Single-cell Bioreactors boost Bioprocess Development: New Insights into Cellular Metabolism

D. Kohlheyer, A. Grünberger, C. Probst, S. Helfrich,

J. Frunzke, L. Eggeling, K. Nöh, S. Noack, W. Wiechert

Forschungszentrum Jülich; Institute of Bio- and Geosciences - IBG-1:Biotechnology, Jülich, Germany

Cell-to-cell heterogeneity caused by biological (intrinsic) and environmental (extrinsic) fluctuations can have severe impact on the productivity of biotechnological process- es. Nevertheless, till now the complex interplay between environmental reactor dy- namics, genetic noise and cellular activity is hardly understood. Specially designed microfluidic systems offer unique possibilities to tackle these problems [1]. Aiming at metabolic engineering and bioprocess development, we developed an innovative mi- crofluidic platform for microbial single-cell investigations [2]. Different reactor types from femto- to pico-litre scale facilitate spatio temporal analysis of single microbial cells under well-defined, externally controlled and even dynamic environmental con- ditions. For the first time several central questions regarding growth, productivity, substrate uptake as well as population heterogeneity and influence of bioreactor in- homogeneity can be addressed within well-defined single-cell cultivation experiments
.

Figure 1: Growth chambers of a parallel single cell cultivation device.

The Jülich single-cell bioreactor technology platform developed in the recent years exhibits several specific advantages for applications in metabolic engineering and bioprocess development:

â?¢ sub-micrometer structures needed for bacterial investigations can be fabricated in a PDMS molding process [3] (Fig. 1)

â?¢ many single-use chips can be replicated at low cost from one master mould [3]

â?¢ new designs can be quickly realized and, thus, tailor-made chips can be adapted to the experimental questions and not vice versa [4]

â?¢ environmental conditions (temperature, pH, medium composition) can be tightly controlled and also dynamically changed on a sub second time scale [2, 5, 6]

â?¢ hundreds of single-cell bioreactors can be operated simultaneously in one chip, thus generating the required statistical data for meaningful results (Fig. 1)

â?¢ microfluidic chips are fixed on an automated x-y stage to enable high throughput microscopy including fluorescence imaging [2, 5, 6, 7] (Fig. 2)

â?¢ hundreds of interlaced time-lapse videos can be automatically evaluated by a specially developed image analysis pipeline (Fig. 2)

Figure 2: Singke cell analysis of L-valine production strains of Corynebacterium glutamicum.

The following applications from our institute demonstrate that microfluidics has be- come mature as a highly valuable tool to understand hitherto non understood meta- bolic phenomena:
â?¢ Strain characterization: Growth, one of the most important performance indicators in biotechnological production processes, is still one of the â??most underexploited assays of cellular heterogeneity studies to dateâ? [8]. The link between metabolism and growth or morphology is currently intensely investigated [9]. Microfluidics in combination with image analysis produces precise lineage-trees and single-cell growth curves revealing population inhomogeneity and cell aging effects [10]. Strong differences in the growth behavior of E. coli [10] and C. glutamicum pro- duction strains could be revealed [6].
â?¢ Population heterogeneity: Genetically encoded fluorescence biosensors and re- porter systems are ideal tools for time-lapse imaging during microfluidic single-cell analysis (Fig. 2). In particular transcriptional regulator based metabolite reporters proved to be a versatile tool in monitoring intracellular metabolites [5, 6, 7, 11]. These reporter systems transform intracellular metabolite concentrations into a detectable fluorescence readout and were used to investigate single-cell produc- tion of amino acids at various environmental conditions. Interestingly, under cer- tain complex environmental cultivation conditions, isogenic micro colonies split up into to producing and non-producing sub populations. Other microfluidic investiga- tion aims at spontaneous prophage induction in C. glutamicum strains and its in- fluence to bioprocess performance [12].
â?¢ Process characterization: C. glutamicum strains exhibiting 1.5 fold improved growth rates were analyzed during microfluidic cultivations [13]. The bioreactor environment could be simulated on chip by applying bioreactor supernatant from distinct cultivation time points. In combination with extensive substrate screening experiments, the constant availability of protocatechuate (PCA), typically added as iron chelator, was identified to be responsible for the elevated growth rates [14]. This reveals that microbial diets in industrial bioreactors can strongly differ from the wanted substrate usage. Thus, using microfluidic chips single-cell per- formance can be allocated to specific process conditions.

â?¢ Cell manipulation for strain screening: Till now most single-cell microbial cultiva- tion systems suffer from limited control during cell inoculation and sampling meth-

ods during cultivation. To enable individual cell selection and manipulations we combined laser tweezers with microfluidic cell cultivation environments specifically tailored for micrometre sized bacteria. For the first time a filamentous E. coli WT (MG1655) was safely relocated from its growing microcolony by laser manipula- tions. The cell was transferred to an empty cultivation site allowing single cell growth and morphology investigations [15].
Summarizing, the advent of tailor-made single-cell bioreactors which can precisely simulate bioprocess relevant conditions and allow the exact monitoring of growth, morphology and biosensor signals related to metabolism and gene expression will most certainly become an indispensable tool for strain characterization and screen- ing, metabolic engineering and bioprocess development.

