Cultivation of unculturable soil bacteria

Van H.T. Pham and Jaisoo Kim
Kyonggi University, College of Natural Sciences, Department of Life Science, Suwon, Gyeonggi-Do 443-760, Republic of Korea
Despite the abundance of bacterial species in soil, more
than 99% of these species cannot be cultured by tradi-
tional techniques. In addition, the less than 1% of bacteria
that can be cultured are not representative of the total
phylogenetic diversity. Hence, identifying novel species
and their new functions is still an important task for all
microbiologists. Cultivating techniques have played an
important role in identifying new species but are still low-
throughput processes. This review discusses the issues
surrounding cultivation, including achievements, limita-
tions, challenges, and future directions.
The need to culture the unculturable
Current estimates indicate that there are about 61 distinct
bacterial phyla, of which 31 cannot be cultured [1]. To date,
only 54 species of Archaea have been cultured among the
49 different lineages [1,2]. Although many new molecular
approaches have been applied for studying the biodiversity
and role of microorganisms in the environment, cultivation
is still useful in understanding the detailed metabolism
and functions of those organisms, and makes them possible
for genetic manipulation, confirming certain functions.
Unfortunately, we do not have sufficient knowledge to
create natural conditions for all microorganisms. Artificial
media are often incapable of mimicking the endogenous
abiotic and biotic conditions required for microbial growth
[3], and microorganisms cannot immediately adapt to
these changes in conditions.
Microbial diversity has been measured by the cultiva-
tion of bacteria on laboratory media and molecular
approaches based on 16S rRNA methodologies (see Glos-
sary) [4]. However, during the past 25 years, many
advances have been made in cultivation-based techniques,
which have significantly contributed to our understanding
of living microbes. These advances are: (i) use of modified
media [3,5–19], (ii) changes of growth conditions
[3,9,10,12,13,16,20–24], (iii) community culture and cocul-
ture [1,18,25–32], (iv) use of transwell plates [33], (v)
optical tweezers and laser microdissection [6], (vi) high-
throughput microbioreactor [8,34–36], (vii) simulated nat-
ural environments using diffusion chambers [11,37–40],
and (viii) assistance of culture-independent methods
[6,10,41–57].
Molecular-based approaches have shown that approxi-
mately 1% of the total microbial population can be cultured
under a restricted range of media and cultivation condi-
tions [58] and the diversity of the uncultured majority is
vast [59,60]. Cultivation of microbes using solid media in
petri dishes and analysis by 16S rRNA has been used to
confirm the high degree of phylogenetic novelty and a
number of isolates affiliated with so-called unculturable
groups. Through identifying novel isolates, we clearly
know what types of bacteria exist and their possible roles
in the environment, providing further clues to their ecolo-
gical influences and new functions for industrial applica-
tions. For example, the anaerobic ammonium oxidation
pathway was recently discovered based on the cultivation
of anaerobic ammonium-oxidizing bacteria [16]. These
findings indicate that cultivation methods should be devel-
oped further to obtain laboratory cultures of many phylo-
genetically novel soil bacteria [61].
One of the main challenges for microbiologists is to find
new ways of growing these microbes in a laboratory set-
ting. To achieve this goal, we must obtain a comprehensive
understanding of the chemical, physical, and biological
processes of the environments where microbes live and
apply this knowledge to microorganisms for their growth
while considering all their interactions. Further, we may
need to combine molecular techniques such as fluorescence
in situ hybridization (FISH) and metagenomics to gain
better information about their cultivation.
Review
Glossary
16S rRNA methodologies: the 16S rRNA gene is used for phylogenetic studies
due to highly conserved sequences between different species of bacteria and
archaea, and also hypervariable regions as species-specific signature se-
quences. There are two types of methodologies, 16S rRNA gene sequencing
and ribotyping. 16S rRNA gene sequencing has become prevalent in
microbiology because it provides a rapid accurate alternative to phenotypic
methods of bacterial identification. Ribotyping is the fingerprinting of genomic
DNA restriction fragments of the 16S rRNA genes.
Catalyzed reporter deposition fluorescence in situ hybridization (CARD-FISH):
a FISH method that enhances the signal through conjugation of an enzyme
peroxidase to the specific nucleic acid probe instead of a fluorescent dye. It is
useful in phylogenetic studies of prokaryotes that may be small, slow growing,
or starving bacteria.
