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.