Microbioreactors for nutrient-controlled microbial cultures: Bridging
the gap between early bioprocess development and industrial scale use
Kartik Totlania, Rinke J. van
Tatenhove-Pelb, Michiel T.
Kreutzera, Walter M. van Gulikb and
Volkert van Steijna*
a Department of Chemical Engineering, Faculty of
Applied Sciences, Delft University of Technology, van der Maasweg 9,
2629 HZ, Delft, the Netherlands
b Department of Biotechnology, Faculty of Applied
Sciences, Delft University of Technology, van der Maasweg 9, 2629 HZ,
Delft, the Netherlands
Abstract
It is common practice in the development of bioprocesses to genetically
modify a microorganism and study a large number of resulting mutants in
order to select the ones that perform best for use at the industrial
scale. At industrial scale, strict nutrient-controlled growth conditions
are imposed to control the metabolic activity and growth rate of the
microorganism, thereby enhancing the expression of the product of
interest. Although it is known that microorganisms that perform best
under these strictly controlled conditions are not the same as the ones
that perform best under uncontrolled batch conditions, screening, and
selection is predominantly performed under batch conditions. Tools that
afford high throughput on the one hand and dynamic control over
cultivation conditions on the other hand are not yet available.
Microbioreactors offer the potential to address this problem, resolving
the gap between bioprocess development and industrial scale use. In this
review, we highlight the current state-of-the-art of microbioreactors
that offer the potential to screen microorganisms under dynamically
controlled conditions. We classify them into: (i) microtiter plate-based
platforms, (ii) microfluidic chamber-based platforms, and (iii)
microfluidic droplet-based platforms. We conclude this review by
discussing the opportunities of nutrient-fed microbioreactors in the
field of biotechnology.
Keywords Fed-batch, Chemostat, Microbioreactors, Nutrient-limited
growth, Industrially-relevant screening.
1. Introduction
Industrial biotechnology uses microorganisms to transform renewable
resources like agricultural waste into products, resulting in more
sustainable processes than the conventional chemical production from
fossil feedstocks. Microorganisms naturally synthesize antibiotics,
vitamins, proteins, and other valuable products, but typically in
amounts insignificant for industrial scale production. Economic
feasibility of bioprocesses hence hinges on the ability to enhance the
performance of microorganisms. This requires modification of the
microorganisms’ metabolic pathways through genetic modification, either
in a directed or a random way, and optimization of the cultivation
conditions. Possible cultivation conditions are uncontrolled batch,
controlled batch, fed-batch and chemostat (Figure.1 ). A general
challenge in the development of bioprocesses is the identification of
the optimum combination of modified microorganism and cultivation
conditions. This requires studying the performance of a large number of
modified microorganisms under dynamically controlled, nutrient-limited
cultivation conditions, like fed-batch and chemostat cultures[1].
However, tools that afford high throughput as well as dynamic control
over process conditions are only limitedly available.
The current bioprocess development strategy typically starts with
studying a large number of modified microorganisms in the wells of
microtiter plates (uncontrolled batch), with a small fraction of best
performers progressing to the next phase[2]. Further selection is
sequentially performed with tools that afford more control over
cultivation conditions (controlled batch, fed-batch, chemostat), but
have a lower throughput, as illustrated in Figure.2 .
While microtiter plates enable high-throughput experimentation in an
automated fashion[3], the low volume (typically 10 – 2,000 µL) in
the wells of these plates impedes dynamic control over the cultivation
conditions due to the difficulty in supplying minute amounts of
nutrients to the individual wells during cultivation. Experiments in
microtiter plates are hence commonly performed with all nutrients
present from the start and without active control over parameters like
pH and dissolved oxygen, resulting in growth of the microorganisms at
the maximum possible growth rate[4]. However, these uncontrolledbatch conditions are not comparable to the strict
nutrient-limited conditions that are commonly imposed at industrial
scale to control the metabolic activity and growth rate of the
microorganisms[5], thereby enhancing the expression of the product
of interest. Unfortunately, modified microorganisms that perform best
under batch conditions are seldom the ones that perform best under
nutrient-limited conditions. This has been demonstrated by Scheidle and
co-workers, who compared the production of green fluorescence protein
(GFP) by 224 different clones of Hansenula polymorpha under batch
and fed-batch conditions[6]. Clearly, there is no correlation
between best-producing clones under batch and fed-batch conditions, seeFigure.3(a) . Additionally, the average yield achieved under
fed-batch conditions is about 14-fold higher than that achieved under
batch conditions. Similar observations, although less strikingly, were
reported by Keil and co-workers[7], who compared the performance of
32 clones of cellulose producing E.coli under batch and fed-batch
conditions, see Figure.3(b) . Both studies illustrate that
identifying best performers under batch conditions with the purpose to
use them under nutrient-limited conditions at industrial scale leads to
ineffectiveness in bioprocess development.