Clone library analysis: a method to analyze a clone library; a collection of DNA
fragments that is stored and propagated in a population of microorganisms
through the process of molecular cloning.
Colony-forming curve (CFC): a plot of the number of visible colonies on a plate
against incubation time.
Denaturing gradient gel electrophoresis (DGGE): a technique to detect
differences in the melting behavior of small DNA fragments (200–700 base
pairs) that differ by as little as a single base substitution. The separated bands
on the gel are located according to the concentration gradient of denaturants.
Fluorescence in situ hybridization (FISH): a cytogenetic technique that is used
to detect and localize the presence or absence of specific DNA sequences on
chromosomes.
Selective medium-design algorithm restricted by two constraints (SMART): a
strategy to allow growth of targeted microbes through highly selective media
based on two selective agents, a carbon source and an antimicrobial.
Shotgun sequencing: a method used for sequencing long DNA strands.
Because DNA sequencing can only be used for relatively short strands (100–
1000 base pairs), longer sequences must be subdivided into smaller fragments,
and subsequently re-assembled to give the overall sequence.
Stable isotope probing (SIP): a method based on the incorporation of
13
C-
labeled substrate into cellular biomarkers such as nucleic acids, separation of
labeled from unlabeled nucleic acids by density gradient centrifugation, and
molecular identification of active populations carrying labeled nucleic acid.
Corresponding author: Kim, J. (jkimtamu@kgu.ac.kr).
0167-7799/$ – see front matter ß 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tibtech.2012.05.007 Trends in Biotechnology, September 2012, Vol. 30, No. 9 475
This review examines the cultivation methods used in
the past and present (Table 1), and discusses the chal-
lenges and future direction of cultivation.
Lack of knowledge on unculturable bacteria
The important task of cultivation in situ is creating arti-
ficial systems that mimic natural conditions. However,
some natural conditions are difficult to mimic: (i) the
chemistry of a microbe’s natural habitat, (ii) interactions
of biotic and abiotic factors, (iii) the diversity of microbes,
and (iv) the effect of climate change and other functions in
ecosystems at the microbial level [3].
These difficulties have led to many mistakes in cultiva-
tion, such as addition of an inhibitor in a Spirochetes
medium [35], ignorance of K- strategists that require
low concentration of nutrients [16], and no consideration
of density-dependent cell signaling mechanisms [62]. In
addition, some isolates require a long time for growth and
visibility, which we often do not have the patience for. A
typical example of this includes organisms originating
from oligotrophic conditions, where each species will
require different time intervals for growth [3].
Abiotic interactions are often lost in the enrichment–
isolation process of prokaryotic culture. Many biogeo-
chemical factors are disrupted, such as carbon, nitrogen,
oxygen, sulfur, phosphorus, and water, and synthetic
conditions must be designed, including resource type
and concentration.
Some important physicochemical parameters that
directly affect microbial interactions and metabolism are
pH, temperature, pressure, redox potential, oxygen con-
centration, salinity, and time for growth. These conditions
can dictate the characteristics and growth rate of each
species. In some cases, changes in the environment will
also affect their reactions. Therefore, techniques for iso-
lating microorganisms should be designed to consider
physical, chemical, and biological parameters [63].
Another factor that effects cultivation is additional local
communities that may form complex relationships with the
microbial community of interest. For example, Veillonella
spp. may have established metabolic cooperation with
Streptococcus mutans that produce lactic acid as their
carbon source [1].
Modifications to growth media
Energy, nutrients, and proper physicochemical sources are
needed for the growth of any microorganism and the levels
vary depending on the species. Thus, the design of media
for the growth of different microorganisms is a difficult
task and a real challenge for microbiologists. This requires
in-depth insight into the nature and characterization of
samples, including all factors that are directly related to
and mediate growth. A better understanding of these
factors will help us develop methods to culture not-yet-
cultured microbes.
