenclature are constantly being reassessed. Meanwhile, scien-
tists have realized that bacterial and archaeal phyla without a sin-
gle cultivated representative comprise the majority of life’s current
diversity [27,32,34]. Certainly, the current knowledge about the
microbial world, not only the substantial roles played by microor-
ganisms in the function of the biosphere but also their reservoir of
novel bioactive compounds, is profoundly challenged by what have
Culture media
-Plant l extract additives Culturomics Incubation conditions
-Signaling compounds -Aerobic/anaerobic
and coculturing -Different temperatures
-Plant-based culture media -Light/Dark
-Creation of stress
conditions for culturing
been cultivated in the laboratory [35]. So far, physiologic and geno-
mic information has been confined to pure cultures and dominated
extremophiles (pH, salinity,
temperature,...etc)
by representation of the Proteobacteria, Firmicutes, Actinobacteria,
Omics-derived
and Bacteriodetes within the Bacteria and of methanogens and
cultivation information
halotolerant members of the Euryarchaeota within Archaea [36].
Fig. 1. Toolbox of strategies developed for improving culturability of environmental
microbiomes. High throughput culturomics adopt various combinations of the
From synthetic to environmental cultivation of microbiomes
specific methods of the 4 major strategies of in situ and high throughput cultivation,
culture media development, incubation conditions, and genome-derived cultiva-
Today, it is established that culture media tailored for in vitro
tion. For further details, please refer to Table 1.
cultivation of microorganisms, including CP microorganisms, must
provide environmental and nutritional conditions that resemble
centrations in standard media together with longer incubation
their natural habitats, combined with long incubation times [37].
[39], diluting to extinction to minimize the influence of fast grow-
Further attempts towards improving culture media to grow novel
ers and facilitate growth of oligotrophs [40], co-incubating cells
species depended mainly on supplementing macro- and micronu-
individually encapsulated into microdroplets under low flux nutri-
trients in the medium as well as manipulating cultivation condi-
ent conditions [41], adding signaling compounds and/or co-
tions
(Table
1).
Conspicuous
developments
and
higher
cultivation to trigger microbial growth [42,43].
throughput methods have been applied to marine and terrestrial
Novel in situ cultivation techniques, e.g. diffusion chambers,
environments (Fig. 1, Table 2), adopting a number of approaches
have been introduced to mimic natural conditions and provide
reviewed by Epstein et al. [38]: for example, lowering nutrient con-
access to critical growth factors found in the environment and/or
Table 1
Progressive supplements of culture media to improve culturability of environmental microbiomes.
Culture media supplementation
Recovered taxa
Basal medium supplemented with isoleucine and yeast extract [44]a
Aminobacterium mobile
Basal medium supplemented with yeast extract [45]
Acidilobus aceticus
Nitrogen-free LGI-P medium supplemented with sugarcane juice [46]
Burkholderia tropica
10-fold-diluted Difco marine broth 2216 supplemented with yeast extract
Hoeflea phototrophica
[47]
Postgate’s medium B supplemented with yeast extract [48]
Desulfitibacter alkalitolerans
MPN soil solution equivalent (SSE) supplemented with pectin, chitin,
Edaphobacter modestus and Edaphobacter aggregans
soluble starch, cellulose, xylan, and curdlan as carbon sources [49]
Basal medium supplemented with humic acid and vitamin B (HV medium)
Pseudonocardia eucalypti
[50]
TSA, casein-starch, and 869 culture media supplemented with plant
Kaistia sp. and Varivorax sp.
extracts [51]
Peptone-Yeast extract-Glucose medium (PYG) supplemented with
Arthrobacter liuii
Resuscitation-promoting factors (Rpf) [52]
Modified Biebl and Pfennig’s medium [53]
Thiorhodococcus fuscus
Culture media based on extracts of potato, onions, green beans, black beans,
Biomass production of Pseudomonas fluorescence
sweet corn, sweet potato, or lentils [54]
Selective King’s B medium supplemented with lichens extract [55]
Resulted in higher endo-lichenic and ecto-lichenic bacterial CFU counts
Basal medium supplemented with sugarcane bagasse [56]
Higher CFU recovery compared with other standard media
Fastidious anaerobic agar and blood agar media supplemented with
Prevotella sp., Fretibacterium fastidiosum, Dialister sp., and Megasphaera sp.