Over the years, several technologies have been developed to address this
shortcoming and transform the conventional bioprocess development
strategy. The unsurpassed control over fluids in microfluidic channels
offers the potential to overcome the impasse between throughput and
level of control in microbioreactors[8]. These microbioreactors
enable studying a large number of modified microorganisms in parallel
under dynamically controlled conditions. An ideal microbioreactor
should: (1) enable dynamic supply of nutrients and control over pH and
dissolved oxygen, (2) enable integration with analytics to quantify
biomass and products of interest and allow for selection and retrieval
of best performers, (3) be scalable to enable high throughput
experimentation, and (4) be robust and simple for easy adoption by the
industry.
In this review, we present an overview of the emerging microbioreactor
technologies, with the focus on nutrient feeding strategies. For more
general reviews on microbioreactors or larger miniaturized bioreactors,
we refer to Hemmerich et al. [8], Hegab et al. [9]
and Schäpper et al. [10]. We start by illustrating feed
strategies in microtiter plates. Next, we highlight microfluidic systems
in which microorganisms are studied inside chambers. Finally, we
elucidate the developments in the field of droplet-based microfluidics.
After reviewing these three classes separately, we discuss the
opportunities in the final section, highlighting where emerging
miniaturized fermentation platforms can play a drastic role in the field
of industrial biotechnology and beyond.
2. Microbioreactors for nutrient-controlled cultivation of
microorganisms
2.1 Microtiter plate-based platforms
In recent years, significant effort has been made to modify microtiter
plates to enable cultivation of microorganisms in fed-batch mode with
passive and active control over nutrients. One strategy to continuously
supply glucose to cells is by producing glucose inside the wells through
enzymatic conversion of starch by glucoamylase, both added to the
culture medium at the start of cultivation[15,16]. Different supply
rates of glucose can be achieved by using different concentrations of
the enzyme. A different strategy is to connect pairs of wells by a
microchannel, with one well acting as a reservoir for nutrients, and the
other as a cultivation chamber, see Figure.4(a) . The use of a
polyacrylamide gel inside the connecting microchannel enables the slow
and steady supply of nutrients to the culture well, with the feed rate
depending on the diffusional properties of the gel[11]. Another
strategy for nutrient release into the culture chamber is by loading
wells with a silicon elastomer that contains crystals of glucose, seeFigure.4(b) . In these so-called FeedPlates, release of glucose
from the elastomer into the culture well is driven by a difference in
osmotic pressure[12][17][18]. Since the silicon elastomer at
the bottom of the wells hinders optical monitoring during cultivation,
Habicher and co-workers[13] designed glucose-releasing rings to make
the wells accessible for online monitoring, see Figure.4(c) .
Apart from passive diffusion-based release strategies, active supply
strategies have been developed such as the microfluidic BioLector system
by m2p-labs[14][19][20]. Figure.4(d) illustrates
two wells of a microtiter plate that are connected through a
microchannel, with the flow from the nutrient well to the culture well
actively controlled through pneumatic actuation. Controlled dosage is
achieved by first filling the pump chamber with nutrient solution
through opening and closing parts of the microchannel with an integrated
micropump that inflates/deflates an elastic membrane, and subsequently
emptying the pump chamber into the culture well. This technology allows
fed-batch fermentations with a predetermined feeding profile[14].
Besides, it enables actively pH-controlled cultivations. A commercially
available modified microtiter plate system (Micro-matrix) developed by
Applikon Biotechnology (http://www.applikon-bio.com, Delft, the
Netherlands) offers 24 parallel fed-batch fermentations with the
possibility to feed nutrients whilst controlling parameters such as pH,
temperature, and dissolved oxygen. While some of the modified microtiter
plates discussed above integrate microfluidic channels, microbioreactors
constructed as complete microfluidic devices present an interesting
alternative and are discussed next.
