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Critical Reviews in Biotechnology
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Co-culturing microbial consortia: approaches

for applications in biomanufacturing and
bioprocessing
Rahul Vijay Kapoore, Gloria Padmaperuma, Supattra Maneein &

Seetharaman Vaidyanathan
To cite this article: Rahul Vijay Kapoore, Gloria Padmaperuma, Supattra Maneein &
Seetharaman Vaidyanathan (2022) Co-culturing microbial consortia: approaches for
applications in biomanufacturing and bioprocessing, Critical Reviews in Biotechnology, 42:1,
46-72, DOI: 10.1080/07388551.2021.1921691
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CRITICAL REVIEWS IN BIOTECHNOLOGY
2022, VOL. 42, NO. 1, 46–72
https://doi.org/10.1080/07388551.2021.1921691
REVIEW ARTICLE
Co-culturing microbial consortia: approaches for applications in

biomanufacturing and bioprocessing
Rahul Vijay Kapoorea,b, Gloria Padmaperumaa, Supattra Maneeina,c and Seetharaman Vaidyanathana
a
Department of Chemical and Biological Engineering, The University of Sheffield, Sheffield, UK; bDepartment of Biosciences, College
of Science, Swansea University, Swansea, UK; cDepartment of Pharmaceutical, Chemical & Environmental Sciences, The University of
Greenwich, Kent, UK
ABSTRACT ARTICLE HISTORY

The application of microbial co-cultures is now recognized in the fields of biotechnology, ecol- Received 21 April 2020
ogy, and medicine. Understanding the biological interactions that govern the association of Revised 4 January 2021
microorganisms would shape the way in which artificial/synthetic co-cultures or consortia are Accepted 24 February 2021
developed. The ability to accurately predict and control cell-to-cell interactions fully would be a
KEYWORDS
significant enabler in synthetic biology. Co-culturing method development holds the key to stra- Co-culturing techniques;
tegically engineer environments in which the co-cultured microorganism can be monitored. microbial consortia; info-
Various approaches have been employed which aim to emulate the natural environment and chemicals; metabolites;
gain access to the untapped natural resources emerging from cross-talk between partners. metabolomics; encapsula-
Amongst these methods are the use of a communal liquid medium for growth, use of a solid–li- tion; membrane separation;
quid interface, membrane separation, spatial separation, and use of microfluidics systems. spatial separation;
Maximizing the information content of interactions monitored is one of the major challenges microfluidics; biofilms
that needs to be addressed by these designs. This review critically evaluates the significance and
drawbacks of the co-culturing approaches used to this day in biotechnological applications, rele-
vant to biomanufacturing. It is recommended that experimental results for a co-cultured species
should be validated with different co-culture approaches due to variations in interactions that
could exist as a result of the culturing method selected.
Introduction potential for biotechnological application due to their

versatility, robustness, and ability to undertake sophisti-
Microbial communities have evolved and shaped the
face of the Earth from the beginning of time [1–3]. cated tasks [12]. The synthetic/artificial co-culture sys-
Humans have co-evolved with microbes, assimilating tems surpass the limitations of monocultures or
them within their bodies to carry out complex tasks, consortia in nature with the added advantages in
and one can say the first examples of biotechnology exploring allelopathic interactions [13] in food indus-
used combinations (consortia) of microbes for the fer- tries involving fermentation [4] and natural product/
mentation and production of food and drinks [4,5]. drug discovery [14]. However, a full understanding of
Learning from the past, the study of co-cultures, in microbial molecular networks is still largely needed [9].
which two or more populations of cells are grown with To date, fully deciphering the communication networks
some degree of contact between them [6] in symbiosis, has been the focus of co-culture research. A deeper
has been seen today as a method to enhance current understanding of microbial interactions can benefit bio-
biotechnological processes [7]. technological and synthetic biology advancements, and
Co-culturing microorganisms have further evolved, provide a more sustainable and economical method for
finding their way into biomanufacturing, for the pro- bio-productions [5].
duction of pharmaceuticals, nutraceutical, food, and Microbial networks involve macromolecules and
drinks on a large scale [8,9], and plays a prominent role small molecules, such as metabolites, used in communi-
in the bioremediation and bioenergy sectors [10,11]. cation during intra or inter-species microbial interac-
Successful co-culture systems have shown great tions [15]. The symbiotic/antagonistic/allelopathic
CONTACT Seetharaman Vaidyanathan [email protected] Department of Chemical and Biological Engineering, The University of
Sheffield, Mappin Street, Sheffield S1 3JD, UK
ß 2021 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits
unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
CRITICAL REVIEWS IN BIOTECHNOLOGY 47
interaction between microorganisms can be a combin- best designs for a given context that presents the
ation of physical interactions [16], info-chemicals [17], potential for being taken further.
special signaling molecules (quorum sensing), adhesion In this review, we provide an overview of the current
factors (biofilms), and metabolites [18]. Info-chemicals co-culturing techniques for microbial consortia and
include both hormones (conveys information within an explore the associated advantages and challenges with a
individual) and semio-chemicals (mediates information specific focus on biotechnology applications, in particular
between individuals), collectively known to influence biomanufacturing and bioprocessing. The overview, poten-
the behavior, physiology, and structure of individuals of tial, and challenges of the co-culturing techniques for bio-
another species [19]. Alternatively, one partner can medical engineering applications have been extensively
induce the production of de novo products or induction reviewed elsewhere in recent times [28–32] and hence is
of de novo cryptic biosynthetic pathways in others not covered here. The techniques evaluated include meth-
[14,20]. A better understanding of these interacting ods such as communal liquid medium growth (microor-
cues or functions of particular microorganisms can ganisms come into direct physical contact); solid–liquid
enable the construction of high-performance consortia interface systems (involves encapsulation of microorgan-
to accomplish the desired tasks [21]. Elucidation of the isms which are co-cultured in a liquid media); membrane
interplay at the molecular level can benefit applications separation (microorganisms are separated using perme-
in the field of synthetic engineering, allowing for the able substances/membranes); spatial separations (involves
creation of engineered synthetic communities for eco- no direct physical contact, instead monocultures are inocu-
logical, industrial, and medical applications [22,23]. lated separately and are allowed to interact in space) and
Co-culturing techniques are designed with a few microfluidic systems (commonly employed in mammalian
goals in mind (biomass generation, bio-production, or research with better control over fluids and
clean-up systems), which will shape the choice of microenvironments).
microorganisms and growth parameters. A better
understanding of the trigger-response mechanisms [7]
Current techniques for co-culture
will shape the way in which to improve a bioprocess.
biotechnology
However, detecting and interpreting microbial cues has
proven to be difficult, due to the dynamic nature of the This section will provide a compendium of techniques
system and the complexity of microbial communities. currently used to study co-cultures. Broadly, these
As the synergistic interaction that exists between co- methods are classified as communal liquid medium
cultured microorganisms is species-specific, the same growth, solid–liquid interface, membrane separation,
effects will not be obtained by species from similar gen- spatial separation, and microfluidics systems. An over-
era, indicating that each partnership has to be eval- view of key co-culturing techniques used currently in
uated singularly [24]. Additionally, microbial biotechnology is given in Table 1.
communities are highly susceptible to abiotic and biotic
stresses [6], changes that will be reflected in their intra
Communal liquid medium growth
and extracellular metabolomes. Moreover, high turn-
over rates, physicochemical diversity, and low concen- Microorganisms co-cultured in a communal liquid medium
trations (due to poor co-culture designs) present (CLM) allow for a better understanding of the underlying
additional analytical challenges which often lead to effects that govern microbial interactions. With this
poor coverage, detection, and quantification of these method, the changes in biochemical components and
info-chemicals [25,26]. overall growth of the interacting species can be investi-
Various co-culturing methods have been developed gated thoroughly. For example, it can be used to identify
to address these challenges. Small co-culturing vessels over-yielding (higher biomass compared to its component
and targeted metabolite profiling are deemed to be monoculture) or under-yielding (lower biomass compared
ideal for trapping metabolic dependencies at a high to its component monoculture) effects between the co-
resolution [27]. Finding a balance between various stra- cultured partners at the different time frames and phases
tegic propositions would allow for better resolution and [57]. CLM systems, to an extent, emulate conditions in the
coverage of the untapped/novel natural product real world, if microorganisms from the same niche are iso-
resources. By evaluating each co-culturing method, it is lated and grown together, or in the case of artificial co-cul-
possible to address the shortcomings that need to be tures, it provides a way to attest a relationship if these
overcome in future designs. The availability of this organisms were to find themselves in a shared environ-
information in a concise review helps to visualize the ment. For this to succeed, various parameters such as
48 R. V. KAPOORE ET AL.
Table 1. A survey of key co-culturing techniques used in biotechnology.