[1] Grünberger, A,: Wiechert, W.; Kohlheyer, D.

Single-Cell Microfluidics: Opportunity for Bioprocess Development. Current Opinion Biotechnology. (2014) In Press.

[2] Grünberger, A.; Paczia, N.; Probst, C.; Schendzielorz, G. ; Eggeling, L. ; Noack, S.; Wiechert, W.; Kohlheyer, D.

A disposable picolitre bioreactor for cultivation and investigation of industrially relevant bacteria on the single cell level.

Lab on a Chip 12, 2060 - 2068 (2012)

[3] Grünberger, A.; Probst, C.; Heyer, A.; Wiechert, W.; Frunzke, J.; Kohlheyer, D.

Microfluidic Picoliter Bioreactor for Microbial Single-cell Analysis: Fabrication, System Setup, and

Operation.

Journal of Visualized Experiments 82, 50560 (2013)

[4] Probst, C.; Grünberger, A.; Wiechert, W.; Kohlheyer, D.

Polydimethylsiloxane (PDMS) Sub-Micron Traps for Single-Cell Analysis of Bacteria. Micromachines 4(4), 357 - 369 (2013)

[5] Mustafi, N.; Grünberger, A. ; Kohlheyer, D. ; Bott, M.; Frunzke, J.

The development and application of a single-cell biosensor for the detection of L-methionine and branched-chain amino acids.

Metabolic Engineering 14, 449 - 457 (2012)

[6] Mustafi, N.. ; Grünberger, A. ; Mahr, R.; Helfrich, S.; Nöh, K.; Blombach, B. ; Kohlheyer, D.; Frunzke, J.

Application of a Genetically Encoded Biosensor for Live Cell Imaging of L-Valine Production in Py- ruvate Dehydrogenase Complex-Deficient Corynebacterium glutamicum Strains.

PLoS One 9(1), e85731 (2014)

[7] Schallmey, M.; Frunzke, J.; Eggeling, L.; Marienhagen, J.;

Looking for the pick of the bunch: high-throughput screening of producing microorganisms with bi-

osensors.

Current Opinion in Biotechnology 26, 148-154 (2014).

[8] Lecault,V.; White, A.K.; Singhal, A.; Hansen, C.L.; Microfluidic single cell analysis: from promise to practice. Current Opinion in Chemical Biology 16, 381-390 (2012).

[9] Klumpp, S.; Hwa, T.;

Bacterial growth: global effects on gene expression, growth feedback and proteome partition. Current Opinion in Biotechnology 28, 96â??102 (2014).

[10] Wang, P.; Robert, L.; Pelletier, J.; Dang, W.L.; Taddei, F.; Wright, A.; Jun, S.

Robust growth of Escherichia coli. Current Biology 20, 1099-1103 (2010).

[11] Schendzielorz, G.; Dippong, M.; Grünberger, A.; Kohlheyer, D.; Yoshida, A. ; Binder, S.; Nishiyama, C. ; Nishiyama, M. ; Bott, M.; Eggeling, L.

Taking Control over Control: Use of Product Sensing in Single Cells to Remove Flux Control at

Key Enzymes in Biosynthesis Pathways.

ACS Synthetic Biology. 130715122931005 - (2013)

[12] Nanda, A.; Heyer, A.; Kramer, C.; Grünberger, A.; Kohlheyer, D.; Frunzke, J.

Analysis of SOS-Induced Spontaneous Prophage Induction in Corynebacterium glutamicum at the

Single-Cell Level.

Journal of Bacteriology 196(1), 180 - 188 (2014)

[13] Grünberger, A.; van Ooyen, J.; Paczia, N.; Rohe, P.; Schendzielorz, G.; Eggeling, L.; Wiechert, W.; Kohlheyer, D.; Noack, S.

Beyond growth rate 0.6: Corynebacterium glutamicum cultivated in highly diluted environments. Biotechnology & Bioengineering 110(1), 220 - 228 (2013)

[14] Unthan, S.; Grünberger, A.; van Ooyen, J.; Gätgens, J.; Heinrich, J.; Paczia, N.; Wiechert, W.; Kohlheyer, D.; Noack, S.

Beyond growth rate 0.6: What drives Corynebacterium glutamicum to higher growth rates in de- fined medium?

Biotechnology & Bioengineering 111(2), 359â??371 (2014)

[15] Probst, C.; Grünberger, A.; Wiechert, W.; Kohlheyer, D.

Microfluidic growth chambers with optical tweezers for full spatial single-cell control and analysis of evolving microbes.

Journal of microbiological methods 95(3), 470 - 476 (2013)