2.2 Microfluidic chamber-based platforms
One of the first microfluidic bioreactors for carrying out
nutrient-controlled cultivation was developed by Balagaddé and
co-workers[21], who made a microfluidic chemostat. The design of one
of the six microfluidic circuits integrated onto a single chip is shown
in Figure.5(a) . Cells are continuously circulated in the growth
chamber (loop) through the use of an integrated peristaltic pump
constructed from pneumatically actuated membrane valves, also known as
Quake valves. A solution of nutrients is periodically pumped into the
growth chamber by opening and closing parts of the circuit using such
valves, while effluent is removed from the growth chamber. This allows
continuous cultivation of cells under nutrient-limited conditions, with
the cell growth rate directly controlled by the rate at which the
chamber volume is refreshed by the nutrient solution (the dilution
rate). An alternative strategy that does not require the integration of
peristaltic pumps to supply nutrients and induce mixing was presented by
Jensen[27]. They developed a microbioreactor with an external
syringe pump for nutrient supply, while reactor effluent was collected
in a pressurized water reservoir and mixing in the culture chamber was
achieved by a ringed magnetic stir needle.
In addition to chemostats, fed-batch microbioreactor were developed. An
example by Bower and co-workers[22] is shown inFigure.5(b) . The device comprises of three independent input
channels which are connected to growth chambers via pressurized fluid
reservoirs. The fed-batch process is achieved by partially filling up
the chambers with cell solution, followed by the periodic supply of
nutrients to the cells in the chambers through the actuation of the
on-chip valves, until the maximum working volume is occupied, and the
fed-batch process is complete.
A different strategy that requires less advanced integrated micro-pumps
and valves is to culture cells inside perfusable chambers. An example of
such a device, developed by Groisman and co-workers[23], is shown inFigure.5(c) . The chambers in which the cells are trapped are
perfused by two surrounding nutrient supply channels. The shallow vias
that connect the chambers to the supply channels ensure fluid to be
exchanged, while cells remain trapped. The rate of diffusion of
nutrients through these vias is much faster than the rate of nutrient
consumption by the cells, such that the nutrient concentration in the
chamber equals that in the supply channels, allowing direct control over
nutrient-limited growth conditions. The use of an elastomeric material
such as PDMS provides the means to load cells into the chambers, by
injecting a cell solution into the device and subsequently pressurizing
the device allowing cells to enter the chambers through the inflated
vias. As cells remain trapped inside the chambers and do leave as
effluent, this type of perfusable device results in chemostat-like
cultivation.
Continuous regulation of the number of cells inside the chambers during
cultivation can be achieved by controlled inflation of the vias. This
can be done by depressurizing a separate channel above the chambers, seeFigure.5(d) . Automatic dilution of cells is then achieved using
a feedback loop, with the pneumatic actuation controlled based on online
measurements of the number of cells in the chambers[24][28].
While the primary feed strategy in the above two examples is based on
diffusion, flow-through chambers have also been developed, notably by
Grunberger and co-workers[25]. Cells are trapped inside shallow
chambers located in a main channel, while nutrient solution is flown
around and through the chamber, as illustrated in Figure.5(e) .
Nutrient-limited growth conditions are primarily controlled through the
concentration of nutrient solution, supplied using an external pump. The
shallow nature of the chambers facilitates cells to remain trapped and
to grow in a two-dimensional fashion, enabling accurate monitoring at
single cell resolution. Besides in- and outflow, the perforations in the
chambers also allow cells to leave the chambers once they are populated.
Similar strategies even enable studies on co-cultures of cells in
chemostat-like conditions, see Figure.5(f) [26]. For
extensive reviews on applying chamber-based microfluidic devices for
studying morphology, heterogeneity, growth, and communication of
microorganisms in a high-throughput manner and at single-cell
resolution, we refer to [29-31].
2.3 Microfluidic droplet-based platforms
The potential to use droplets as cultivation environments has been
outlined decades ago[41]. The precise generation and control of
droplets in microfluidic devices led to the development of droplet-based
microbioreactors. Most efforts so far focus on batch processes, with all
components encapsulated at the start. The typical workflow then
comprises the generation of millions of droplets with cells and
nutrients encapsulated, incubation of the droplets off-chip, and
reinjection in a separate chip to analyse and sort the
droplets[42-44]. While providing a high throughput, this type of
workflow with off-chip incubation makes it cumbersome to periodically
supply nutrients to all individual droplets and turn the system into a
fed-batch or a chemostat[45]. An alternative workflow that may
facilitate fed-batch or chemostat cultivation is based on on-chip (or
in-tube) incubation. While operations on droplets can be precisely
performed when operating microfluidic devices under steady state
conditions, the inherent non-steady nature of such microbioreactors with
different droplet operations to enable regular nutrient supply
nevertheless makes their development an outstanding challenge.