Co-culturing Info-chemicals Field of
Co-cultured microorganisms technique of interest study Ref.
Colletotrichum lagenarium, Agar System Antifungal proteins Food Technology [33]
Bacillus amyloliquefaciens
Botrytis cinerea, Pseudomonas sp. Agar System x Agriculture [34]
E. coli, Salmonella typhimurium Agar System x Food Technology [35]
Fusarium sp., Aspergillus strain Agar System de novo production of 18 Biotechnology [14]
metabolites (natural products)
Sarocladium strictum, Fusarium oxysporum Agar System Fusaric acid Medical [20]
Streptomycetes from rhizosphere of Agar System 24 anti-fungal compounds Ecology [36]
Araucariaceae, Neofusicoccum parvum
Shewanella putrfaciens, Brochothrix Agar Systems Formic acid and 2 Food Technology [37]
thermosphacta, Pseudomonas sp. unidentified organic acids
Candida albicans, Clostridium perfringens, Biofilms Upregulation of WOR1 Medical [38]
K. pneumoniae, E. coli, E. faecalis
Aspergillus nidulans, actinomycetes Dialysis tube Lecanoric acid, orsellinic Ecology [16]
membrane acid, polyketides
Chlorella vulgaris, Microcystis aeruginosa Dialysis tube Linoleic acid and Ecology [39]
membrane nitrous oxide
Denitrifying anaerobic methane oxidation Direct mixing Nitrate and nitrite Ecology [40]
(DAMO) and anaerobic ammonium
oxidation (Anammox)
Chlorella vulgaris, Direct mixing Chlorellin Ecology [13]
Pseudokirchneriella subcapitata
E. coli, Bacillus megaterium Direct mixing Peptide-based signaling: Biotechnology [41]
auto-inducing peptides
Fusarium tricinctum, Bacillus subtilis Direct mixing Inducing secondary Biotechnology [18]
metabolites production
(78 fold increase)
Ignicoccus hospitalis, Direct mixing Increase in CO2 fixation and Ecology [42]
Nanoarchaeum equitans nitrogen assimilation
enzymes (Glutamine and
asparagine synthase)
C. sorokiniana, Encapsulation x Bioremediation [43]
A. brasilense
Delftia acidovorans, Arthrobacter sp. Encapsulation x Bioremediation [44]
Klebsiella oxytoca, Bacillus subtilis, Encapsulation x Ecology [45]
Rizoctonia solani
Rhodosporidium toruloides, Encapsulation x Biofuels [46]
Saccharomycopsis fibuligera
Zymomonas mobilis, Encapsulation Suggest metabolites present Biofuels [47]
Pichia stipitis (not investigated) for
efficient ethanol
production
Chlorella protothecoides (Heterotrophic and Gas separation CO2 and O2 exchange Biofuels [48]
autotrophic)
Oocystis marsonii, Microcystis aeruginosa Membrane separation Allelopathic metabolites Biotechnology [49]
not identified
Microcystis aeruginosa (mycrocystins Membrane separation Bioactives, toxins Biotechnology [50]
producing and non-producing) (microcystins)
and peptides
Rhodotorula glutinis, Chlorella vulgaris Membrane Separation Propionic acid, pyruvic acid, Biofuels [51]
acetic acids
Lactobacillus brevis subsp. lindneri or L. Membrane Separation Amino acids such as valine Food technology [52]
plantarum with S. cerevisiae or S. exiguus and isoleucine
Lactobacillus, S. cerevisiae or Z. florentina Membrane Separation Amino acids Food technology [53]
P. aeruginosa, A. fumigatus Microfluidics x Biotechnology [54]
R. solanacearum, A. flavus Chlamydospores (A. flavus ) (natural products)
Sphingobium chlorophenolicum, Microfluidics x Bioremediation [55]
Ralstonia metallidurans
Chlorella protothecoides, Tetraselmis suecica. Pelletization and Bio-flocculating compounds Bioremediation [56]
flocculation
priority effects, inoculation ratio and the timing at which where enhancing the growth of the main partner
one monoculture is seeded into the other do play would give higher bioproduct yields [8]. Moreover, syn-
an important role in establishing a balance with the co-cul- ergistic or antagonistic partnerships could be exploited
ture [7]. for various biotechnological applications, without the
This type of co-culturing is useful to enhance bio- need to use gene modifications. Systems such as direct
mass yield [58], in a process such as fermentation [4], mixing, pelletization, flocculation, and biofilms, fall into
biofuels, nutraceutical, and chemicals production, this category (Figure 1).
(a) Direct Mixing (b) Pelletization and Flocculation (c) Biofilm
Stage 1
Communal medium
Communal medium Communal medium
Stage 2 EPS network

Microorganism A
Microorganism B
Intra-species interactions
Inter-species interactions
Figure 1. Communal liquid medium growth co-culture system. (a) Direct Mixing: Microorganism A and B come into direct contact
allowing them to exchange info-chemicals at a close proximity. (b) Pelletization and Flocculation: Microorganism B releases bio-
flocculants, which induce Microorganism A to form aggregates. This process is not 100% efficient, as shown by the non-floccu-
lated cells. (c) Biofilm: both microorganisms secrete EPS compounds creating an intertwined network (filaments, orange-
Microorganism A, blue-Microorganism B).
Direct mixing the plant pathogen [33]. A symbiotic interaction or

Direct mixing (Figure 1(a)) refers to co-cultures grown cross-talk between Chlorella sp. (algae) and
in the same environment, where microorganisms come Saccharomyces cerevisiae (yeast) in a bioreactor, showed
into physical contact with each other. These microor- enhanced CO2 bio-fixation with a simultaneous increase
ganisms interact in close proximity, exchanging signal- in biomass and lipid productivity with co-culture com-
ing molecules and metabolites. Co-culturing pared to microalgal monoculture [67]. Similarly,
experiments involving the direct mixing of microorgan- Rhodotorula glutinis (yeast) and Scenedesmus obliquus
isms have been shown to have enhanced functions and (algae) grown in communal media showed synergistic
accomplished tasks difficult to be achieved with mono- interactions where higher biomass and lipid productiv-
cultures [15]. These include processes such as bio- ity was observed compared to each monoculture.
remediation [59,60], hydrogen production [61], These results indicated that a combination of gas
acetone-butanol-ethanol production via fermentation exchange (O2 and CO2) and a source of trace elements
[62], the production of nondairy probiotic [4], and bio- from naturally lysed cells played a vital part in the syn-
active compounds with antifungal properties superior ergism [65]. A combination of both synergistic and
to those obtained with monocultures [63]. antagonistic interactions between Prorocentrum min-
Direct mixing co-culturing methods have been used imum (algae) and Dinoroseobacter shibae (bacteria) was
to study the interactions between fungi and bacteria illustrated with this method [66], backing up the pro-
[33,64], yeast and algae [65], algae and bacteria [66], posed “Jekyll and Hyde” lifestyle [68]. Briefly, the
and between algae species [13]. Compared to its mono- authors investigated the population dynamics of co-cul-
culture, the marine fungus, Emericella sp. secreted ture and demonstrated that co-culture reproducibly
emericellamides A and B (a secondary metabolite of went from mutualistic phase (where both bacteria and
marine cyclic depsipeptide with antimicrobial proper- algae profit) to pathogenic phase (where bacteria-
ties) in much higher concentrations when co-cultured induced algal death). With respect to the inter-species
with the bacterium Salinispora arenicola [64]. Similarly, interactions, the co-culture of two microalgae Chlorella
Bacillus amyloliquefaciens, when co-cultured with vulgaris and Pseudokirchneriella subcapitata resulted in
Colletotrichum lagenarium (plant pathogenic fungus), higher levels of extracellular chlorellin (a mixture of
secreted an antifungal protein, as a result of being fatty acids and hydrocarbons), responsible for inhibitory
exposed to the fungus. This secreted protein by bac- effects on both species. This investigation showed the
teria exhibits b-1,3-glucanase activity on fungi (decom- application of direct mixing as a tool to analyze the
position of fungal hyphal walls), thereby acting as an evolution of allelopathic chemicals [13]. Furthermore,
effective biocontrol candidate and antagonist against the population density of the starting inoculum
(inoculation ratio) needs to be assessed prior to setting hydrophobicity, pH, salinity, temperature, divalent cati-
up the co-culture. This has been true for studies con- ons concentration (calcium and magnesium ions),
ducted using Spirulina platensis and Rhodotorula glutinis population density, the initial ratio of co-cultured part-
[69] and Scenedesmus obliquus and Candida tropicalis ners, timing for triggering flocculant formation, and the
[70], where the growth rate of the yeast/bacteria concentration of the flocculant releasing microorgan-
exceeded that of the alga. By adjusting the population isms. The use of synthetic flocculants on a commercial
density to alga:bacteria (3:1) and alga:yeast (2:1) it was scale is being widely criticized due to their toxicity to
possible to construct a balanced co-culture with humans and the environment. In contrast, bio-floccu-
enhanced alga biomass output. Later, a study with co- lants produced by a variety of microorganisms are con-
cultures of Chlorella pyrenoidosa and Rhodotorula gluti- sidered as good alternatives. However, their large-scale
nis, confirmed the importance of inoculation ratios/ production is limited by factors such as lower concen-
population density, where a ratio of alga:yeast (3:1) is tration, lower flocculating efficiency, and associated
identified as optimal for achieving the highest biomass high production costs. The overall yield and flocculation
concentration and the lipid productivity [71] and to efficiency of bio-flocculants can be substantially
improve nutrient removal from wastewater and protein improved by co-culturing optimal strains. This method
productivity [72]. has been successfully used to decrease the capital costs
Direct mixing co-culture can be used to identify and associated with microbial harvesting and dewatering
understand the effects of secreted metabolites by [56,75,76], for screening of optimal strains for co-cultur-
microorganisms on each other. However, as shown by ing and in bioremediation [73].
Oh and coworkers [64], when analyzing the supernatant Harvesting microalgae biomass that contains prod-
of Emericella sp. for emericellamides A and B, the con- ucts of value has been achieved with the aid of natural
centration of these depsipeptides in the media can be
pelletization and flocculation, by co-culturing microal-
very low for their isolation, structural elucidation, and
gae with fungi or bacteria. In the case of fungi-assisted
detection by LC-MS. This finding suggested that direct
algae harvesting, the co-culturing of Chlorella protothe-
mixing is not an ideal way to trap extracellular metabo-
coides and Tetraselmis suecica with fungal strains
lites. Similarly, the various extraction and concentration
resulted in higher biomass, lipid productivity, and bio-
steps of the compound could result in loss or degrada-
remediation efficacy compared to monocultures [56].
tions of compounds. This method is, therefore, limited
Similar trends were observed with co-cultures of
to the analysis and production of larger molecules such
Chlorella vulgaris and two species of Aspergillus sp. [73].
as exopolymeric substances (EPS) and/or info-chemicals
The influence of rotation speed, culture time of
with higher extracellular concentration. In addition, dir-
Pleurotus ostreatus (an edible fungi) pellets and pH on
ectly mixed cultures in the same communal media are
harvesting efficiency of Chlorella sp. was recently inves-
not suitable for microorganisms that have slightly dif-
tigated, where authors reported 100 rpm rotation speed
ferent demands in culturing conditions or in circum-
with lower pH values resulted in a maximum harvesting
stances where microorganisms cannot exist in direct
contact [43], necessitating other approaches, as dis- efficiency of 65% in 150 min [77]. In the case of bac-
cussed below. teria-assisted algae harvesting, Bacillus sp. (bacterium)
at pH above 9 showed a flocculation efficiency of up to
Pelletization and flocculation 95% with Nannochloropsis oceanica (algae) in a liquid
Alternative methods of co-culturing such as pelletiza- medium [74]. Similarly, co-culturing of C. vulgaris with
tion and flocculation (Figure 1(b)) involve a naturally bacteria (with direct physical contact) caused the micro-
close association of microorganisms. During co-culture, algae to flocculate, a phenomenon not seen in either
flocculating compounds (bio-flocculants) released by axenic C. vulgaris culture or even when grown in the
one partner cause the other microorganism to agglom- bacterial culture supernatant [78], suggesting that the
erate and form pellets. The mechanism for aggregation presence of the bacterium is essential for microalgal
has been attributed to cell surface charge and/or fila- flocculation. However, the effects of the bacteria on the
ments of the bacteria/fungus [70,73–75]. This method growth and biochemical composition of the microalgae
has several added advantages such as improved set- were not explored in this study. In the case of bacterial
tling ability and optimized symbiosis within the micro- co-cultures, the consortium of Halomonas sp. and
bial community through mutually beneficial Micrococcus sp. [79] and Staphylococcus sp. and
associations. Key parameters that govern the bio-aggre- Pseudomonas sp. [80] triggered the production of the
gation/bio-flocculation are surface charge, novel bioflocculant, CBF-F26 and MMF1 respectively.
The screening involving the individual co-cultures of interface by a single species or a combination of spe-
Aspergillus fumigatus (fungi) with eleven different cies [82]. An extracellular matrix in biofilms, where the
strains of microalgae showed variations in bio-floccula- microbiome resides and communicates, is composed of
tion efficiencies. Furthermore, the biochemical analysis hydrated EPS. EPS are mainly comprised of proteins,
showed that synergistic interactions with A. fumigatus polysaccharides, amino acids, nucleic acids, lipids, and
were evident only with few microalgal strains out of other biopolymers (humic substances). These EPS,
eleven. This was indicated by the increase in lipid pro- immobilize biofilm cells by providing mechanical stabil-
duction that was similar or higher than the sum of the ity and keeping them in close proximity, thereby form-
monoculture of the microalgae and fungus [81]. ing an inter-connected cohesive three-dimensional
However, these observations were only limited to cells polymer network where cross-talk between cells results
grown using glucose as the carbon source, and not in in the formation of synergistic micro-consortia [83]. The
cells grown using pretreated wheat straws as the alter- secretion and uptake of substances within a biofilm
nate carbon source. Hence, the benefits of this co-cul- may be analyzed by gene activation or inactivation to
ture were shown to depend on both the deduce how they influence each other, however, their
microorganisms being co-cultured and the carbon molecular level interactions are yet to be sufficiently
source provided. This was also evident in results found defined [38,83]. Appropriate co-culturing methods are
during the co-culture of Aspergillus niger (fungi) and C. required for a better understanding of regulatory fac-
vulgaris (microalgae) [75], where the heterotrophic co- tors for EPS production and assessing molecular level
culture conditions lowered the flocculation efficiency interactions between different partners in multispecies
when compared to autotrophic conditions. This demon- biofilms. Biofilms have found application in biomedical,
strated that co-culture conditions are important to reap bioremediation, and bioenergy-related fields [84].
the full benefits of the synergistic interaction. Similarly, As has been emphasized by other investigators in
the co-culturing of C. vulgaris and A. niger [76] high- the medical context [85], knowledge of interspecies
lighted the importance of population density, inoculum interaction within the biofilm is vital for an understand-
size, and timing during pelletization. In this case, the ing of biofilm physiology and the treatment of biofilm-
concentration of the flocculant and its binding strength related co-cultivation strategies in biomanufacturing.
was proven not to be effective at very high microalgae An illustration of biofilm-associated induction has been
biomass concentrations, resulting in variations in pellet shown, where microorganisms within the biofilm can
morphology, however, a co-culturing ratio of 1:300 cause activation of genes for biofilm production in
(fungi:microalgae) yielded >90% cell harvest efficiency. another strain, therefore enabling them to survive in
The trigger-response mechanism can be manipu- environmentally challenging conditions [38]. Briefly, the
lated by variations in the growth environment and by interactions between the bacteria and Candida albicans
selecting the optimal organisms with varying degrees within the gut microbiome have been shown to sup-
of bio-flocculant producing capacity [79,81]. The use of port each other’s growth and survival via modulation of
pelletization and flocculation, however, is limited only the local chemistry of their environments in multiple
to microbial co-cultures where the mechanism of bio- ways. Bacteria-induced biofilm formation in yeast has
aggregation/bio-flocculation can be triggered and also been investigated, where co-culture of S. cerevisiae
maintained. The nature of the bio-flocculant and its and LAB (lactic acid bacteria) or monoculture of S. cere-
binding capacity would also be a limiting factor, as the visiae exposed to bacterial supernatant resulted in bio-
duration of this would need to factored in when har- film formation [82]. Recently, mycoalgae biofilms
vesting the biomass. However, using bio-flocculants (lichen type) on a supporting polymer matrix have
would decrease the costs of centrifugation and the been investigated for various bioremediation and bio-
environmental impact of synthetic chemicals. Overall, processing applications such as biomass harvesting
the strategy of using palletisation/flocculation for co- [84,86], which stemmed from previous knowledge of
culturing has been shown to be effective not only for fungi and algae interactions [87]. Plastic composite sup-
microbial harvesting and downstream processing but port biofilm reactor was used for simultaneous sacchari-
also to improve biomass productivity and product yield fication and fermentation of ethanol in a potato waste-
in such processes compared to monocultures. based medium by co-cultures of A. niger and S. cerevi-
siae, where the influence of temperature, pH, and aer-
Biofilms ation rates on ethanol production was investigated.
Biofilms (superficial microbial colonies) (Figure 1(c)) can Maximum ethanol production was reported at pH 5.8,
be naturally formed on solid surfaces at the solid–liquid 35 C with no aeration [88]. The advantage of using this
co-culture method in this instance is due to the induc- Solid–liquid interface