siderophores-like molecules [57]
Minimal medium supplemented with peels of orange, potato, or banana
Biomass production of Bacillus subtilis
[58]
PBS buffer supplemented with pig fecal slurry or dried grass hay as carbon
Streptococcus caviae
sources [59]
MRS and TSB supplemented with Titania (TiO2) nanoparticles [60]
Enhanced biocontrol performance of PGPR strains against Fusarium culmorum
Modified 80% ethanol soil extract culture media [61]
18 novel species including isolates belonging to Verrucomicrobia and Elusimicrobia
a Numbers between brackets refer to related references.
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Culturomics of the plant prokaryotic microbiome and the dawn of plantbased culture media – A review. Improving cultivability of a wider range of bacterial and archaeal community members, living natively in natural environments and within plants, is a prerequisite to better understanding plant-microbiota interactions and their functions in such very complex systems. Sequencing, assembling, and annotation of pure microbial strain genomes provide higher quality data compared to environmental metagenome analyses, and can substantially improve gene and protein database information. Despite the comprehensive knowledge which already was gained using metagenomic and metatranscriptomic methods, there still exists a big gap in understanding in vivo microbial gene functioning in planta, since many differentially expressed genes or gene families are not yet annotated. Here, the progress in culturing procedures for plant microbiota depending on plant-based culture media, and their proficiency in obtaining single prokaryotic isolates of novel and rapidly increasing candidate phyla are reviewed. As well, the great success of culturomics of the human microbiota is considered with the main objective of encouraging microbiologists to continue minimizing the gap between the microbial richness in nature and the number of species in culture, for the benefit of both basic and applied microbiology..

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Journal of Advanced Research 19 (2019) 15–27 Contents lists available at ScienceDirect Journal of Advanced Research journal homepage: www.elsevier.com/locate/jare Review Culturomics of the plant prokaryotic microbiome and the dawn of plant-based culture media – A review Mohamed S. Sarhana, Mervat A. Hamzaa, Hanan H. Youssefa, Sascha Patzb, Matthias Beckerc, Hend ElSaweya, Rahma Nemra, Hassan-Sibroe A. Daanaad, Elhussein F. Mourada, Ahmed T. Morsia, Mohamed R. Abdelfadeela, Mohamed T. Abbase, Mohamed Fayeza, Silke Ruppelf, Nabil A. Hegazia,⇑ a Environmental Studies and Research Unit (ESRU), Department of Microbiology, Faculty of Agriculture, Cairo University, Giza 12613, Egypt b Algorithms in Bioinformatics, Center for Bioinformatics, University of Tübingen, Tübingen 72076, Germany c Institute for National and International Plant Health, Julius Kühn-Institute – Federal Research Centre for Cultivated Plants, 38104 Braunschweig, Germany d Department of Genetics, School of Life Science, The Graduate University for Advanced Studies (SOKENDAI), Shizuoka 411-8540, Japan e Department of Microbiology, Faculty of Agriculture & Natural Resources, Aswan University, Aswan, Egypt f Leibniz Institute of Vegetable and Ornamental Crops (IGZ), Großbeeren, 14979, Germany h i g h l i g h t s The plant microbiome culturomics is substantially lagging behind the human microbiome. Conventional chemically-synthetic culture media recover < 10% of plant-associated microbiota. Plant-based culture media (PCM) are introduced as a novel tool for plant microbiome culturomics. PCM extended the microbiota culturability to recover unculturable bacterial taxa. Streamlined- and large-genomes conspicuously contribute to the dilemma of unculturability. g r a p h i c a l a b s t r a c t Hello fellow “Endo”! We have an invitation on dinner tonight Warm Petri dish of meat extract + pepton, they call it “Nutrient Agar” NA LB BAP TSA Oh my God! Those people!!! They don‘t realize that we are vegetarians! a r t i c l e i n f o a b s t r a c t Article history: Received 18 January 2019 Revised 11 April 2019 Accepted 12 April 2019 Available online 19 April 2019 Keywords: Plant microbiome