Jakiela and co-workers[32] presented the first example of a
droplet-based chemostat by developing a device that comprises different
circuits that can be isolated from each other with the use of off-chip
solenoid valves. Cells were encapsulated inside droplets, which were
transported back and forth in the main channel of the device. Each
droplet regularly entered a circuit in which effluent was removed from
the droplets through controlled break-up, as illustrated in the top ofFigure.6(a) . The resulting droplets were supplied with
nutrients in another circuit in which they were coalesced with nutrient
droplets that were generated on demand, as illustrated in the bottom of
Figure.6(a). More recently, Jian and co-workers[33] developed a
similar automated droplet-based chemostat, including a sorting step
based on monitoring biomass growth via OD measurements (seeFigure.6(b) ).
Droplet-based fed-batch reactors can be made by spatially immobilizing
the cell-containing droplets and supplying them with nutrients. One of
the first examples of such a strategy was by presented by Leung and
co-workers[34]. The device comprises 95 chambers, which can all be
individually addressed through the pneumatic actuation of integrated
Quake valves. After loading a cell-containing droplet inside each
chamber of this multiplex device, nutrient droplets can be generated on
demand and guided to the chambers as illustrated inFigure.6(c) , enabling studies under fed-batch conditions. A
similar strategy based on immobilization of droplets in chambers and
controlled supply of reagents through pneumatic actuation of integrated
valves has been used by the group of Chang-Soo Lee[35,46,47] for the
cultivation of cells and the development of bio-chemical assays, seeFigure.6(d) ).
The above examples show a trend towards enhanced control over
cultivation environment by developing sophisticated devices with
integrated/external values, operated through multi-step actuation
schemes. There is also another, almost opposite, trend visible in
literature, in which control is achieved using (passive) geometrical
features to keep device architecture and operation as simple as
possible. An elegant example was demonstrated by Ismagilov and
co-workers[36][48], who made a droplet-based fed-batch by
compartmentalizing droplets inside chambers, which can be joined by
manually sliding the top and bottom half of these so-calledSlipChips towards each other, as illustrated inFigure.6(e) . Baroud and co-workers[37] explored the use of
cavities in the floor/ceiling of a microchannel in which droplets
squeezed between the floor and ceiling can relax their shape, and
thereby remain trapped. These cavities can be designed such that each
allows immobilization of a cell-containing droplet, while leaving room
for another droplet to be loaded for controlled supply, as illustrated
in the left panel of Figure.6(f) . After loading those droplets
(middle panel), supply is achieved by imposing coalescence (right
panel). This is done by flowing a solvent through the channel in which
the surfactant used for stabilization of the interfaces is less soluble.
This strategy so far has been used for drug toxicity studies of cells
with a single delivery of drugs[37]. Whether it can be used for
repeated supply of nutrients to enable nutrient-controlled growth
experiments under fed-batch conditions is yet to be explored. A recent
example of a droplet-based fed-batch microbioreactor that does allow the
repeated and controlled supply of nutrients to a cell-containing droplet
immobilized in a chamber was demonstrated in the lab of the
authors[39]. The design of the geometry in which the nutrient
droplets are produced allows a robust periodic on-demand supply of
droplets in a device free of valves, just through the use of a
commercially available pressure pump[38], see Figure.6(g) .
The authors demonstrated the cultivation of cells under fed-batch
conditions, with the growth rate of the cells inside the immobilized
cell-containing droplet controlled by the concentration of the
nutrient-containing droplets.
Besides the droplet-based approaches in microfluidic channels, we
conclude by highlighting a channel-free fed-batch approach in which
cell-containing droplets are bio-printed in a yield-stress fluid. Nelson
and co-workers[40] developed a bio-printing method to study the
response to drugs injected into the droplets after 24h of incubation,
see Figure.6(h) . Since the print-head enables injection of
nutrients or extraction of effluent, this relative unexplored strategy
is also potentially interesting for high-throughput studies of
microorganisms under nutrient-controlled growth conditions.
3. Challenges of microbioreactors
technologies
3.1 Microtiter plate-based platforms
While different modifications to existing microtiter plate-based
platforms enable carrying out nutrient-controlled fed-batch cultivation
experiments at microscale, there are several challenges that these
platforms face before they are ready for adoption in biotechnological
screening routines. Firstly, enzymatic glucose release-based systems are
strongly influenced by operation parameters such as pH and temperature
as the activity of the enzyme depends on them. Additionally, enzymatic
release can only be used for feeding of glucose-controlled fed-batch
cultivations. Diffusion-based feeding also depends on environmental
factors such as media, pH, temperature, and geometrical factors as in
the case of PAA filled microchannel [11]. Establishing specific
nutrient feeding profiles is difficult in diffusion-based release
strategies, let alone establishing active control with online feedback.