tion of biofilm formation on a support matrix, with the
The solid–liquid interface systems involve trapping a
attachment efficiency dependent on the species of co-
monoculture or a co-culture within a porous vessel,
cultivation and the material of the matrix. In summary,
usually in soft beads or cell droplets. The bead/droplet
the potential usefulness of this co-culture method is
is then suspended in a liquid or a gaseous medium. The
evident but requires a further understanding of how
medium composition of the bead or capsule can differ
these microorganisms interact, which will facilitate
from the suspension fluids. Extra-cellular metabolites
future couplings of synergistic microorganisms for their
intended applications as biofilms. interaction is facilitated through the porous membrane.
However, it is also evident that similar to co-cultur- Amongst these methods are encapsulation and cell
ing by pelletization and flocculation, biofilm formation droplet formation techniques (Figure 2), useful for co-
is limited to microorganisms that can form biofilms culturing microorganisms that require protection
and/or those that can induce biofilm production. For against environmental stresses, have dissimilar growth
example, monocultures of yeast or LAB were unable to characteristics, nutritional requirements, and hinder
form biofilms [82]. This could be due to the inability to substrate competition [43], for which direct mixing or
form the required components for biofilms such as EPS membrane separation methods are not suitable.
or the requirement for other regulatory signals. Solid–liquid interface systems have been used to pro-
Likewise, the trigger-response stimulus that will be duce nondairy probiotic drinks, such as during the
established between the biofilm-forming microorgan- fermentation of peanut-soy milk using P. acidilactici
isms will vary the outcome of the assemblage, there- and S. cerevisiae [4], and in increasing lipid content
fore, each biofilm is unique to itself making in microalga Chlorella sp. by entrapping it with
reproducibility a challenge. Additionally, since metabo- Trichosporonoides spathulata in glass beads [89]. These
lites and signaling molecules are not secreted only methods are useful for co-culturing microorganisms
through the biofilm, other methods of co-culture are that require an uninterrupted supply of nutrients with
required to investigate other means of communication. relatively low competition, especially when co-culturing
(a) Encapsulation (b) Encapsulation (co-immobilization)
Beads
Beads
Microorganism A medium Microorganism B medium Medium (Liquid/Gas) Communal