Metagenomics Plant-based culture media Culturomics Unculturable bacteria Candidate Phyla Radiation (CPR) Improving cultivability of a wider range of bacterial and archaeal community members, living natively in natural environments and within plants, is a prerequisite to better understanding plant-microbiota inter-actions and their functions in such very complex systems. Sequencing, assembling, and annotation of pure microbial strain genomes provide higher quality data compared to environmental metagenome analyses, and can substantially improve gene and protein database information. Despite the comprehen-sive knowledge which already was gained using metagenomic and metatranscriptomic methods, there still exists a big gap in understanding in vivo microbial gene functioning in planta, since many differen-tially expressed genes or gene families are not yet annotated. Here, the progress in culturing procedures for plant microbiota depending on plant-based culture media, and their proficiency in obtaining single prokaryotic isolates of novel and rapidly increasing candidate phyla are reviewed. As well, the great suc-cess of culturomics of the human microbiota is considered with the main objective of encouraging micro-biologists to continue minimizing the gap between the microbial richness in nature and the number of species in culture, for the benefit of both basic and applied microbiology. The clear message to fellow Peer review under responsibility of Cairo University. ⇑ Corresponding author. E-mail addresses: nabil.hegazi@agr.cu.edu.eg, hegazinabil8@gmail.com (N.A. Hegazi). https://doi.org/10.1016/j.jare.2019.04.002 2090-1232/ 2019 The Authors. Published by Elsevier B.V. on behalf of Cairo University. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 16 M.S. Sarhan et al./Journal of Advanced Research 19 (2019) 15–27 plant microbiologists is to apply plant-tailored culturomic techniques that might open up novel proce-dures to obtain not-yet-cultured organisms and extend the known plant microbiota repertoire to unprecedented levels. 2019 The Authors. Published by Elsevier B.V. on behalf of Cairo University. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). The birth and development of in vitro cultivation and pure culture studies Since the discovery of microorganisms, in vitro cultivation and isolation of bacteria in pure cultures has represented one of the major pillars in developing the science of microbiology. Introduc-ing their pioneer work on the germ-disease theory, both Louis Pas-teur and Robert Koch, and their associates, were able to present their nutrient broth ‘‘Bouillon, Nährflüssigkeit” and solid culture media, together with single colony isolation and pure cultures studies [1]. The well-known solid culture media consisting of meat extract, peptones and agar, were developed by the 1890s. With extensive progress in selectivity profiles, diagnostic properties, chromogenic reactions, pre- and selective enrichment power, cul-ture media were the main tools to estimate viable counts, enrich, select and differentiate groups of bacteria. In addition, individuals were isolated in pure cultures to identify, study properties, test for secondary metabolites, and determine the genetic composition (britannica.com/science/pure-culture) [2,3]. Further environmen-tal adaptation techniques are discussed in the section ‘‘From syn-thetic to environmental cultivation of microbiomes”. From plate count anomaly to candidate phyla Nutrient agar and many other derived culture media, with their major components of meat extract and peptone developed for the isolationofpureisolatesofhumanpathogens,havebeencontinually used for culturing various types of microbiomes irrespective of the nature of their environments, whether humans, animals or plants [4–6]. Additionally, many of the earlier methods continued to be used, whilediscoveringthe majordifferencesbetween the numbers of cellsfrom naturalenvironmentsthat form viable colonies on agar media and the numbers observed by microscopy. This observation noted at the dawn of microbiology [7] was called ‘‘the great plate count anomaly” by Staley and Konopka [8], and continued to be researched by microbiologists over the years [9–12]. The phe-nomenon was brought