Additionally, multiple glucose crystals can release due to the osmotic
pressure difference leading to uneven glucose concentration due to this
burst release. Finally, embedding microfluidic channels at the bottom of
the standard microtiter plate as in the case of Microfluidic
BioLector[14] poses difficulties in fabrication. The combination of
Microtiter plates and microfluidic channels replacing the base of the
plate with embedded micro-pumps and valves increases the complexity of
the system. Additionally, systems like Microfluidic Biolector offer a
maximum of 44 to 48 fed-batch experiments per plate, which could be
treated as relatively low-throughput in screening routines.
3.2 Microfluidic chamber-based platforms
A challenge of microfluidic chamber-based platforms arises from the
large surface to volume ration when miniaturizing, leading to biofilm
formation at the solid walls. Other challenges arise from the chambers
not being completed isolated, which may lead to cross contamination or
to difficulties in screening based on secreted extracellular products.
Besides, the large number of inlet ports required for operation in some
of the platforms poses difficulty in scale out and accessibility of the
technology to non-experts.
3.3 Microfluidic droplet-based platforms
A challenge of microfluidic droplet-based platforms is associated with
the analytics and process control in microdroplet format. On-line
measuring of process parameters such as pH, dissolved oxygen, nutrient
or metabolite concentration, which forms the heart of any screening
routine, can be difficult. One way to achieve this is by using different
fluorescent-based readouts. Further on, implementing control over
dissolved oxygen and assuring that the cultivation does not run under
oxygen limitations can be a challenge. Fluorinated oils which often
serve as the continuous phase can be used as oxygen source during
cultivation[49]. Another challenge involves creating a simple and
robust nutrient feeding strategy, making the technology accessible to
non-experts[50]. As aforementioned, microfluidic droplet
arrays are useful in carrying out non-steady assays in which nutrients
can be added semi-continuously. However, these droplet arrays use
multiple membrane based pneumatic valves which make the devices less
robust, difficult to fabricate and possibly inhibit its easy adaptation
by biotechnology and bioprocess engineering community. The barrier of
adoption of droplet microfluidic methods by non-experts can be reduced
by embedding the complicated chip operation workflow and associated
experimental paraphernalia in “chip-in a box” type of systems[51].
Another challenge in implementation of droplet-based microfluidic
platforms for long-term nutrient-limited fermentation is the possible
leakage of molecules through the interface of the
droplets[52][53]. Leakage of nutrients or secreted metabolites
could not just lead to uneven growth rates but also selection of false
positives during strain selection. Several studies have been performed
to investigate mass transport through the oil-water interphase where
droplets incubate inside PDMS devices. The nature of the molecules
inside the droplets and the surfactants at the oil-water interphase are
hypothesized as the most important parameter in understanding and
controlling the leakage of molecules through microdroplets.
4. Opportunities for emerging
microbioreactor technologies
Nutrient-limited microbioreactors can play a key role in the development
of sustainable industrial biotechnology. Their small volume and
therefore the possibility for high throughput-screening can accelerate
the identification of robust and productive strains[54], while
screening under nutrient-limited conditions ensures that industrially
relevant strains are identified[6][7]. In addition, the
screening-process itself is also more sustainable and cheaper, as less
reagents and consumables are needed[55].
The nutrient-feeding strategies described in this review also allow
separation of growth- and production-phases by changing the medium,
something that is not possible in batch-cultures. This is for example
relevant for lipid-production in algae, where different media
compositions for growth (stage 1) and lipid-production (stage 2) are
used to improve lipid-production[56]. This separation of growth- and
production is also relevant for the production of recombinant protein,
as protein-production is typically induced after growth, by adding
compounds like isopropyl β-d-1-thiogalactopyranoside (IPTG)[57]. To
ensure selection of industrially relevant mutants, it is essential to
incorporate these media-changes already at the start of the
screening-procedure.
In the chemostat-like microbioreactors, the ability to remove part of
the medium and the cells from the microbioreactor can also be used to
miniaturize sequential batch evolution experiments. When the cells are
kept in the exponential phase, such experiments select for mutants with
a higher maximum growth rate in the imposed condition and are for
example used to adapt cells to high concentrations of toxic compounds
and low pH values[58]. When the cells are pulse-fed, sequential
batch reactors can be used to select for mutants that produce large
amounts of storage-compounds[59]. Miniaturizing such experiments can
increase the number of parallel experiments that can be done, while
decreasing the costs.
Overall, microbioreactors with nutrient-feeding strategies allow for
high-throughput and low-cost cultivation in a broad range of cultivation
set-ups (batch, fed-batch, chemostat, but also retentostat and
sequential batch). Therefore, they have a high potential as new
cultivation-tool for screening and selecting mutants in industrial
biotechnology.
Data availability statement: The data that support the findings
of this study are available from the corresponding author upon
reasonable request.
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