medium
(c) Cell Droplets
Microorganism A
Trapped in
Microorganism B
Droplets
Microorganism C
Grow and select the best Co-culture interactions
Microorganisms pool co-culture/consortia Porous bead/droplet
Figure 2. Solid–liquid interface co-culture system. (a) Encapsulation: Microorganism A is grown in liquid culture, whilst
Microorganisms B is trapped within beads. The info-chemicals diffusing from the beads aid Microorganism A (for example in
growth). (b) Encapsulation (co-immobilization): Microorganism A and B are both trapped within the beads. The info-chemicals dif-
fusing into the growth chamber can affect the outer media (e.g. fermentation or compound digestion). (c) Cell droplets: droplets
are used to isolate sub-cultures of species from within a microorganism pool. The best performing/surviving microorganism co-
culture/consortia is chosen for further application.
microorganisms with very dissimilar nutritional require- reinhardtii (microalgae) to improve the growth and lipid
ments, as there still may be competition for gaseous production of the microalgae. In the case of co-immo-
compounds diluted within the media/flowing across bilization, co-encapsulation of algae and bacteria has
the capsule membrane. great potential in bioremediation applications, such as
reduction of ammonium and phosphorous from the
Encapsulation wastewater, however, a realization of this potential is
Encapsulation is a method of co-culture that can over- limited by growth suppression by native wastewater
come the challenges posed by variations in the growth bacterial community. This limitation can be overcome
environment. This method involves the immobilization by immobilization of algae and bacteria in alginate
of microorganisms in substances such as alginate, agar, beads [43], where beads inhibit both liberation of
and j-carrageenan structures [43,45,89,90]. Often, one immobilized microorganisms into wastewater and
of the two microorganisms is trapped in beads and co- penetration of outside microbiome into the beads.
cultured with the other microorganism in the liquid Similarly, co-encapsulation of yeast and microalgae has
medium (Figure 2(a)). This method does not allow them been shown to result in similar lipid productivity com-
to come into physical contact with one another pared to their directly mixed co-culture, however, the
[43,89,91]. Alternatively, co-immobilization (Figure 2(b)), added advantage of this method is reduced cost and
where both microorganisms are encapsulated within simplification of downstream harvesting process [89].
the same bead is used to facilitate biomass harvesting This method has several drawbacks, nevertheless,
and promote closer interactions [89,92]. It enables a one of which includes the reduced growth shown by a
more effective transfer of info-chemicals and metabo- decrease in biomass production during co-culture com-
lites between interacting species with minimal loss in pared to the direct mixing method [89]. The fragility of
the bulk medium due to diffusion. This isolation from the beads is also an issue that leads to leakages of the
the environment also makes them less affected by the trapped microorganism (in a period of few days) into
culturing conditions outside the bead. This has been the culture environment [47,89]. The economic feasibil-
demonstrated to be beneficial for co-cultures that have ity of this method is another challenge, as for industrial
the potential to replace sequential processes such as applications, mass production of uniform alginate
fermentation [47], direct oil conversion from starch [46] beads is required which is costly.
and bioremediation [43,44].
The immobilization of Zymomonas mobilis (bacter- Cell droplets
ium) in beads and its co-culture with free-flowing cells Monocultures and co-cultures can be isolated in drop-
of Pichia stipitis (yeast) yielded 96% more bioethanol lets, micro- or macro-droplets, where the info-chemicals
than the theoretical value [47]. The immobilization are exchanged between the isolated droplets via diffu-
relieved oxygen competition between the two microor- sion [95,96]. Droplets can be made using a microfluidic
ganisms whilst mitigating the inhibition of the bacteria device that could encapsulate and co-cultivate subsets
caused by the yeast when directly mixed. Observations of a community by dispersing aqueous droplets in a con-
of their interactions confirmed some level of inhibition, tinuous oil phase [97] or by encapsulating microorgan-
however, evidence shows that Z. mobilis was also utiliz- isms within microdroplets composed of agar and single
ing an additional source of nutrient/or carbon, other cells, forming microcolonies that could still exchange
than glucose when co-cultured with P. stipitis. Another substances between each other [95]. Alternatively, an
example is the immobilization of Aureobasidium pullu- aqueous two-phase system environment can be used
lans (yeast link fungus) to polyurethane foam with where microcolonies can be relocated by using magnetic
encapsulated S. cerevisiae in calcium-alginate beads, in remote control [96]. The cell droplets technique (Figure
co-culture, where an improved purity and yield of 2(c)) has been highlighted for its ability to enable the
fructo-oligosaccharides was demonstrated, compared culturing of microorganisms that often cannot be easily
to monocultures [93]. Similarly, yeasts Rhodosporidium cultured under laboratory conditions.
toruloides and a mutant version of Saccharomycopsis Microdroplets were used as a method to isolate sym-
fibuligera were co-immobilized in polyvinyl alcohol biotic interactions from within a microbial community
(PVA) and alginate beads that allowed for the conver- [97]. Later separation of the microorganism’s assem-
sion of cassava starch to cell lipids in a single process blage into smaller portions will facilitate a better under-
[46]. Additionally, Magdouli and coworkers [94] high- standing of the subset communications that govern
lighted the possibility of recycling Synechococcus sp. complex systems. Microdroplets were achieved by dis-
(cyanobacterium) beads during co-culturing with C. persing aqueous droplets in a continuous oil phase
within a microfluidic device. This method allowed for the population density of each microorganism and their
the isolation of symbiotic microorganisms only as these allelopathic interactions. Several types of signaling mol-
would keep generating with time. This work presents ecules have been identified so far using different types
itself as a method used to isolate natural symbionts of membrane separation co-culture systems (Table 1).
from complex ecological systems to be studied for bio- This technique is primarily employed to investigate dif-
technological applications. Encapsulating cells in gel fusible molecular mechanisms used for interactions
microdroplets (made up of agarose) was recently within co-culture and their ultimate effects. These
described as an alternative to surrounding cells with oil include the use of a dialysis tube membrane [39,100];
[98]. This method described high-throughput screening vessel chambers [49,50,101,102] and a TranswellV sys-
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of cell to cell interactions (HiSCI) in isolating the algae tem [53]. Amongst the biomolecules identified are
growth supporting bacteria. The porous nature of the amino acids [52], fatty acids [39], and sugar derivatives
gel matrix allowed the free flow of nutrients, metabo- [51] (Table 1). Gas chromatography-mass spectrometry
lites, and gases to and from the encapsulated cells. Byun (GC-MS) and liquid chromatography-mass spectrometry
and coworkers [96] designed an aqueous two-phase sys- (LC-MS) are common analytical techniques employed to
tem that trapped bacterial colonies within magnetic dex- identify metabolites secreted within the growth
tran phases (DEX). This DEX phase was then suspended medium [16,39].
as cell droplets and patterned within a polyethylene gly-
col (PEG) phase. With such magnetized droplets, it was TranswellV system
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possible to observe how the microorganisms interacted The isolation and identification of extracellular mole-
over varying distances by relocating the cell droplets at cules can also be achieved using a TranswellV system
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different time intervals compared to a stationary loca- (Figure 3(a)). This system comprises a six-well plate with
tion. Their results indicate that relocation can enhance two separated parts, and a lower compartment (reser-
communication between the droplet colonies. This voir), and an upper compartment (insert) each of which
method proved to be advantageous for microorganisms can hold different co-culture partners separated by a
subjected to changing environmental conditions. As permeable membrane (polycarbonate) which only
opposed to other co-culture methods, where microor- allows the diffusion of metabolites [53,103]. A small-
ganisms remain in one environment, this method scale TranswellV system has been developed within the
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enabled tracking changes that can occur when the assay plates, required for low cell and media volumes,
microorganisms were exposed to slightly different sur- and enabling replication and multiple studies to be
roundings. A limitation of this technique is that not all
conducted simultaneously [53]. As used in biomedical
bacterial species partitioned well in the cell droplets. In
research, this method can be used to understand secre-
addition, the phases pose limitations for different types
tion factor profiles and their levels as a physiological
of microorganisms that can be negatively affected by
response during cross-talk between different cell types,
the substances constituting the phases. This method-
in particular mammalian cell lines [104,105].
ology was further developed by Han and coworkers [99],
In the past, this method of co-culture has been used
where authors used a density adjusted PEG/DEX aque-
to understand the trophic relations between LAB and
ous two-phase system which can generate various size-
yeast co-culture (Table 1) that occur during sourdough
controlled spheroids in a conventional multi-well plate.
leavening, which are otherwise difficult to understand
This method offers the added advantage of simple cul-
due to the complex proteolytic activity taking place in
ture mode switching from spheroid to a surface-attached
sourdough [52]. Briefly, higher growth rates and final
adhesion culture with the addition of few drops of the
yields were demonstrated for both LABs compared to
polymer-free medium, thereby avoiding conventional
their monocultures, where yeasts were unaffected and
laborious spheroid manipulation steps and errors associ-
were found to compete partially with LAB for nitrogen
ated with it. Nevertheless, the approach is more suited
sources and are also responsible for the synthesis and
to studying interactions in co-cultures more than
secretion of amino acids (valine and isoleucine). These
employing it as a strategy for large-scale manufacturing,
given the limitations of the volumes employed. secreted amino acids are responsible for enhancing the
growth of LAB. In contrast, the lower diffusion rates and
accessibility in the Transwell system were highlighted
Membrane separation
in reducing the overall toxicologic impact and improv-
A membrane can form a separation barrier between ing growth profiles as demonstrated with co-cultures of
microorganisms during co-culture. It has the added A. niger and Nostoc sp. (cyanobacteria), where Nostoc
benefit of easing the task associated with monitoring sp. grown on wastewater was known to produce
(a) Transwell System (b) Vessel Chambers (c) Dialysis tube
Communal
Gasket/O-Ring Communal
medium Dialysis Tube Communal medium
medium
Microorganism A
Microorganism B
Membrane
Figure 3. Membrane separation co-culture system. The co-culture microorganisms grown in communal media can only mediate
through info-chemicals. Direct contact is not possible. (a) TranswellV systems: a horizontal oriented permeable membrane is
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placed between the two microorganisms allowing for an exchange of info-chemicals. This method only allows the cultivation of
low concentration of cultures. (b) Vessel chambers: a vessel is sectioned into half by placing a vertically oriented permeable
membrane which allows the diffusion of info-chemicals. (c) Dialysis tube: Microorganism A is grown in liquid culture, whilst
Microorganisms B is trapped with a dialysis tube. The info-chemicals diffuse through the permeable membrane of the dialy-
sis tubing.
signaling molecules toxic to A. niger [106]. More were cultivated in the reservoir and lactobacilli in
recently, the role of amino acid metabolism in synergis- the insert.
tic interactions between LAB and yeasts (Table 1) iso-
lated from water kefir has been described, where Vessel chambers