sharply into focus, leading to the realization just how diverse and unexplored microorganisms are, as a result of analyzingmicrobialsmallsubunitribosomalRNA(SSUor16SrRNA) gene sequences directly from environmental samples [13]. Historically, until the mid-1980s, most of the available micro-bial ecology knowledge was based on cultivation techniques and microscopy or enzyme activities measured in laboratories after substrate induction [14]. Then, Muyzer et al. [15] introduced the denaturing gradient gel electrophoresis (DGGE) technique, designed to separate specific PCR-amplified gene fragments, to analyze microbial communities without the need of culturing microorganisms. As a procedure, DNA samples extracted directly from the environment were targeted to amplify gene regions such as 16S rRNA for bacterial or ITS regions for fungal communities. Concomitantly, terminal restriction fragment length polymor-phism (T-RFLP) was introduced to produce fingerprints of micro-bial communities [16]. The emergence of improved sequencing techniques, and the entailed increase of database-stored sequence information in combination with the development of in situ hybridization probes provided new methods for microbial commu-nity profiling, especially in the 90s, like the full-cycle or cyclic rRNA approach [17–19].The major limitation of these methods, including the 16S rRNA gene-based high throughput sequencing of PCR amplicon libraries and the PhyloChip microarray technology of 16S rRNA amplicons to oligonucleotide probes hybridization [20], is the PCR-biased amplification efficiency. This is affected by sam-ple origin, DNA extraction method, primer specificity, and the pro-portion of target genes within the sample background, which usually favor highly abundant targets [21]. Nevertheless, data obtained by these methods revealed that members of the ‘‘rare” biosphere are actively attracted by specific environments, and may play an important role despite their low abundance [22]. Newer next generation sequencing techniques (NGS) did enable and simplify metagenomic and metatranscriptomic approaches that partially alleviate the PCR-related problems for just a single or a combination of taxonomic/phylogenetic marker genes by sequencing all genomic variants within an environmental sample [23]. This results in a highly comprehensive dataset of sequenced microbialreadsrepresenting genomicfragmentsor transcripts,that aimed to be assigned to operational taxonomic units (OTUs) and/or specific genes, to describe microbial taxonomic diversity and to estimate functional variety or activity of a certain taxonomic level, optimally of single strains. Although progresses have been achieved in extracting DNA/RNA from environmental samples to reduce con-tamination and increase purity, there are still limiting factors: (i) restrictions in sequencing methods (e.g. error rate); (ii) direct assignment of reads to their corresponding genes; (iii) gene assem-bly with the risk of chimaera production among other problems, and (iv) the quality and availability of annotated genes and gene families in the databases; which often lead to genes of unknown functions and consequently to unknown taxa [24]. To overcome the issues above, a huge variety of bioinformatic tools have been developed to prioritize read quality control and processing (e.g. FastQC, FastX, PRINSEQ, Cutadapt), contamination filtering (e.g. BMTagger), and chimaera detection (e.g. Uchime2). Further tools are applied to assign a specific read to its correspond-ing gene or protein, function or taxon, that can be alignment-based (e.g. BLASTn/x, DIAMOND, LAST, RAPSearch2) or alignment-free (e.g. KRAKEN); the latter mostly uses k-mers to minimize database inadequacies. Currently, comprehensive tools for taxonomic and/or functional classification of reads are exemplified by MEGAN6, MG-RAST, MetaPhlAn2 and Qiita. Notably, some of these metagenomic tools (e.g. MEGAN-LR) deal with the output of long-read sequenc-ing techniques, such as of Pacific Biosciences (PacBio) or Oxford Nanopore Technologies (ONT) [25]. Those gains of interest in metagenomic research are due to the fact that taxonomic and its functional annotation do not rely anymore on single genes covered by multiple short reads (approx. 