Several recent studies have examined the effect of dif-
ferent components and concentrations in media formula-
tions on the cultivationof unculturedmicrobes (Table 2). For
example, Tannerella forsythia needs N-acetyl muramic acid
for growth, whereas Abiotrophia spp. and Granulicatella
Table 1. Summary of new approaches in cultivation
New approach Description Refs
Modification of growth media Includes change of composition, addition of some specific chemical(s), and dilution of media. [1,20]
Modification of growth conditions Modifies incubation time, inoculum size, air conditions (CO
2
/O
2
level), temperature, and pH for
mimicking natural conditions.
[16]
Community culture Uses mixed bacteria to cultivate the unculturable bacteria that require their cooperation. [28]
Coculture Cultivates the unculturable with the assistance of helper bacterium/bacteria. [1,18,29]
Transwell plates with membranes A modified plate method where the bottom layer (i.e., the membrane filter) does not allow
passage of bacterial cells. The transwell plate is then placed on the soil slurry in a container,
allowing diffusion of soil solution into the agar layer on the membrane.
[33]
Micromanipulator Uses a capillary tube or a microneedle to pick up a single bacterial cell out of a mixed
community under visual control with an inverse microscope.
[75]
Optical tweezers A highly focused laser beam used to trap and manipulate microscopic neutral objects such
as microbial cells. Using this method, a bacterial cell can be isolated from a mixture of cells.
[75,76]
Laser microdissection Involves visualization of the cells of interest via microscopy, transfer of laser energy to a
thermolabile polymer with formation of a polymer–cell composite (IR system) or
photovolatilization of cells surrounding a selected area (UV system), and removal of the
cells of interest from the heterogeneous tissue section.
[75]
High-throughput microbioreactor A robotically-driven cell culture bioreactor system that enables rapid screening and cell
culture/fermentation process parameter optimization with process monitoring technologies,
feedback control, and real-time powerful data acquisition systems.
[34]
Simulated natural environments
using diffusion chambers
Allows the uncultivated microorganisms to grow in pure culture through provision of the
required chemical components from their natural environment using diffusion chambers.
[11,37–40]
Single cell encapsulation
combined with flow cytometry
By emulsifying a mixture of diluted cell suspension and preheated agarose, gel microdroplets
(GMDs) including single cells are formed and incubated in media. This method allows low
nutrient flux into GMDs and creates proper conditions for slow-growing microorganisms.
Then, GMDs containing colonies are separated from free-living cells and empty GMDs
using a flow cytometer.
[3,77,78]
Multiwell microbial culture chip A micro-petri dish that is composed of a unique ceramic having millions of compartments
in which cultures can be separately grown.
[79]
Entrapped gelating agent coated
with polymer
Gelating agent spheres containing the entrapped microorganisms are coated with a natural
or synthetic polymer to form a polymeric membrane. They are then incubated in the
original environment.
[80]
Review
Trends in Biotechnology September 2012, Vol. 30, No. 9
476
spp. require pyridoxal or L-cysteine [1]. By using traditional
media, previously unculturedbacterial groups were isolated
on VL55 medium formulated with low concentrations of
inorganic ions [20]. To examine the respiration and meta-
bolic process of microbes, microbiologists have considered
different electron donors, electron acceptors, and carbon
sources whose efficiency have been demonstrated in culture
methods for many kinds of microbes such as Proteobacteria,
Bacteoidetes, Fusobacteria, Actinobacteria, Firmicutes,
Bubrobacteidae, Acidobacteria, Verrucomicrobia, and Gem-
matimonadetes [16,64,65]. In these studies, energy and
carbon are often obtained from inorganic and organic com-
pounds [16,64,65].
Some studies have examined the effect of anaerobic
oxidation of methane to sulfate or nitrate reduction. These
approaches required refined media containing hydrocar-
bon energy sources and nitrate, iron, and sulfate as elec-
tron acceptors [3]; some phylogenetic types were shown to
grow on this type of media.
Some other studies using samples from the North Sea
showed that the quality of the carbon sources were deter-
minant factors of cultivation efficiency. Media containing
various carbon sources and complex compounds yielded
higher numbers and more diverse isolates than similar
media with only one carbon source [3,5]. Conversely, many
autotrophic organisms failed to grow in the presence of a
fixed carbon source [6].