higher biomass yields were obtained compared to their Vessel chambers (Figure 3(b)) consist of two vessels
monocultures [53]. connected through an O-ring junction (made up of sili-
This method of co-culture is very easy to set-up and cone for leak-proof sealing) equipped with a permeable
is convenient for studies requiring small culture vol- membrane filter (a 0.22 mm hydrophilic polyvinylidene
umes (up to 5 ml). Moreover, the porosity of the poly- fluoride (PVDF) or 0.45 mm cellulose nitrate). Each micro-
carbonate membrane can be selected depending on organism is cultured in its own half of the vessel. The
the ultimate aim of the study. For example, 8 mm poros- membrane allows for the diffusion of the metabolites
ity has been selected as the main aim of the model was from one chamber to another. This method has been
to assess the invasion of metastatic cancer cells through used to assess ecological systems such as the predator-
the structural blood-brain barrier [104], whereas 0.25 to prey interaction [101] and interactions within phyto-
0.4 mm porosity has been used to study the cross-talk plankton communities [102].
between bacteria and yeasts [52,53]. The set-up can be In case of predator-prey interactions, co-culturing of
ideal in screening co-culture partners. However, this Pseudomonas fluorescens (bacterium) with Dictyostelium
setup is not suitable for larger culture volumes thereby discoideum (ameba) resulted in high levels of the bac-
limiting its application to planktonic research (due to terial alkaloids, pyreudiones A–D being produced by P.
low cell abundance) and studies involving time-course fluorescens to protect itself from the ameba. The perme-
sampling for metabolomic and proteomic investiga- able membrane allowed predator-prey signaling mole-
tions (due to low biomass availability). Additionally, this cules to diffuse between the chambers, activating the
method requires pre-optimization of overall setup with self-defense mechanism of the bacterium, thereby
respect to compartment suitability for each co-culture decreasing growth rates or causing cell lysis of ameba
partner as demonstrated by Stadie and coworkers [53],
[101]. In planktonic research, vessel chambers were
where best effects were only obtained when yeasts
used to study the effect of Dinoroseobacter shibae (bac-
terium) on the metabolic profile of the diatom,
Thalassiosira pseudonana [102]. The study showed that natural habitats within the laboratory setting. Using
the intracellular amino acid levels of T. pseudonana comparative methods of co-culturing, in this case, dir-
were upregulated when in co-culture with no improve- ect mixing and membrane, demonstrated that other
ments in microalgal growth rates. The authors high- factors come into play in microbial communication,
lighted the application of this co-culturing technique opening the door to more avenues to explore!
for the investigation of various plankton interactions
and understanding the metabolic fluxes within plank- Dialysis tube system
ton communities. In the case of bacterial interactions, Dialysis tube systems (Figure 3(c)) involve the use of
co-cultures of Streptomyces sp. and Pseudomonas sp. in semi-permeable dialysis membrane/bags to separate
a glass vessel separated by a 0.22 mm PVDF led to upre- microorganisms in co-culture. One microorganism (a
gulation of several metabolites. Such a co-fermentation guest strain) is inoculated within the dialysis bag, usu-
approach induced the expression of cryptic indole alkal- ally held together with a mechanical spring, to prevent
oid BGC in Streptomyces sp. and later characterization it from collapsing. The bag is then re-suspended in a
of indolocarbazole alkaloid, a phenomenon not large vessel containing the other microorganism (the
observed with their monocultures [107]. With respect to host strain) in free liquid media [100]. Both microorgan-
intraspecific interactions, Briand and coworkers [50] isms are in liquid media, however, the composition of
studied the effects of three types of Microcystis aerugi- the media can differ. The porous membrane of the dia-
nosa on each other. The aim of the study was to eluci- lysis tube is biocompatible and made up of polycarbon-
date the factors that regulate the production of ate/cellulose (molecular weight ranging 8–14 kDa,
secondary metabolites and toxins (during co-culture) enough to separate fungi and bacteria), allowing for
essential for cyanobacterial blooms. With this co-culture info-chemical interactions but preventing direct cell-to-
technique, the authors demonstrated quantitative cell contact.
changes in the production of major extracellular pepti- A novel methanotrophic process was described with
des (regulatory factor) as a physiological response to this method for the consortia of Methylocystis sp. M6
co-culturing when compared to that of monocultures. and Hyphomicrobium sp. NM3. Such a membrane sys-
In contrast to the TranswellV setup, vessel chambers
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tem allowed the cross-feeding of methane-derived car-
allow larger culture volumes (up to 500 ml), thereby bon species from Methylocystis sp., thereby improving
permitting sampling for omics investigations even in the methanotrophic performance and the biomass yield
cases with limited biomass availability. Also, this of Hyphomicrobium sp. [108]. This method of co-culture
method supported equal growth conditions for both along with biochemical analysis and -omic approaches
partners in contrast to the dialysis tube system (dis- (proteomics and metabolomics) has been successfully
cussed in Section “Dialysis tube system”), where un- implemented to elucidate novel interspecies allelo-
equal growth conditions were used. Vessel chambers pathic interactions. An underlying interspecies molecu-
are a good method for assessing predator-prey interac- lar mechanism was briefly described, where Microcystis
tions and in assessing allelopathic activities. However, aeruginosa mediated negative allelopathic effects
the success of this method in illustrating the allelo- (inhibits growth) on Chlorella vulgaris, via the release of
pathic interactions strongly depends on the nature of linoleic acid [39]. Moreover, the role of nitric oxide (cell
the molecules exchanged (as these need to be able to signaling compound produced by C. vulgaris) in stimu-
diffuse readily through the membrane) and cannot be lating the positive feedback mechanism of linoleic acid
applied to the microorganisms which require physical released by M. aeruginosa and its toxicity was demon-
contact to elicit the response. An example of hindered strated. Similarly, Shi and coworkers [100] employed
interaction, when using vessel chambers was witnessed this method with LC-MS based metabolomics platform
when associating green algae Oocystis marsonii with to illustrate and define chemically mediated interac-
Microcystis aeruginosa. These microorganisms were tions (mainly secondary metabolites) between not only
investigated with respect to algae blooms and fungal-bacterial (Cladosporium sp. and B. subtilis) com-
eutrophication of waters. The use of membrane-diffu- munity but also between two microbial strains of the
sion, however, hindered the allelopathic activity of the same background (Streptomyces sp. WU20 and
cyanobacteria on the green algae, when compared to Streptomyces sp. WU63). LC-MS analysis of the fungal-
the direct mixing method, where direct cell-to-cell con- bacterial community revealed production of diphenyl
tact was necessary for the toxic effects of the cyanobac- ether with polyhydroxy side chains including six novel
teria to play a part [49]. This highlights the importance antibiotics as a result of defense mechanism (of
of the use of the right co-culturing system to study Cladosporium) against the growth inhibition resulting
from surfactins (antifungal cyclopeptides) secreted by competition for nutrients as the cells are inoculated in
B. subtilis. separate vessels, as in gaseous separation, or attached
Another type of encapsulation involves the entrap- on solid matrices as seen in matrix immobilization and
ment of microbes in a hydrogel within a dialysis tube agar systems (Figure 4).
[109]. An example of such a co-culture technique
involves the co-culturing of Synechococcus elongatus Gaseous separation
and Azotobacter vinelandii, where S. elongatus was In contrast to direct mixing and membrane separation,
trapped within a polyacrylate hydrogel matrix, which gaseous separation (Figure 4(a)) allows only for the
facilitated the secretion of sucrose to be consumed by exchange of gases between the co-cultured microor-
A. vinelandii. This method allowed to cater to the ganisms. Here, the microorganisms are grown in separ-
growth and nutritional requirements of each micro- ate vessels connected via a port. The two species in co-
organism. The advantage of this method is the ability culture are not exposed to the nonvolatile metabolites
to optimize environmental conditions of the two differ- present within the culturing liquid or solid media pro-
ent species that have different environmental require- duced by either of the species, thereby reducing com-
ments for their particular functions. In this instance, the petition for nutrients. The exchange of gases, resulting
S. elongatus was subjected to osmotic stress by the for example from respiration, facilitated by the connec-
hydrogel, causing the release of compounds that tion port, can, however, affect the growth mechanism
enhanced the growth of the co-cultured species. and consequently the intercellular and/or extracellular
Although this stress response of S. elongatus was spe- metabolome of the receiving organism. Therefore, this
cies-specific, the further use of the dialysis tubing pre- method can be used to only assess the effect of volatile
vented direct physical contact between the two metabolites on microorganisms.
microorganisms. Santos and coworkers [48] demonstrated the symbi-
This method of co-culture offers faster diffusion rates otic association via a gaseous exchange between the
(for secondary metabolites/info-chemicals), quick equi- heterotrophic and photoautotrophic cultures of
librium conditions, easy set-up, and larger culture vol- Chlorella protothecoides. The heterotrophic C. protothe-
umes (1.5 to 5 L) that allows sampling for omics coides cultured in a photo-bioreactor were fed off-gas
investigations and the further isolation of target com- from the outlet autotrophic reactor, and vice-versa. The
pounds. Furthermore, this method allows different symbiotic bioreactor demonstrated that the enriched
growth spaces for both partners in contrast to vessel air with off-gas from the other bioreactor increased
chambers, where equal growth conditions were used. both the biomass and oil productivity of the microal-
This added advantage minimizes the impact of guest gae. Similarly, autotroph C. protothecoides (microalgae)
strain signaling molecules while discriminating the was co-cultured with heterotroph R. toruloides (yeast) in
interactions of co-culture from that of monocultures. In a vertical-alveolar-panel (VAP) photobioreactor, thereby
contrast, Paul and coworkers [102] criticized this taking advantage of their symbiotic association via
method for not allowing identical growth conditions of complementary nutritional metabolism, that is, respir-
the interacting partners or sufficient diffusion between ation and photosynthesis [110]. The VAP facilitated the
both culturing chambers. In summary, this method in exchange of carbon dioxide and oxygen between the
combination with systems biology approaches has a two microorganisms, resulting in greater microalgal bio-
great potential in understanding the functioning of a mass and lipid production.
microbial ecosystem, allelopathic interactions and in Gaseous separation methods are ideal for assessing
the discovery of novel drugs/natural biomarkers within the role of volatile molecules within co-culture systems
the co-culture community. which can be used as a tool to untangle and validate
the possible effects of microorganisms on each other.
The upscaling or perhaps expansion of this concept to
Spatial separation
validate gaseous exchanges within a consortium is feas-
Spatial separation consists of methods where the part- ible [111]. However, spatial separation methods are not
ners are spatially separated not allowing direct a true reflection of how the microorganisms interact in
exchange of materials as seen in the co-culturing meth- nature. For example, if yeast and algae were co-cultured
ods discussed earlier, but allows the indirect exchange together in the same medium, the number of gases
of chemicals, through contact of different phases, for produced may be lower than in monoculture. Also, the
example, gas-liquid and liquid-solid phases. This composition of the gases may differ. In nature, as the
method provides an effective way for eliminating microorganisms come into contact, other interactions
(a) Gaseous separation Gas exchange (b) Matrix Immobilization