50–300 bp) and their gene copy number issues (e.g. 16S rRNA) but on multiple genes covered by long reads, with an average read length of 5 to 50 kb, whereof approx. 50% of the reads are larger than 14 kb [26]. Continuous advances in high throughput genomic sequencing technologies, metagenomics and single cell genomics, have con-tributed new insights into uncultivated lineages. Several of the known microbial phyla, 120 bacterial and 20 archaeal phyla, con-tain few cultivated representatives (ncbi.nlm.nih.gov/Taxonomy/ Browser/wwwtax.cgi). Moreover, phyla composed exclusively of uncultured representatives are referred to as Candidate Phyla (CP) [27,28]. Such uncultivated majority, approx. 90 bacterial can- didate phyla, defined as microbial dark matter and exist in various M.S. Sarhan et al./Journal of Advanced Research 19 (2019) 15–27 17 environmental microbiomes [6,29–31]. Remarkably, metage- nomics and microbiome analyses have detected so many candidate phyla, and phylogenetic analyses have revealed such a close rela-tionship among many of them that the term ‘‘Candidate Phyla Radiation” (CPR) was coined for a group of uncultured bacteria that share evolutionary history [32–34]. The number of newly discovered candidate phyla is increasing due to further developments in metagenomic techniques and con-tinual updating of genomic databases, and representing a striking challenge to the scientific community [27,35]. With increased metagenomic sampling and analysis, taxonomic boundaries and nomenclature are constantly being reassessed. Meanwhile, scien-tists have realized that bacterial and archaeal phyla without a sin-gle cultivated representative comprise the majority of life’s current diversity [27,32,34]. Certainly, the current knowledge about the microbial world, not only the substantial roles played by microor-ganisms in the function of the biosphere but also their reservoir of novel bioactive compounds, is profoundly challenged by what have been cultivated in the laboratory [35]. So far, physiologic and geno-mic information has been confined to pure cultures and dominated by representation of the Proteobacteria, Firmicutes, Actinobacteria, and Bacteriodetes within the Bacteria and of methanogens and halotolerant members of the Euryarchaeota within Archaea [36]. From synthetic to environmental cultivation of microbiomes Today, it is established that culture media tailored for in vitro cultivation of microorganisms, including CP microorganisms, must provide environmental and nutritional conditions that resemble their natural habitats, combined with long incubation times [37]. Further attempts towards improving culture media to grow novel species depended mainly on supplementing macro- and micronu-trients in the medium as well as manipulating cultivation condi-tions (Table 1). Conspicuous developments and higher throughput methods have been applied to marine and terrestrial environments (Fig. 1, Table 2), adopting a number of approaches reviewed by Epstein et al. [38]: for example, lowering nutrient con- throughput cultivation -Diffusion chamber -Isolation chip (Ichip) -Microfluidic Streak Plate (MSP) -Double encapsulation technique -Soil Substrate Membrane System (SSMS) -Hollow-Fiber Membrane Chamber (HFMC) Culture media -Plantlextract additives Culturomics Incubation conditions -Signaling compounds -Aerobic/anaerobic and coculturing -Different temperatures -Plant-based culture media -Light/Dark -Creation of stress conditions for culturing extremophiles (pH, salinity, temperature,...etc) Omics-derived cultivation information Fig. 1. Toolbox of strategies developed for improving culturability of environmental microbiomes. High throughput culturomics adopt various combinations of the specific methods of the 4 major strategies of in situ and high throughput cultivation, culture media development, incubation conditions, and genome-derived cultiva-tion. For further details, please refer to Table 1. centrations in standard media together with longer incubation [39], diluting to extinction to minimize the influence of fast grow-ers and facilitate growth of oligotrophs [40], co-incubating cells individually encapsulated into microdroplets under low flux nutri-ent conditions [41], adding signaling