There has been some success in cultivating unculturable
soil bacteria by using low substrate concentrations (Table
2) [3,7–11]. The bacterial phyla Actinobacteria, Acidobac-
teria, Proteobacteria, and Verrucomicrobia were obtained
using low-nutrient media and increased incubation times
[12]. Changing the culture conditions (e.g., nutrient level,
oxygen concentration, addition of humic acids and signal-
ing molecules) increased the presence of some novel micro-
organisms in soil [13].
In 2005, a new medium, called the soil-extract agar
medium, was used to cultivate microorganisms from soil;
some novel bacteria and Actinomycetes were identified
using this medium [14]. Recently, a novel strategy for
designing highly selective media called selective medium-
design algorithm restricted by two constraints (SMART)
was developed (Figure 1) [15]. This method was used to
develop selective media for Burkholderia glumae, Acido-
vorax avenae, Pectobacterium carotovorum, Ralstonia sola-
nacearum, and Xanthomonas campestris [15].
The addition of humic acids and signaling molecules (for
transition to growth forms of dormant forms such as spores
and cysts) in media also improved the cultivability of
unculturable soil bacteria [13,16], as well as the addition
of enzyme to cope with reactive oxygen species [3,13] and
the addition of some inhibitors to remove undesired micro-
organisms [3,17].
Some microbiologists have also demonstrated that gel-
lan gum was effective for the cultivation and isolation of
hitherto-uncultured microbes in freshwater sediment
because its plates are clearer than agar for finding very
tiny colonies [12,18,19]. Gellan gum may also enhance the
growth of bacteria whose growth is inhibited by agar [66].
Colony-forming unit (CFU) counts from gellan gum med-
ium were 10-fold higher than those from agar medium.
Approximately 60% of microbes grown on gellan medium
were considered novel, a number 2-fold higher than agar
medium. More than half of the novel isolates did not form
colonies on agar medium under the same conditions.
Modifications of growth conditions
Growth conditions such as incubation period, inoculum
size, temperature, pH, and air conditions are important
for microbial cultivation, in particular for growing uncul-
turable soil bacteria [16].
Incubation time
Increased incubation periods are one of the most important
factors when attempting to identify lower-growth and
fastidious isolates [3,9,10,13,20–22]. One strategy to culti-
vate and detect uncultured microbes involves extending
incubation time. For example, extended incubation periods
Table 2. Composition of different diluted media used for culturing unculturable soil bacteria [15]
Ingredient Medium type (g/l distilled water)
0.01 Â NB WSS
a
VL55 VL70
2-[N-morpholino] ethanesulfonic acid 1.95 2.09
Beef extract 0.03
Casein
Peptone 0.05
Soya bean
CaCl
2
Á2H
2
O 44.11 Â 10
–3
44.11 Â 10
–3
K
2
HPO
4
0.40
MgSO
4
Á7H
2
O 0.13 49.29 Â 10
–3
49.29 Â 10
–3
MnSO
4
ÁH
2
O 1.52 Â 10
–3
NaCl
NH
4
NO
3
0.50
(NH
4
)
2
HPO
4
26.41 Â 10
–3
26.41 Â 10
–3
(NH
4
)
2
SO
4
0.13
Selenite-tungstate solution
b
1 ml 1 ml
Trace element SL-10
c
1 ml 1 ml
a
Winogradsky’s salt solution.
b
NaOH, 0.5 g; Na
2
SeO
3
Á5H
2
O, 3 mg; Na
2
WO
4
Á2H
2
O, 4 mg; distilled water, 1000 ml.
c
HCl, 10 ml; CoCl
2
Á6H
2
O, 190 mg; CuCl
2
Á2H
2
O, 2 mg; FeCl
2
Á4H
2
O, 1.5 g; NaBO
3
, 6 mg; MnCl
2
Á4H
2
O, 100 mg; Na
2
MoO
4
Á2H
2
O, 36 mg; NiCl
2
Á6H
2
O, 24 mg; ZnCl
2
, 70 mg; distilled
water, 990 ml.
Review Trends in Biotechnology September 2012, Vol. 30, No. 9
477
(5–12 weeks) have yielded isolates of both known and new
groups [12,13]. New bacterial strains successfully isolated
with long incubation include the following phyla: Acido-
bacteria, Actinobacteria, Chloroflexus, Gemmatinonadetes,
Planctomydetes, and Verrucomicrobia [20,21,23,61].