chamber
Off-gas Matrix (e.g.

(bubbles) agar, metal,
silicon)
Microorganism
A medium
Microorganism B
medium
(c) Agar Systems

Agar 0.5%
Fixed
microorganisms
Agar
Agar 1.5-1.6%
Method (1) Method (2) Method (3)
Agar for Microorganism A and B can differ in composition and consistency.
Microorganism A
Microorganism B
Figure 4. Spatial separation co-culture systems. The co-culture microorganisms can only mediate through info-chemicals. Direct
contact is not possible. (a) Gaseous separation: each microorganism is grown in its own vessel. The vessels are connected
through a chamber that allows for volatile info-chemicals to be exchanged. (b) Matrix immobilization: microorganisms are
trapped within a porous matrix. Overlapping the matrixes allows for info-chemical exchange. (c) Agar Systems. Microorganisms A
and B can be co-culture on agar plates. The composition of the media can be different. The agar diffusible info-chemicals allow
for the species to communicate.
may take place, perhaps also at the expense of gaseous degree of control over interactions. The matrix compos-
exchange. This represents the limitation of this co-cul- ition will vary according to the nature of the microor-
turing method in terms of info-chemical analysis. ganisms in co-culture. The microorganisms attach
themselves to the support because of stress (producing
Matrix immobilization EPS) or within crevices that facilitate binding, as in the
In this method, microorganisms are secured or attached case of hollow-fiber membranes [112]. Additionally, the
to a surface/matrix (Figure 4(b)). Unlike the encapsula- microorganisms can be trapped between thin layers of
tion method discussed in Section “Encapsulation,” this different solidifying agents such as agar [113], hydro-
approach allows a greater degree of separation gels, j-carrageenan, and gelatin or combinations of
between partners and hence potentially a higher these [35,114]. These layers can be superimposed onto
each other to facilitate interaction [115]. Matrix immo- sequence of fungi. This is demonstrated by the lack of
bilization is widely used in tissue engineering applica- response when Aspergillus nidulans and 58 soil-dwelling
tions [116] and has also been developed to investigate actinomycetes were co-cultured using a dialysis tube
the cross-talk between microorganisms in co-culture membrane [16]. Besides, using qRT-PCR analyses, the
systems. In contrast to the use of shakers and bioreac- authors demonstrated no fungal response was initiated
tors, the use of this system enabled the creation of when the fungal culture was treated with the super-
models, which were used to simulate microbial interac- natant of the bacterial culture and when treated with
tions in their local environments [113]. This made this the supernatant of co-culture (of bacterium and fungus
method invaluable for the investigation of microbial lacking the PKS gene) [16]. It is evident, therefore, that
interactions in solid matrices such as food [114]. unlike the use of matrices, the physical interaction that
A hollow fiber matrix bioreactor (HfMBR) was used may exist naturally between two microorganisms was
to enrich denitrifying anaerobic methane oxidation enabled by the directly mixed culture to elicit the fun-
(DAMO) microbes and anammox bacteria consortium gal gene expression. This was further validated by the
for flue gas denitrification purposes [112]. The use of a authors with scanning electron microscopy (SEM) and
direct mixing method for the same consortia resulted in metabolomics platform [16]. On a positive note, mem-
a limited mass transfer of methane due to the forma- branes can also be used as a deduction tool to the
tion of microbial clusters. In contrast to direct mixing, mode of interaction in the co-culture experiments. On
HfMBR allows molecular diffusion of methane through the other hand, 3D bioprinting technology is obtaining
the biofilm’s substratum directly to the biofilm without a wider interest in research communities for studying
any bubble formation. Moreover, compared to direct microbial interactions [118–120]. A recent investigation
mixing, the activity of DAMO archaea in the ternary bio- highlighted several advantages of hydrogel-based
film built by HfMBR was found to be three times higher immobilization for on-demand bioproduction and pres-
[112]. Therefore, attaching an environmental inoculum ervation when compared to direct mixing techniques
within the hollow fiber allows for quick recovery of the [119]. Briefly, this method involves 3D printing of
system as the matrix facilitated methane gas diffusion microbe-laden hydrogels that spatially compartmental-
through the reactor. ize each organism (yeast and bacteria in this case). This
Some matrix systems do suffer from mass transfer minimizes or removes competition for nutrients, where
limitations. However, Smet and coworkers [117] showed authors have reported identical growth rates as that of
that matrix immobilized cells of S. typhimurium and E. monoculture for both partners, partners do not impede
coli growth dynamics were similar to those grown in cell growth of other and overall technique offers more
static communal liquid media. However, growth profiles control over a consortium controlling population
were lower when compared to shaken liquid cultures, dynamics. More importantly, this technique was dem-
where the mass transfer is facilitated. Therefore, better onstrated for the production of both small molecules
nutrient and gas distribution methods should be incor- and active peptides with the ability to repeated re-use
porated into this method. Additionally, the methods and preservation of the consortia for up to 1 year via
employed for metabolite extraction are more complex lyophilization, thereby offering unique advantages over
compared to liquid cultures. Difficulties were encoun- direct mixing techniques.
tered when extracting metabolites embedded or bound
to the matrix, where a stomacher was used to hom- Agar systems
ogenize the samples [37]. Therefore, these metabolites Agar systems are another example of spatial separation
may not be detected or accurate levels of the secreted co-culturing. This technique uses agar of various com-
compounds cannot be determined. positions such as potato dextrose [34] and LB-agar to
Matrix immobilization can be used quite flexibly in a create porous solid support, onto which microorgan-
co-culture system to analyze secreted substances by isms can be inoculated. Unlike matrix immobilization,
microorganisms and to act as a supporting matrix. the cultures here are not trapped in a matrix but rather
However, unlike mixed cultures, the use of such matri- allowed to grow on the surface. The configuration of
ces cannot provide a native environment in which the the agar system may vary according to the purpose of
microorganisms can interact physically. With such the study (as shown (Figure 4(c)).
matrix or spatial separation techniques, the potential of In Figure 4(c), Method (1) shows superimposed agar
consortia partners to produce the secondary metabo- of different compositions, which allow a transversal
lites during cross-talk is greatly underestimated under exchange of molecules with a degree of physical con-
laboratory conditions, as indicated by the genomic tact. In Method (2), longitudinal communication across
the agar is obtained on the boundaries between the secondary metabolites and to uncover the mechanism
two agar phases. The microorganisms at the boundary of silent gene activation. Yao and coworkers [122] used
may come into physical contact and secrete different the agar co-culture method and metabolomics platform
molecules to those away from the boundary. Whereas, to develop an interactive model (using co-culture of
in Method (3), the microorganisms are placed far apart. Trametes versicolor and Ganoderma applanatum) for
This design intends to elicit a response/exchange by activating the silent genes. This work led to the identifi-
relying on traveling-released cues between the species cation of 62 novel features that were either newly syn-
over a distance. thesized or highly produced in the co-culture
The porosity of the agar allows for the exchange of compared to their monocultures.
info-chemical between the microorganisms. This The use of agar plates was criticized by Mouget and
method has been extensively used to elucidate the coworkers [123], pointing out that only agar diffusible
interaction between fungi and bacteria co-culture molecules are permitted to be exchanged. This was
[34,36,121] and as a valuable tool in studying the co- shown by the null-effect when Pseudomonas diminuta
culture cross-talk in ecology, agriculture, medicine, and and P. vesicularis were co-cultured on agar plates with
biotechnological applications [121]. Scenedesmus bicellularis and Chlorella sp. Furthermore,
Agar systems have been used to study the allelo- the volume, porosity and composition of the agar can
pathic interactions between Botrytis cinerea (fungus) also lead to a varying rate of diffusion for info-chemi-
and the rhizobacterium Pseudomonas sp. [34]. Botrytis cals. More importantly, the very low concentration of
cinerea is responsible for gray mold syndrome on extracellular metabolites in a large pool of culture
leaves, whereas rhizobacterium was shown to promote medium makes their isolation, identification and quanti-
plant growth and antagonistic effects on in vitro fungal fication difficult with poor reproducibility. The use of
growth. Co-culturing of fungi and bacteria on the small plates/petri dishes (2 cm) instead of conventional
potato-based agar plate allowed the area of contact plates/petri dishes (9 or 15 cm) may solve the above
between the two species to be observed microscopic- problems. Bertrand and coworkers [14] used 2 cm
ally. This revealed a growth disruption of fungi around multi-well plates inoculated with pre-cultured agar
Pseudomonas sp., where Pseudomonas sp. did not affect plugs of Fusarium and Aspergillus fungi. The limited
the polygalacturonase activity of B. cinerea but inhib- nutrient supply due to smaller size wells increased the
ited its growth by causing coagulation, and leakage of competition between co-culture partners resulting in
protoplasm. Similarly, other studies using agar systems stronger and faster stimuli (increased concentration of
have revealed the secretion of compounds such as anti- de novo metabolites).
fungal, antibacterial substances as well as de novo Ideally, any co-culturing strategy should aim at pro-
metabolites during co-culturing [121]. Toxicological viding the platform that will mimic the naturally occur-
studies using potato dextrose agar were used to under- ring ecology. With the use of agar co-cultures, it is
stand the mechanisms of food poisoning caused by important to note that the microorganisms that are dir-
Burkholderia gladioli (bacterium), when Rhizopus micro- ectly below the spot inoculated area could become
spores (fungus) cultures contaminated with B. gladioli anoxic. Therefore, the compounds released may not
were used for the fermentation/production of Asian reflect the true ecological exchange between the co-
food dish tempe bongkrek [121]. This study not only cultured partners. Hence, this method may work better
identified that the fungus aided the bacterial growth when co-culturing anaerobic microorganisms.
which in turn increased the production of a lethal toxin Therefore, the ultimate method of co-culture using agar
(bongkrekic acid), but also showed that the bacteria would depend on the type of microorganisms being
produced antibiotics of the enacyloxin family. In the co-cultured and may have to be validated by other co-
case of ecological studies, Dalmas and coworkers [36] culture methods if the most number of molecules being
used this method along with the LC-MS platform and secreted are aimed to be detected.
demonstrated that Streptomycetes (from the rhizo-
sphere of Araucariaceae) produce exudates (twenty- Gel cassette system. An upgrade from conventional
four compounds), some of which suppress the growth/ agar systems is the gel cassette system. This method
activity of the fungus Neofusicoccum parvum. Under was first developed by Brocklehurst and coworkers
laboratory conditions, many genes for secondary [124] for monitoring monoculture species, which was
metabolite synthesis are presumably silent as revealed later applied to study the interactions between consor-
by transcriptomic analysis on cultured fungi. Activation tia partners [37]. Gel cassettes consisted of a gelatin
of such silent genes will enable us to discover novel matrix trapped between a gas permeable membrane
enclosed within two transparent windows made of (biomedical applications), where the cells are fragile in
Plexiglas and covered with a plastic film. This method is nature and culture volume requirements are minimal
commonly used to study the behavior of bacteria in a [67,125–128]. However, such systems can also be
solid structure, which emulates solid foods. Tsigaride employed with other cell systems to enable better con-
and coworkers [37] used this method to monitor trol of fluid flow, where low volume operations are pref-
growth and metabolic activity of Shewanella putrfaciens, erable. Recently, the combination of microfluidic
Brochothrix thermosphacta, and Pseudomonas sp. bac- systems with co-culturing designs has gained popular-
teria (in both mono- and co-culture) responsible for ity within various research fields [54,55,129–131].
food spoilage. The cassettes allowed co-culture of vari-
ous population mixes and to observation of their rela- Core-shell fibers
tionship. The findings suggested that changes in Microbial communities that interact in nature optimally
behavior were dependent on the co-cultured species. and perform multifunctions usually have a specific spa-
Furthermore, Pseudomonas sp. strains co-cultured with tially structured arrangement. Such spatial organization
B. thermosphacta propagated, whilst the ones grown is crucial in modulating the degree of co-existence
with S. putrefaciens perished.
[132–135]. Considering this fact, a core-shell fiber
method has been developed [55] in an attempt to con-
Microfluidic systems struct a biomimetic synthetic functional community, as
an alternative approach to genetic engineering (Figure
The conventional cell models and co-culture techniques
5(a)). To demonstrate this concept, the co-culture of
used so far in mammalian cell research do not allow for
trapping paracrine communication between different Sphingobium chlorophenolicum (a pentachlorophenol
cells due to poor spatial control over the cellular micro- (PCP) degrader) and Ralstonia metallidurans (a mercuric
environment and the coexistence of diffusion and con- ion Hg(II) reducer) was used to remove the mixture of
vection, which makes the control of communication for environmental pollutants from the soil. This system was
monitoring difficult. In contrast, the microfluidic system developed by coupling microfluidics with spatially sepa-
offers better control over fluids and microenvironments rated calcium alginate fibers to obtain a co-culture
with the use of integrated valves, where better fluid environment on the 100 mm scale. The degradation of
routing can be achieved along with the ability to separ- PCP and the reduction of Hg(II) was achieved simultan-
ate the defined section of the platform isolated from eously only in a spatial arrangement, which was not
the other sections. This type of culture system is com- achieved by directly mixed liquid cultures (unstructured
monly employed in mammalian cell research communities). This investigation highlighted the
(a) Core-Shell Fibres