compounds and/or co-cultivation to trigger microbial growth [42,43]. Novel in situ cultivation techniques, e.g. diffusion chambers, have been introduced to mimic natural conditions and provide access to critical growth factors found in the environment and/or Table 1 Progressive supplements of culture media to improve culturability of environmental microbiomes. Culture media supplementation Basal medium supplemented with isoleucine and yeast extract [44]a Basal medium supplemented with yeast extract [45] Nitrogen-free LGI-P medium supplemented with sugarcane juice [46] 10-fold-diluted Difco marine broth 2216 supplemented with yeast extract [47] Postgate’s medium B supplemented with yeast extract [48] MPN soil solution equivalent (SSE) supplemented with pectin, chitin, soluble starch, cellulose, xylan, and curdlan as carbon sources [49] Basal medium supplemented with humic acid and vitamin B (HV medium) [50] TSA, casein-starch, and 869 culture media supplemented with plant extracts [51] Peptone-Yeast extract-Glucose medium (PYG) supplemented with Resuscitation-promoting factors (Rpf) [52] Modified Biebl and Pfennig’s medium [53] Culture media based on extracts of potato, onions, green beans, black beans, sweet corn, sweet potato, or lentils [54] Selective King’s B medium supplemented with lichens extract [55] Basal medium supplemented with sugarcane bagasse [56] Fastidious anaerobic agar and blood agar media supplemented with siderophores-like molecules [57] Minimal medium supplemented with peels of orange, potato, or banana [58] PBS buffer supplemented with pig fecal slurry or dried grass hay as carbon sources [59] MRS and TSB supplemented with Titania (TiO2) nanoparticles [60] Modified 80% ethanol soil extract culture media [61] a Numbers between brackets refer to related references. Recovered taxa Aminobacterium mobile Acidilobus aceticus Burkholderia tropica Hoeflea phototrophica Desulfitibacter alkalitolerans Edaphobacter modestus and Edaphobacter aggregans Pseudonocardia eucalypti Kaistia sp. and Varivorax sp. Arthrobacter liuii Thiorhodococcus fuscus Biomass production of Pseudomonas fluorescence Resulted in higher endo-lichenic and ecto-lichenic bacterial CFU counts Higher CFU recovery compared with other standard media Prevotella sp., Fretibacterium fastidiosum, Dialister sp., and Megasphaera sp. Biomass production of Bacillus subtilis Streptococcus caviae Enhanced biocontrol performance of PGPR strains against Fusarium culmorum 18 novel species including isolates belonging to Verrucomicrobia and Elusimicrobia 18 M.S. Sarhan et al./Journal of Advanced Research 19 (2019) 15–27 Table 2 Developed novel methods to increase culturability of environmental microbiomes. Developed methods Diffusion Chamber [62]a Recovered taxa Method illustration Deltaproteobacteria, Verrucomicrobia, Spirochaetes, and Acidobacteria [62] Soil substrate membrane system (SSMS) [63,64] Enrichment of uncultured Proteobacteria and TM7, as well as isolation of Leifsonia xyli sp. nov. [63,64] Hollow-Fiber Membrane Chamber (HFMC) [65] Enrichment of uncultured Alphaproteobacteria, Gammaproteobacteria, Betaproteobacteria, Actinobacteria, Spirochaetes, and Bacteroidetes [65] Single cell encapsulation in gel microdroplets (GMD) [66] Enrichment of uncultured Gammaproteobacteria, Betaproteobacteria, Alphaproteobacteria, Bacteroidetes, and Planctomycetes [67] [66] Isolation chip (Ichip) [68] Single-Cell Cultivation on Microfluidic Streak Plates [69,70] Enrichment of Alphaproteobacteria, Betaproteobacteria, Deltaproteobacteria, Epsilonproteobacteria, Gammaproteobacteria, Actinobacteria, Bacteroidetes, Firmicutes, Planctomycetes, and Verrucomicrobia Enrichment of uncultured Proteobacteria, Firmicutes, Actinobacteria, Bacteroides, Acidobacteria, Planctomycetes, and Verrucomicrobia, in addition to isolation of novel Dysgonomonas sp. [68] [69,70] a Numbers between brackets refer to references related. supplied by neighboring species. This allowed the cultivation of variants that otherwise would not grow ex situ [12]. Some of the resulting chamber-reared populations were spontaneously lab-domesticated to acquire the ability to grow in vitro [65]. Undoubt-edly, the newly advanced cultivation technologies have unraveled the existence