Inoculation size
A novel enrichment and isolation procedure was necessary
for slow-growing microorganisms. One group enriched a
large number of slow-growing microorganisms by repeated
batch culture under high biomass concentration (more
than 10 g biomass/l). Characteristics of the microorgan-
isms were analyzed by colony-forming-curve (CFC) and
large numbers of slow-growing bacteria survived under
starvation conditions [24].
Air conditions
Incubation in the presence of air and hypoxic and anoxic
atmosphere was used to isolate various bacteria that live in
different microhabitats in soil, and an elevated concentra-
tion of CO
2
was added for some bacteria, especially for
autotrophs [13,16]. A successful isolation of novel anaero-
bic bacteria of Verrucomicrobiales was done from anoxic
rice paddy soil using non-oxygenated media [67]. Two
genera of methanogens, Methanosarcina and Methano-
cella, were identified even under oxic/oxygenic conditions
[68]. A wide variety of aerobic facultative anaerobic and
microaerobic diazotrophs from sea water were isolated
using O
2
gradient media [69].
Temperature
Because lower temperature decreases metabolic rate, the
productivity of growth-inhibiting materials decreases,
inducing more colonies for more isolates. Soil bacteria grew
better at a temperature range of 20–25 8C than 30 8C [16].
In particular, if samples are obtained from cold regions,
then the incubation temperature should be lowered. For
example, distinct colony types were successfully isolated
through low-temperature recovery strategies from ancient
permafrost sediments [70]. Extended low-temperature cul-
tivation significantly improved the recovery of previously
unculturable bacteria, which could be members of the
Proteobacteria, Cytophaga-Flavobacteria-Bacteroides,
high-G+C Gram-positive, or spore-forming low-G+C
Gram-positive bacteria [71].
pH
pH is also a critical factor to cultivate unculturable soil
bacteria; the pH can be adjusted to actual sampled soil
values [16]. The most commonly used nutrient media
have a near-neutral pH and a salt content of 1–3 g/l.
Some of these media have conditions that differ drama-
tically from acidic (pH 3.5–5.5) peat water with a mineral
salt content of 5–50 mg/l. This explains why most peat-
inhabiting bacteria do not develop on conventional media.
As a result, a number of peat-inhabiting methanotrophs
were isolated on mildly acidic, low-ionic-strength media
[72]. In addition, many members of the Acidobacteria
were isolated from soil samples at moderately acidic
pH values [73].
Community culture and coculture
Microbes always establish a relationship with other micro-
organisms in a community and these interactions can
either compete for limited resources or cooperate through
an exchange of metabolites and signaling molecules
[25,26]. Typical examples can be found from biofilms or
multicellular assemblages where microbes communicate
with one another. For example, mixed cellular assem-
blages can perform multistep degradation of cellulose
Is genomic information
available in KEGG
Pathcomp?
Is genomic information
available in NCBI entrez
gene?
No No
Yes
Yes
Metabolizable carbon
sources are listed
Carbon source with
high saprophyte
inhibitory rate are
selected by reference
An optimal carbon
source
Determination of composition
of SMART media
Combination of resistant
antimicrobials
Information of relative
species of target
bacteria is used
Information of relative
species of target
bacteria is used
TRENDS in Biotechnology
Figure 1. Flowchart of the selective medium-design algorithm restricted by two constraints (SMART) method, which develops highly selective media based on two
selective agents, an optimal carbon source and antimicrobials.
Review
Trends in Biotechnology September 2012, Vol. 30, No. 9
478
and the intermediates can be utilized as carbon sources
[3,74].
Community culture is an effective way to cultivate
facultative or syntrophic organisms and other community
members. To date, two successful examples of such cul-
tures have been reported: enrichment cultures of (i) a
thermophilic syntrophic anaerobic glutamate-degrading
consortium in anaerobic sludge [28], and (ii) mixed bacteria
(a community in sludge) in anaerobic and aerobic alternate
batch reactors [28].
New microorganisms were successfully identified
through either cocultures with H
2
-consuming organisms
or by using different substrates in a pure culture that was
appropriate for their growth [18]. The removal of H
2
is
required because of its inhibitory action against the oxida-
tion process of many anaerobic bacteria. A novel Psychro-
bacter sp. was cultivated after repeated coculture using
helper strains together with tissue culture inserts and agar
plates [1,29]. The helper strains produce essential growth
factors, an important finding for cultivation of an unculti-
vable strain with artificial media.