Microorganism A
Microorganism B
Alginate
In
Fibres
IInter-species interactions
In
Membrane
Encasing Interactions extracted
(b) Microfluidics plus Agar
Solvent
Membrane interface Matrix
Figure 5. Microfluidics systems. (a) Micro-scale systems: coupled with agar allow for co-culturing and extraction of metabolites in
the same device (micro-metabolomics platform). (b) Core-shell fibers: consists of microorganisms trapped in the filamentous
alginate fibers. These microorganisms do not come into contact directly.
importance of spatial arrangements when developing This method offers several advantages, such as (a)
co-culturing techniques. The co-culture was only suc- ease of use; (b) one step metabolite extraction; (c)
cessful when S. chlorophenolicum was placed at the retrieval of organic phase without any aqueous media
center of the core-shell fiber, whereas having S. chloro- components carryover (an essential step for subsequent
phenolicum in the other outer cortex of the core-shell LC-MS platform); (d) rapid workflow with smaller extrac-
fiber decreased biodegradation efficacies by 50% [55]. tions volumes; and (e) versatility in the choice of sol-
The application of such techniques is gaining momen- vents used for metabolite extraction. As the device is
tum in biomedical applications, as demonstrated coated in Parylene C (high solvent resistance) it permits
recently for the proliferation of co-cultured C2C12 cells for the detection of un-interrogated segments of the
(mouse myoblasts) [136]. metabolome, unattainable with conventional extraction
solvents. This system was stated to have the advantage
Microfluidic system and agar of enabling the use of two different media for species
Another novel microfluidic device (made up with trans- that grow optimally in different media and to enable
parent polydimethylsiloxane (PDMS)) developed else- equidistance diffusion of metabolites, which was not
where to study the underlying molecular mechanism in the case with the use of direct mixing and agar co-cul-
Parkinson’s disease, where cross-talk between two dif- ture methods.
ferent cell populations was monitored by soluble fac-
tors (either by perfusion or by diffusion) [129] can also Critical considerations/challenges
be developed for biomanufacturing. The device con-
A summary of the approaches discussed in this review
sisted of two separate culture chambers connected by
is presented in Table 2. Establishing a co-culture
three channels and integrated pneumatic valves for iso-
approach that will facilitate obtaining the desired infor-
lating one cell population from another where required
mation is a feat in itself. Alongside the choice of which
(Figure 5(b)). This device allows for closer replication of
microorganisms to be co-cultured and the method to
in vivo conditions where paracrine signals are effective,
be used, the following factors need to be taken into
as the two culturing chambers are separated by a short
consideration: inoculation ratios, inoculation timing
distance of 250 mm, facilitating rapid molecular
[6,7], priority effects [137], and history of the micro-
exchange and better control over the cellular micro-
organism [138]. Each will have an impact in its own way
environment. Additionally, the chamber isolation tool
on the dynamics established between the co-cultured
encourages the concentration of the molecules in one
microorganisms. This will consequently influence the
area, facilitating isolation and detection. However, the
availability of molecular cues to be detected. Ideally, we
use of external pumps in such microfluidic devices want a method at both laboratory and industrial scale,
makes screening experiments nearly impossible and which will allow us to emulate the natural environment
fabrication challenging, effectively preventing wide- and noninvasive direct investigation of all possible
spread integration into biology labs. forms of dynamic interactions in real-time that emerges
The concept of an open micro-metabolomic method naturally in the microbial consortia. Currently employed
was recently developed by Barkal and coworkers [54]. A co-culturing methods appear to be useful in under-
device comprised of cultured micro-agar pad or liquid standing only a fraction of these interactions. Moreover,
well within an open microfluidic channel, where organic this fractional knowledge obtained does not reflect true
solvents (used for metabolite extractions) can be natural interactions that may be taking place, as such
directed to flow over the aqueous culture area. This associations comprise numerous organisms thriving
results in the formation of biphasic interfaces, allowing together [53]. The other critical considerations/chal-
for the integrated and passive extraction of metabolites lenges that require attention are;
over a defined period after which an organic solvent
can be recovered by a simple pipetting step. Later, the i. Many genes for these interactive cues are silent
micro-metabolomics platform was used to trap the under laboratory conditions, adding further limita-
chemical diversity of co-cultured fungal and bacterial tion [14,54,122]. Furthermore, the competition for
secondary metabolomes in response to changing nutrients in artificial consortia disturbs the
microenvironments. Here, the two micro-metabolomics homeostasis, as partners try to out-compete one
platforms were placed (opposing face) between a another and exhaust their available resources in a
thermoplastic layer with diffusion pores in-between to microenvironment, which is not the case with
allow an exchange of metabolites. microbial communities living in nature [15].
Table 2. Key co-culturing approaches discussed in the review and their bioprocess applicability.
Co-culturing approach Applicability Scalabilitya Factors to consider
Communal liquid medium growth Industrial production/ bench Inoculation ratio;
Direct mixing scale studies þþþ Timing of introducing co-
Pelletization and flocculation þþ culture partners.
Biofilms þþ
Solid–liquid interface Maximal surface for effective
Encapsulation Bench scale studies þ contact and molecular exchange;
Cell droplets þ Appropriate culture densities for
maximal effect
Membrane separation Membrane permeability to allow
TranswellV system þ
R
molecular exchange;
Vessel Chambers Bench scale studies/ þþ Pre-optimization of set-up
Screening for partners
Dialysis tube system þ
Spatial separation Matrix and medium composition to
Gaseous separation þþ enable and not restrict mass
Matrix immobilization Industrial production/ bench þ transfer for molecular exchange
scale studies and interactions
Agar systems þ between cultures
Gel cassette system
Microfluidic systems Spatial orientation and fluid flow to
Core-shell fibers Bench scale studies þþ maximize interactions
Microfluidic system and agar þ
a
þþþ: scalable; þþ: less scalable; þ: least scalable.
However, such nutrient limitation might be useful Understanding these parameters in terms of
as it can cause induction of de novo metabo- behaviors of the monoculture vs co-culture will
lites [14]. shed light on interactions that will govern the
ii. The available techniques are not designed to trap final co-culture [7]. Having the wrong starting
all forms of interactive cues. For example, the use ratio of two microorganisms at the co-inoculation
of nanospray desorption electrospray ionization or adding the inoculant of one to the other at
imaging mass spectrometry on agar co-cultures the wrong time (stage of growth of the other),
has allowed for real-time analysis of only agar dif- could lead to an unbalanced system, where one
fusible molecular signals (few forms of these interspecies overtakes or triggers an adverse reaction
actions), with low disruption to the microbial in the other.
interaction [139]. v. A lack of appropriate sampling strategies, analyt-
iii. Abiotic and biotic stress factors hugely affect ical workflows, techniques, and data analysis tools
these interactive cues, creating doubt in their reli- presents an additional major challenge in the
able resemblance to that of the natural interplay. detection and quantification of these interactive
For example, stress factors arising from co-culture cues [100]. The interactive cues emerging from
designs include physical restriction (cell confine- available small-scale spatial configuration are
ment, immobilization, and limited/no molecular often having a very dilute concentration. This
diffusion) and chemical restriction (nutrients), might be due to the poor co-culture designs
which usually results in a generation of nutrient offering very small sample volume for analyses,
and/or metabolite concentration gradients [140]. the existence of very dilute communities as in the
Biotic stress factors such as a selection of suitable case of phytoplankton, and contribution from the
co-culture partners, population dynamics, biovo- biological sample matrix such as salts, proteins,
lume variability, media selection, nutrient source, cell debris, and rich media components. Owing to
inoculum/seeding (ratio, densities, location, and the high turnover rate, dynamic nature, and
timing), pH, and salinity affect the growth kinetics diverse physicochemical properties of these inter-
[7,51,76]. To the most extent, these stress factors active cues, the identification of an optimal ana-
are interlinked, as diffusion limitation will result in lytical workflow (sampling, quenching, extraction,
difficulties in nutrient supply, thereby affecting and analytical platform) represents a major chal-
growth. Overall, these stress factors elicit an lenge, as there is no single platform that is cur-
unwanted response, thereby impairing the overall rently available, which is capable of identifying
aim of the co-culture research. and quantifying these interactive cues, in an
iv. Inoculation ratios and timing of the monocultures unbiased and reproducible way [25,26,141].
need to be factored into the equation. Moreover, the proposed developed and optimized
analytical workflow, for a given consortia partners and nonvolatile signals. The filter allowed for the
are always species-specific [15,142] and might not flow of molecules and the culture parameters
be valid for other partners, thereby requires inde- enhanced the dissolution rate of oxygen, for the
pendent evaluation and validation. yeast to uptake, which in turn generated the car-
bon dioxide necessary for the algae to grow.
iv. The main drawback of laboratory co-cultures is
Future optimization strategies and co-culture
the fact that these are limited in the extent to
design development
which they can mimic the real world. The use of
The current systems outlined in this review have great a multifunctional bioreactor that will allow a
potential to trap the fraction of interacting cues emerg- three-dimensional culture of cells, where co-cul-
ing from microbial interactions, however, to make a real tured partners are spatially separated, is gaining
sense of the soup, further development to co-culture popularity in tissue engineering [111,143].
designs and analytical workflows is vital. Circumventing Whereas few efforts have been made to adapt
this problem is not an easy feat, but the following con- methods used in monoculture systems for co-
siderations hold promise for future optimization strat- cultures studies, as demonstrated with the appli-
egies, concerning co-culture designs: cation of gel cassettes for microbial interactions
[37]. Adoption and further developments of such
i. The application of more than one technique to a methods for microbial consortia hold great
particular co-culture system would indeed pro- potential, as spatial structures will allow us to
vide more rounded conclusions, however, it may mimic the behaviors of cells as it happens
not be a practical approach in terms of time in nature.
and logistics. v. Similarly, the adoption of methods such as diffu-
ii. The environment in which the co-cultures are sion chambers (Figure 6), which are mainly
cultivated will inevitably affect the interactions. developed to isolate microorganisms from the
The analyst should consider the mode of trigger- environment and to acclimatize these to labora-
response mechanism (either physical, diffusion, tory conditions [144,145] may be a viable
adhesion, or gaseous) intended for desired appli- method for co-culture studies. This method is
cations, as this will help in the selection of opti- comprised of a thin film of agar that encapsu-
mal co-culture technique [7]. Additionally, if lates the microorganism on a bottom base layer.
microbes were affected by the media’s structural Initially, the monoculture/co-cultured species
make-up then it would be more logical to test could be inoculated onto the thin film of agar
trigger-response, in an environment most similar and left to incubate in the environment. This will
to its natural local environment. For example, it allow us to capture the true representation of
would be logical to culture fungi on agar, as it the interplay that exists in nature. This set-up
exists naturally on surfaces such as wood, rather would also allow to trap and concentrate metab-
than in liquid media. However, due to the adapt- olites facilitating their identification.
ability of microorganisms, it may be possible vi. Integration of microfluidic single-cell cultivation
that they are able to grow in several different systems with traditional methods is emerging as
media matrices. a valuable tool in exploring the microbiome
iii. The extracellular environment in co-cultures interactions in both natural and synthetic con-
strongly influences cell-cell interactions. This is sortia. For example, coupling of microfluidics
heavily reliant on the experimental set-up, such devices with agar systems has been fruitful
as the bioreactor design, use of separation mem- [54,55]. Novel designs integrating membrane
branes, perturbation within the reactor, tempera- separation techniques with co-culture plates
ture, pH, and other abiotic factors. The collection [146] have great potential for the simultaneous
of these parameters will dictate the mass transfer study of various forms of interactions and
of volatile and nonvolatile compounds [6,101]. growth dynamics. A recent review highlighted
The development of a system that allows moni- the pros and cons of microfluidic systems along
toring more than one form of interactive cues is with an overview of different microfluidic sys-
thus necessary. For example, the development of tems and their integration with traditional meth-
a double system bubble column photo-bioreac- ods used in environmental biotechnology [147].
tor [51], allowed the exchange of both volatile With such integration, cultivations can be
Cover (plastic film)
Agar matrix
with cultured microorganisms
O-Ring
Microorganism A
Microorganism B
Figure 6. Diffusion Chambers – used mainly for isolation of hard to grow species from the environment and can be adapted to
be used to study the behavior of artificial co-cultures in nature.
performed at population (3D) or single-cell level viii. For understanding the spatial distribution of
(2D, 1D, or 0D) with direct cell-to-cell contact or interacting partners and their metabolic state,
indirect contact via permeable membranes. For methods such as fluorescence in situ hybridiza-
example, a novel microfluidic 2D co-cultivation tion (FISH) [150] and fluorescently-tagged pro-
system with spatially separated cultivation cham- teins [41,151] hold great potential. Additionally,
bers was developed, allowing faster metabolite C14-labelled sodium carbonate labeling was
exchange due to short diffusion distances via used to investigate biofilm formation [152].
sieve structure [148]. Such chip-based techniques ix. The use of metabolomics platform with high-
allow systematic investigation of microbial inter- resolution mass spectrometry (HRMS) coupled to
actions at a single-cell resolution. This provides a chromatographic techniques is gaining popular-
one-to-one perspective. However, it must be ity for the study of microbial interactions
noted that microorganisms thrive in “families,” [9,14,54,100,102,122] However, its broad deploy-
thus the behavior of a single cell cannot be ment to biotechnology is not yet as widespread
taken as a representation of the whole. The cell- as desired due to several challenges in the quan-
to-cell effect that was observed in direct mixing titative metabolomics workflow that remain [26].
experiments, where some level of separation hin- Coupling of metabolomics platform with the use
dered communication, is a good indicator of of stable isotope tracers could serve as a gold
this. Another good example is quorum sensing standard for metabolic pathway discovery and
in bacteria, where communication molecules are also for identifying the flow of metabolites in
triggered by an increasing population. microbial consortia studies [27].
Furthermore, biotechnological applications look x. Metabolic modeling is a useful tool to study and
into co-cultures as tools to maximize biomass predict the behaviors of co-cultures and provides
growth, thus further investigation is required an insight into which type or combination of
into the use of “single-cell” methods, to attest if techniques should be used to maximize our
these indeed are a good way of studying micro- understanding of microbial interactions
organisms for biotechnological applications. [153,154]. Metabolic modeling was used to simu-
vii. Concerning analytical tools, so far, metagenomics late the co-culture of respiratory-deficient S. cere-
in combination with transcriptomics and proteo- visiae and wild-type Scheffersomyces stipites, to
mics offers great potential as a guide for inter- maximize the co-culture growth rate [155]. To do
action discovery. For example, methods such as this, the genome-scale metabolic reconstructs of
functional genomic responses (changes in gene each organism were necessary. Dynamic models
expression using RNAseq, microarray analysis) and substrate uptake kinetics were developed
have been used for a deeper understanding of for each organism separately, to be later com-
interacting cues [42,66,149]. bined to predict the outcomes at different
microaerobic growth conditions. On the same processes and commonalities is crucial in mini-
note, the stoichiometric model-based approach mizing the barriers between scientific commun-
was used to construct a synthetic anaerobic co- ities and improving our knowledge of microbial
culture and integrate the metabolism of processes [161].
Clostridium acetobutylicum and Wolinella succino-
genes. Such a model can interact via interspecies
Conclusion
hydrogen transfer/applied different environmen-
tal conditions to infer metabolic-exchange fluxes It is evident from this review that different co-culture
[156]. The development of co-culture databases methods are suitable for different microorganisms and
containing valuable experimental information on for different goals that the co-culture experiment aims
metabolites and metabolic pathways involved in to achieve. Spatial separation methods are useful for
co-cultures is a valuable tool for metabolic mod- the detection of metabolites and the identification of
eling [7]. This was recently demonstrated, where secreted molecules but would not be beneficial for co-
researchers have developed a “Metabolic cultured species that require physical interaction. On
Support Index” for quantifying the metabolic the other hand, encapsulation methods are more suited
interactions in microbial co-cultures [157]. for microorganisms that require different environmental
Modeling the interactions between the microor- conditions. Furthermore, co-culture methods can also
ganisms in consortia presents many obstacles be combined such as using a combination of hydrogel
because of the complexity of the network and matrix and a membrane for spatial separation of the
changes in growth parameters will further add co-cultured microorganisms. These co-culture methods
to the complexity [6]. By dissecting the interac- have highlighted different advantages and challenges
tions into smaller manageable co-culture sys- depending on the aim of the experiments. Therefore, it
tems, with targeted goals, a step-by-step is recommended that the co-culture methods are
approach can be modeled and expanded to chosen based on their advantage for the characteristics
cover the bigger picture. For example, a novel of the co-culturing species. Different co-culture meth-
mathematical biofilm model that can be applied ods should also be utilized to validate experimental
to any bacterial species/environmental condi- results obtained as different environmental structures
tions was proposed [158]. Interactions between and conditions can have effects on communication
Porphyromonas gingivalis and Streptococcus gor- between microorganisms.
donii biofilms were studied with this model,
where independence between species, substrate
Disclosure statement
competition, and production of toxic molecules
can be explored. However, the application of the No potential conflict of interest was reported by
model is not universal to all systems and needs the author(s).
to be developed per bioprocess. However, data
collection and characterization with the aim of Funding
bioprocess optimization will pose a challenge in
This work was supported by Engineering and Physical
itself, that to date needs to be overcome with Sciences Research Council [EP/E036252/1 and DTA 1623367]
the development of high-throughput methodol- and Biotechnology and Biological Sciences Research Council
ogies and better mapping systems. The develop- [BB/K020633/1].
ment of live-cell tracking methods may be a
solution that can be extended to chemical cues
tracking [159]. The potential of differential equa-
ORCID
tion models, constraint-based stoichiometric
models, and later integrative approaches were Seetharaman Vaidyanathan http://orcid.org/0000-0003-
4137-1230
highlighted in exploring the complex interac-
tions between microbial communities [160].
Authors recommended recognition of key References
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Critical Reviews in Biotechnology