of new species en masse. However, microbiologists should be able and continue to minimize the gap between the microbial richness in nature and the number of species in culture, for the benefit of both basic and applied microbiology [12]. Culturomics in place and the progress achieved Realizing the imperative importance of bringing more bacterial isolatesofenvironmentalmicrobiomesintocultivation,thestrategy of ‘‘culturomics” was introduced by the group of Didier Raoult and Jean-Christophe Lagier [5,71–73]. They developed a high through-put strategy of cultivation to study the human microbiota using matrix-assistedlaserdesorption/ionizationtimeofflightmassspec-trometry (MALDI-TOF-MS) and/or 16S rRNA amplification and sequencing to identify the growing colonies. The principals of cul- turomics are based on the diversified and multiple combinations of various growth media, culturing conditions, atmospheres and timesof incubation,that werereducedto only18cultureconditions to standardize culturomics, and to complement the culture-dependent and culture-independent analyses (reviewed in Lagier et al. [72]; Table 3). The extensive application of MALDI-TOF-MS for rapid and high throughput identification of rare and new species allowed the group to dramatically extend the known human gut microbiome to levels equivalent to those of the pyrosequencing repertoire. Lagier et al. [71] identified > 1000 prokaryotic species, thereby adding > 500 species that represent > 50% increase in the total number of microorganisms known in the human gut. Further-more, they were able to extend culturability of archaea without an externalsourceofhydrogentorecoverhumanarchaealspecies[74]. The dawn of plant-based culture media Although the results obtained with culturomics of human gut microbiome are immense and represent a success story, it did not draw much attention from research groups of the plant micro-biome. Here, the compelling question is ‘‘should plant microbiolo- gists follow the steps of human microbiome culturomics and M.S. Sarhan et al./Journal of Advanced Research 19 (2019) 15–27 19 Table 3 The basic principles and techniques of culturomics of human microbiota and results obtained at URMITE, Marseille, France.a 1. Out of 70 culturing conditions, 18 were defined for culturomics standardization, based on the following: Various combinations of culture media used for: – pre-enrichment in broth cultures, followed by – inoculating onto different agar plates for single colony isolation Culture conditions Incubation temperature Incubation time 2. Challenges faced and specific answers to isolate rare species Growth of bacteria having different physiological properties Overgrowth of fast growers Fastidious bacterial species 3. Performance of identification of thousands of developed colonies Majority of colonies Confirmatory analyses for unidentified colonies Colonies representing potential new taxa 4. Total of 531 species were added to the human gut repertoire Major phyla reported Species known in humans but not in the gut Species not previously isolated in humans Potentially new species a Source [71,72]. Various combinations of: – blood culture, rumen fluid, sheep blood, stool extract – Tryptic Soy Broth (TSB), marine broth – Aerobic, anaerobic atmospheres – Thermic shock at 80℃ – Specific supplements (e.g. lipids, ascorbic acid) Ranging from 4 to 55℃ From 1 to 30 days Various incubation temperatures and gas phases (aerobe, anaerobe, microaerophile) Kill the winners by: – diverse antibiotics, and inhibitors (e.g. bile extract, sodium citrate, sodium thiosulphate) – heat shock (65℃ and 80℃) – active and passive filtration – phages Pre-incubation (in selective blood culture bottles, rumen fluid) MALDI-TOF and comparisons with URMITE databases 16S rRNA gene or rpoB sequencing Taxonomogenomics: polyphasic approach of both phenotypic (e.g. primary phenotypic characteristics) and genotypic data (e.g. genome size, G + C content, gene content, RNA genes, mobile gene elements...etc) and compared with closely related type strains Firmicutes, Actinobacteria, Bacteriodetes, Proteobacteria, Fusobacteria, Synergisetes, Lentisphaerae, Verrucomicrobia, Dinococcus-Thermus, and Euryarchaeota 146 bacteria 187 bacteria, 1 archaeon 197