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

Gruenberger, Alexander; Probst, Christopher; Heyer, Antonia; Wiechert, Wolfgang; Frunzke, Julia; Kohlheyer, Dietrich

doi:10.3791/50560-v

Cited by 2 publications

(2 citation statements)

References 20 publications

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“…A silicon wafer mold was fabricated with a two‐layer photolithography process and served as a mold for PDMS (poly(dimethylsiloxane)) soft lithography (Grünberger et al, 2013). The construction and development of a siliconwafer was carried out according to the protocol of Täuber et al (2020).…”

Section: Methodsmentioning

confidence: 99%

Experimental k_S estimation: A comparison of methods for Corynebacterium glutamicum from lab to microfluidic scale

Steinhoff

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Osthege

et al. 2023

Biotech & Bioengineering

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Knowledge about the specific affinity of whole cells toward a substrate, commonly referred to as kS, is a crucial parameter for characterizing growth within bioreactors. State‐of‐the‐art methodologies measure either uptake or consumption rates at different initial substrate concentrations. Alternatively, cell dry weight or respiratory data like online oxygen and carbon dioxide transfer rates can be used to estimate kS. In this work, a recently developed substrate‐limited microfluidic single‐cell cultivation (sl‐MSCC) method is applied for the estimation of kS values under defined environmental conditions. This method is benchmarked with two alternative microtiter plate methods, namely high‐frequency biomass measurement (HFB) and substrate‐limited respiratory activity monitoring (sl‐RA). As a model system, the substrate affinity kS of Corynebacterium glutamicum ATCC 13032 regarding glucose was investigated assuming a Monod‐type growth response. A kS of <70.7 mg/L (with 95% probability) with HFB, 8.55 ± 1.38 mg/L with sl‐RA, and 2.66 ± 0.99 mg/L with sl‐MSCC was obtained. Whereas HFB and sl‐RA are suitable for a fast initial kS estimation, sl‐MSCC allows an affinity estimation by determining tD at concentrations less or equal to the kS value. Thus, sl‐MSCC lays the foundation for strain‐specific kS estimations under defined environmental conditions with additional insights into cell‐to‐cell heterogeneity.

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Section: Methodsmentioning

confidence: 99%

Experimental k_S estimation: A comparison of methods for Corynebacterium glutamicum from lab to microfluidic scale

Steinhoff

Finger

Osthege

et al. 2023

Biotech & Bioengineering

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“…Then inlets and outlets for the fluid and the gas flow connections were manually punched using a punching tool (ϕ = 0.75 mm, World Precision Instruments, USA). Single PDMS chips were finally bonded to glass substrates (D263 ® Bio, 39.5 mm × 34.5 mm × 0.175 mm, Schott AG, Germany) after an O 2 plasma treatment (Femto Plasma Cleaner, Diener Electronics, Germany) for 25 s. For full fabrication details, the reader is referred to Gruenberger et al ( 2013 ) and Täuber et al ( 2021 ).…”

Section: Materials and Equipmentmentioning

confidence: 99%

Enabling oxygen-controlled microfluidic cultures for spatiotemporal microbial single-cell analysis

et al. 2023

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Microfluidic cultivation devices that facilitate O2 control enable unique studies of the complex interplay between environmental O2 availability and microbial physiology at the single-cell level. Therefore, microbial single-cell analysis based on time-lapse microscopy is typically used to resolve microbial behavior at the single-cell level with spatiotemporal resolution. Time-lapse imaging then provides large image-data stacks that can be efficiently analyzed by deep learning analysis techniques, providing new insights into microbiology. This knowledge gain justifies the additional and often laborious microfluidic experiments. Obviously, the integration of on-chip O2 measurement and control during the already complex microfluidic cultivation, and the development of image analysis tools, can be a challenging endeavor. A comprehensive experimental approach to allow spatiotemporal single-cell analysis of living microorganisms under controlled O2 availability is presented here. To this end, a gas-permeable polydimethylsiloxane microfluidic cultivation chip and a low-cost 3D-printed mini-incubator were successfully used to control O2 availability inside microfluidic growth chambers during time-lapse microscopy. Dissolved O2 was monitored by imaging the fluorescence lifetime of the O2-sensitive dye RTDP using FLIM microscopy. The acquired image-data stacks from biological experiments containing phase contrast and fluorescence intensity data were analyzed using in-house developed and open-source image-analysis tools. The resulting oxygen concentration could be dynamically controlled between 0% and 100%. The system was experimentally tested by culturing and analyzing an E. coli strain expressing green fluorescent protein as an indirect intracellular oxygen indicator. The presented system allows for innovative microbiological research on microorganisms and microbial ecology with single-cell resolution.

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

Cited by 2 publications

References 20 publications

Experimental k_S estimation: A comparison of methods for Corynebacterium glutamicum from lab to microfluidic scale

Experimental k_S estimation: A comparison of methods for Corynebacterium glutamicum from lab to microfluidic scale

Enabling oxygen-controlled microfluidic cultures for spatiotemporal microbial single-cell analysis

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

Cited by 2 publications

References 20 publications

Experimental kS estimation: A comparison of methods for Corynebacterium glutamicum from lab to microfluidic scale

Experimental kS estimation: A comparison of methods for Corynebacterium glutamicum from lab to microfluidic scale

Enabling oxygen-controlled microfluidic cultures for spatiotemporal microbial single-cell analysis

Contact Info

Product

Resources

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Experimental k_S estimation: A comparison of methods for Corynebacterium glutamicum from lab to microfluidic scale

Experimental k_S estimation: A comparison of methods for Corynebacterium glutamicum from lab to microfluidic scale