SIMPLE MICROFLUIDICS FOR COMPLEX ORGANISMS:
A MICROFLUIDIC CHIP SYSTEM FOR GROWTH AND
MORPHOGENESIS STUDIES OF FILAMENTOUS FUNGI
Alexander Grünberger1, Katja Schmitz2, Christopher Probst1,
Wolfgang Wiechert1,2, Stephan Noack2, and Dietrich Kohlheyer1*
1
Microscale Bioengineering Group, 2Bioprocesses and Bioanalytics Group,
IBG-1: Biotechnology, Forschungszentrum Jülich GmbH (Juelich Research Centre), Germany
ABSTRACT
Filamentous fungi are of special interest for the production of various economically important metabolites. Up to now
only a limited number of microfluidic systems for handling fungal organisms have been reported. Here a simple and
disposable microfluidic chip-system for high-throughput growth and morphogenesis studies of filamentous fungi is
presented. The device enables trapping of multiple single fungal cell forms at different development stages and allows for
real-time growth analysis under constant environmental cultivation conditions. As a model organism the industrially
relevant production organism Penicillium chrysogenum was investigated. We analyzed different morphological stages,
ranging from simple spores, over multi-branched hyphal structures to complex mycelia. This allowed for quantitative
measurements of industrial important parameters such as the specific growth rate. The presented work paves the way for
future investigations on cell-to-cell heterogeneity as well as advanced morphology engineering of fungal systems.
KEYWORDS: PDMS, single cell analysis, bioprocess optimization, filamentous fungi, Penicillium chrysogenum
INTRODUCTION
Filamentous fungi are of special interest as “industrial cell factories” for various economically important metabolites
used for detergents, food, beverages and pharmaceutical compounds. Unfortunately, the morphogenesis of filamentous
microorganisms is often the bottleneck for productivity in industrial production processes [1]. To improve bioprocesses, a
better understanding of the underlying mechanisms inducing fungal morphogenesis is required.
Microfluidic systems enable the cultivation of microcolonies and single cells of different organisms at constant
environmental conditions [2]. This allows specific studies of various substrates affecting growth and morphology.
Microfluidic systems were reported for several eukaryotic cells (including yeast) and bacteria [3]. All these systems have
in common that cells are trapped inside defined structures to observe them during cultivation. This is possible, due to the
low variability of size and shape of all these organisms during growth and division.
Unfortunately, filamentous fungi significantly change their size and morphology during a typical life-span. Fungal
growth starts from simple spherical spores (typically smaller than 10 µm in diameter) which then form germ tubes
developing into multi-branched hyphal structures (< 100 µm). Finally, these structures can form complex mycelia, which
can reach sizes up to several millimeters in diameter. So far only few microfluidic systems for the analysis of single fungi
have been reported. The first system described by Spohr et al. [4] was used for the online monitoring of fungal growth.
Advances in automated life-cell imaging and microfluidic fabrication techniques, led to the development of advanced
microfluidic systems for fungi. Nicolau and coworkers presented a device for probing dynamic behavior of filamentous
fungi in a microfluidic system [5]. A first microfluidic device for growth analysis of spores was reported recently [6],
focusing on the investigation of spore germination at different environmental conditions.
CHIP DESIGN AND WORKING PRINCIPLE
A microfluidic system suitable for systematic studies of fungal growth needs to be elastic in terms of the applied
material to cope with the continuous changes in shape and size. The chip necessitates the immobilization of various cell
shapes for microscopic studies, while keeping optimal environmental conditions enabling long-term cultivation.
Figure 1: PDMS-glass microfluidic perfusion system for the cultivation of filamentous fungi. (A+B) Illustration of the device and
cultivation principle. (C) Bird perspective of the chip system after several days of cultivation. (D) 100 x magnification of P.
chrysogenum pellet after several days of cultivation.