Other researchers have used free-living amoebae as a
tool for the isolation of novel intracellular microorganisms
from environmental samples including soil [30,31]. Pre-
viously uncultured soil bacteria were cultivated in cocul-
ture with a fungus; the fungus may choose a bacterial
population as its partner, and its exudates may serve as
nutrients for those bacteria [32].
New culture methods
Since the petri dish was discovered, it has been widely used
for cultivation in laboratories. Colonies can be isolated by
repetitive streaking on solid medium (or by performing
pour plate or agar shake tubes). In addition, cells can be
isolated by dilution in liquid medium. The enrichment and
pure culture are selective for the desired organisms. How-
ever, the resources and conditions used for laboratory
culture often do not mimic natural conditions, which pre-
vent proliferation of microorganisms. This classical
method seems to work best for fast-growing organisms,
which only represent a minor fraction of all natural micro-
bial communities. Thus, new methods need to be developed
for the growth of a wider range of microorganisms.
Transwell plates with membranes have recently been
used for the discovery of new species that were previously
considered ‘uncultured microorganisms’ (Figure 2a). For
example, a natural medium membrane system was used to
plate soil solutions [33]. All microorganisms were then
placed in a rack in a gas air-tight jar. The jar was aerated
and re-flushed with methane every 3–5 days and incubated
at 22 8C for up to 47 days. By using this approach, many
colonies of methane-oxidizing bacteria were observed.
Other methods used for cultivation are single cell iso-
lation techniques using micromanipulators [75] or laser
manipulation systems such as optical tweezers (Figure 2b)
[75,76] and laser microdissection [75]. Some novel
microbes from a marine environment were cultured using
the optical tweezers [10].
A novel high-throughput microbioreactor (a robotically
driven cell culture bioreactor system), SimCell
TM
platform
for fed-batch cultivation, allows for pH, dissolved oxygen,
and glucose control (Figure 2c) [34]. This method was used
to successfully simulate a shake flask and performed
better than traditional methods, supporting viable cell
concentrations up to at least 12 Â 10
6
cells/ml. High-
throughput cultivation was successful for the recovery
of previously uncultured marine bacterioplankton Planc-
tomyces and Bacteroidetes [35]. Using this method, up to
14% of the cells collected from the coastal seawater were
cultured [8]. The number of microorganisms obtained was
14- to 1400-fold higher than the number obtained by
traditional techniques. Similarly, diffusion chambers
and high-throughput strategies were used to find the first
member of a previously-uncultured microorganism from
soil [36].
Other researchers have examined the cultivation of
unculturable microorganisms in simulated natural envir-
onments using diffusion chambers with successful results
(Figure 2d) [11,37–40]. They hypothesized that there were
two different reasons for the successful cultivation of these
microorganisms on a petri dish: (i) representatives of these
isolates were too rare at the beginning of the experiment
and needed two rounds of enrichment in the diffusion
chamber before appearing in the petri dishes in numbers
sufficient for isolation; (ii) their adaptation to growth in a
petri dish required several events in the simulated natural
environment of the chamber.
Another technology for massive cultivation was a single
cell encapsulation combined with flow cytometry under low
nutrient flux conditions that yielded numerous isolated
clades (Figure 3a) [3,77,78].
Another cultivation method involved a multiwell micro-
bial culture chip (Figure 3b) [79]. This method used a
porous membrane that allowed the nutrients to diffuse
for bacterial growth, and enabled the growth of separate
microbial samples at an unprecedented density.
A new method for isolating and culturing unculturable
microorganisms was recently developed in which microbial
samples were diluted in the appropriate medium and a
gelating agent was added to entrap microorganisms within
a sphere of the gelating agent (Figure 3c) [80]. These
spheres containing the entrapped microbes were then
coated with a natural or synthetic polymer to form a
polymeric membrane. After incubation for an appropriate
time, the spheres were cut, scanned for microorganisms
colonies, and the microorganisms were isolated. These
steps were repeated until an unculturable microorganism
was obtained as a pure clone.