ISSN: (Print) (Online) Journal homepage: www.tandfonline.com/journals/ibty20

Co-culturing microbial consortia: approaches

Rahul Vijay Kapoore, Gloria Padmaperuma, Supattra Maneein &

To link to this article: https://doi.org/10.1080/07388551.2021.1921691

© 2021 The Author(s). Published by Informa

Published online: 12 May 2021.

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Co-culturing microbial consortia: approaches for applications in

ABSTRACT ARTICLE HISTORY

Introduction potential for biotechnological application due to their

Table 1. A survey of key co-culturing techniques used in biotechnology.

(a) Direct Mixing (b) Pelletization and Flocculation (c) Biofilm

Communal medium Communal medium

Stage 2 EPS network

Direct mixing the plant pathogen [33]. A symbiotic interaction or

co-culture method in this instance is due to the induc- Solid–liquid interface

(a) Encapsulation (b) Encapsulation (co-immobilization)

Microorganism A medium Microorganism B medium Medium (Liquid/Gas) Communal

(c) Cell Droplets

(a) Transwell System (b) Vessel Chambers (c) Dialysis tube

(a) Gaseous separation Gas exchange (b) Matrix Immobilization

Off-gas Matrix (e.g.

(c) Agar Systems

Method (1) Method (2) Method (3)

Agar for Microorganism A and B can differ in composition and consistency.

(a) Core-Shell Fibres

(b) Microfluidics plus Agar

Membrane interface Matrix

Cover (plastic film)

experiments. Comput Struct Biotechnol J. 2020;18: [160] €h O. Review and perspective on

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