978-0-9798064-6-9/µTAS 2013/$20©13CBMS-0001
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17th International Conference on Miniaturized
Systems for Chemistry and Life Sciences
27-31 October 2013, Freiburg, Germany
Therefore, we have developed a simple microfluidic system, containing a glass plate and a PDMS slab with a single
inlet and outlet (Figure 1). Cells are pipetted onto the glass plate before the PDMS slab is sealed on it. Spores are
sandwiched between the glass plate and PDMS slab, and growth can be followed until mycelia and pellets are formed
(Figure 1A). During cultivation the growth and morphological changes lead to local deformation of the elastic PDMS
ceiling and thus the fungus itself defines the height of the cultivation region (Figure 2B). This allows trapping, cultivation
and time-lapse imaging of the growing fungus at different development stages.
Figure 2: Schematic illustration of different morphological development stages during cultivation inside the described chip. (A)
Typical morphological states during fungal growth; (B) Trapping and cultivation of a fungus in different morphological states within
the flexible PDMS glass device.
EXPERIMENTAL RESULTS AND DISCUSSION
As model organism the industrially relevant production organism P. chrysogenum was analyzed. Growth over a whole
live time at different morphological development stages was followed during cultivation. Figure 3A displays the volume
growth of swelling spores. Here a maximum growth rate of µmax,spore swelling = 0.19 ± 0.02 h-1 (Figure 3B) was determined.
Figure 3: Quantitative growth analysis of P. chrysogenum spore swelling. (A) Time-lapse pictures of spores during swelling
(I+II) and germination (III). (B) Volume increase of three individual spores.
Figure 4A illustrates hyphal growth after the initiation of germination. Image sequences were analyzed to determine
growth rates based on the cell area. The maximum growth rate of µmax,hyphal area = 0.1 ± 0.02 h-1, is slightly higher than
during typical bioreactor cultivations (µmax = 0.06 ± 0.02 h-1 [7]), indicating that the developed device is biocompatible.
Apart from growth rates, other valuable information such as the hyphal tip elongation rate (Figure 4B) as well as septum
formation events (see Figure 4A right), can be derived.
Figure 4: Time-lapse analysis of single P. chrysogenum hyphal growth. (A) Representative picture sequence showing tip
extension and hyphal growth. (B) Tip extension rate of P. chrysogenum.
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Figure 5 shows the development of higher-order structures following continuous hyphal growth and morphogenesis.
Starting from multi-branched structures, P. chrysogenum formed complex mycelia (Figure 5A) which then further
developed into densely packed pellets (Figure 5B).
Figure 5: Development stages of higher-order structures of P. chrysogenum. (A) Mycelium formation, starting from a branched
hyphal structure; (B) Pellet formation starting from a mycelium.
CONCLUSIONS
We have described a microfluidic system for real-time growth and morphogenesis studies of filamentous fungi. The
device enables comprehensive growth analysis during all morphological development stages, and thus offers new
opportunities for investigating related industrial processes. This includes the real-time analysis of fungal responses
(associated with growth and morphology changes) to altering cultivation parameters such as temperature, pH and medium
composition. Combined with automated time-lapse microscopy and novel image analysis methods [8], statistically
reliable data can be complied, leading to an improved understanding of fungal growth and development. Finally, the
presented system opens up new possibilities for characterizing fungal producer strains under well-defined conditions
related to large-scale bioprocesses.
ACKNOWLEDGEMENTS
This work was partly performed at Helmholtz Nanoelectronic Facility (HNF) of Forschungszentrum Jülich GmbH.
We acknowledge their help and support. We also want to express our gratitude to Sandoz (Sandoz GmbH,Kundl/Tyrol,
Austria) for supplying us with P. chrysogenum spores.
REFERENCES
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[6] Demming, S., et al., Disposable parallel poly(dimethylsiloxane) microbioreactor with integrated readout grid for
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[7] Schmitz, K., et al., Simultaneous utilization of glucose and gluconate in Penicillium chrysogenum during overflow
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[8] Posch, A.E., et al., A novel method for fast and statistically verified morphological characterization of filamentous
fungi. Fungal Genetics and Biology, 2012. 49(7): p. 499-510.
CONTACT
*D. Kohlheyer, phone: +49-2461-2875; E-mail: [email protected]
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