Assistance of culture-independent methods
In addition to culture-dependent methods, culture-inde-
pendent methods have been developed to improve the
cultivating process. The combination of various factors
such as increasing the incubation time, more inoculation
plates for each dilution, sonication, selection of microcolo-
nies with the help of magnifying lens, using half strength
R2A agar and other modifications, improve cultivation
results [41,42].
To improve the cultivation of fastidious bacteria, the soil
substrate membrane system was combined with immuno-
fluorescent viability staining and advanced micromanipu-
lation to target viable microbes and microcolonies formed
Review Trends in Biotechnology September 2012, Vol. 30, No. 9
479
from soil bacteria. This method resulted in the isolation of
many recalcitrant bacteria in microbial communities [43].
Both culture-dependent and culture-independent
approaches have been used to identify N
2
O reducers in
rice paddy soil [44]. For culture-dependent analysis,
microorganisms were cultured under N
2
O reducing
conditions in the presence of cell division inhibitors. They
were captured using a micromanipulator and transferred to
a low-nutrient medium. For culture-independent analysis,
an approach called stable isotope probing (SIP) was
Buffer chamber 1
Buffer chamber 2
Reservoir 1
Sample chamber
Electrokinetic focusing
Particle B
Particle A
Function area
Optical tweezer
Reservoir 2
Optical sorting
Bacteria
+
Agar
Step 1
Step 2 Step 3
0.03 µm pore-size
polycarbonate membrane
Growth chamber
0.03 µm pore-size
polycarbonate membrane
(a) (b)
(d)
(c)
Shake 15 min
sedimentation 30 min
Spreading of soil suspensions
on cell culture insert
Cell culture inserts transferred to
culture plates with soil suspension
Sampling
module
Fluidic module
Incubation
modules
Optical
sensing
module
Loading cell
Central
robot
Incubation with CH
4
Membrane with
visible colonies
10
−3
10
−2
10 µl 100 µl
x
y
z
TRENDS in Biotechnology
1
0
0
X
N
A
=
0
.8
Figure 2. The new culture methods introdued in this article. (a) A schematic overview of the transwell plate method for membrane growth of methane-oxidizing bacteria
[33]. (b) Microfluid systemwith optical tweezers for cell sorting using a laser beam[76]. (c) The systemof the SimCell
TM
platform, which is a robotically driven cell culture
bioreactor system[34]. (d) Diagramof the diffusion growth chamber for in situ cultivation of soil microorganisms with assembling steps in which microbes obtain nutrients
that come from slurry [11].
Review
Trends in Biotechnology September 2012, Vol. 30, No. 9
480
(a)
(c)
(b)
Agar sphere
Polymeric membrane
pH control
Culture
medium
Gas controller
Mineral cartridge
GMDcc
Culture in 384 channel
parallel microbioreactor
Plasma
Shadow mask
Laminate
Laminate
Compartment
Porous
aluminium oxide
Porous
aluminium oxide
Nutrients
Platinum layer
Wafer
TRENDS in Biotechnology
Figure 3. The additional newculture methods introduced in this article. (a) A model of the experimental setup for a single cell encapsulation combined with flowcytometry,
which consists of flow-through culture in parallel microbioreactors nourished by community culture medium and metabolite products [3]. (b) Schematic figure of the
multiwell microculture chip illustrating microbial growth in the central compartment and the supply of nutrients frombeneath [79]. (c) Schematic model of a sphere of the
gelating agent coated with polymer in which one or more microorganisms are entrapped and then incubated in the original environment [80].
Review Trends in Biotechnology September 2012, Vol. 30, No. 9
481
employed where
13
C-labeled succinate was used to identify
succinate-assimilating microbes. Denaturing gradient gel
electrophoresis (DGGE) andclone library analysis targeting
the 16S rRNA and N
2
O reductase genes were performed.
Some strains that played an important role in the reduction
of N
2
O in rice paddy soils were found.
Similarly, the functional single cells (FSC) method
combines substrate-responsive direct viable counts
(DVC), live cell staining with 5-carboxyfluorescein diace-
tate acetoxymethyl ester, and micromanipulation. Cap-
tured single cells were incubated in media containing
substrates (nitrate and succinate). A total of 37 bacteria
were identified as denitrifiers using this method [45].
In addition, microarray was used in combination with
high spatial resolution in situ measurements of concentra-
tions of inorganic and organic ions, pH, and redox poten-
tial. Using this method, the functions of species
membership and changes in environmental conditions
could be determined, allowing us to more fully understand
the roles of different organism types and their interactions.
A 2,4-dichlorophenoxyacetic acid (2,4-D)-degrading bacter-
ium, Ralstonia eutropha, was monitored in mixed micro-
bial community using the microarray method [81].
Metagenomics is a strategy that involves using the
metagenomes directly isolated from natural samples in
the environment. This approach has proved to be techni-
cally feasible for exploring novel microbial resources [46–
48]. It is based on the complete sequencing of clones con-
taining cosmids or bacterial artificial chromosomes (BACs)
with inserts, and addresses the genetic potential of a
sample irrespective of whether the microorganisms can
be cultured or not [49]. This approach has led to a signifi-
cant increase in our knowledge of genetic diversity. There
are many genes stored in databases that have unknown
functions. Among them are members that are referred to as
‘not-yet-cultured’ or ‘uncultured’ microbes. For example, by
using large insert BAC libraries, proteorhodopsins were
discovered as a functional gene in an uncultivated lineage
of marine Gammaproteobacteria. The advantage of this
method is that it provides contiguous sections of DNA from
a single organism [6]. Metagenomic sequence information
should facilitate the design of better culturing strategies to
link genomic analysis with pure culture studies [50].
A more recent approach is shotgun sequencing. Micro-
biologists use this method to study a low diversity acid
mine drainage biofilm and, by using this method, more
than 95% of the genomes of five dominant organisms were
recovered [6,51]. In other attempts, a few dominant sam-
ples from the Sargasso Sea were used in a vast sequencing
effort to assemble genomes and a massive inventory of
nearly 1.2 million genes from many hundreds of organisms
was obtained [6,52]. Culture-dependent approaches have
some limitations; however, there is no denying the con-
tribution these methods have made in the discovery of new
species. Methods will be continuously developed to identify
new functions, new characteristics, and fastidious require-
ments for growth. Moreover, by using culture-independent
approaches, methods to resolve fundamental ecological
processes may be developed.
For example, with the development of FISH, detection
and enumeration of methanotrophs under methane
oxidation conditions were achieved. Autoradiography com-
bined with FISH was used to identify and culture some
marine planktonic archaea. In addition, free amino acids at
trace levels were identified, implying that these bacteria
have heterotrophic capability [53]. Catalyzed reporter
deposition fluorescence in situ hybridization (CARD-FISH)
for added sensitivity target-specific probes can detect cells
of previously ‘unculturable’ taxa among mixed populations
[1,54–57].
Concluding remarks
Improved culture media formulation that more closely
mimic natural conditions will be needed to study and
identify new roles and functions of some microorganisms.
Currently, pure culture still represents the most widely
used method for studying microbial physiology with regard
to the roles of genes, proteins, and metabolic pathways.
The design of devices that support cell–cell communi-
cation and the development of robots for automatic sys-
tems are also needed to improve current cultivation
methods. The study of soil microorganisms will require
an open system, including all soil layers, which is impor-
tant to the development and functioning of particular
ecosystems. Many studies have considered the microbial
diversity at the biogeocenotic level [82]. Some studies have
examined the diversity of soil bacteria and demonstrated
with DNA–DNA reassociation that the soil was 100-fold
more diverse than could be accounted for by culturing.
These findings indicate that the diversity of the uncul-
tured world exceeds previous estimates [53]. To facilitate
the development of the next generation of cultivation
methods, we must study the biochemical processes, redox
reactions, adaptation of physiology, and relationships
between all microorganisms in more detail. We have a
strong grasp on the strength and weakness of current
cultivation methods, and combining these withtraditional
approaches will allowfor many future successes in biology
and other related fields.
Without cultivation, how can we detect and identify
novel organisms? How can we gain any phenotypic and
functional information? How can we determine the func-
tions of unknown genes?
Acknowledgments
This work was supported by the Basic Science Research Programthrough
the National Research Foundation of Korea (NRF) funded by the Ministry
of Education, Science and Technology (2011-0010144), and also by
Kyonggi University Research Assistant Fellowship 2012.
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