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. 2025 Oct 1;10(40):47355-47362.
doi: 10.1021/acsomega.5c06552. eCollection 2025 Oct 14.

Online Monitoring of Chip-Based Microscale Perfusion Fermentations

Sabrina M Cramer  1   2 , Shubham Gurav  1   3 , David Glinsner  1   3 , Sven Kochmann  1   2 , Diethard Mattanovich  1   3 , Stephan Hann  1   2 , Tim Causon  1   2
Affiliations

Online Monitoring of Chip-Based Microscale Perfusion Fermentations

Sabrina M Cramer et al. ACS Omega. .

Abstract

As part of established biomanufacturing development, screening and early phase bioprocess development occurs at bench scale (microplates and shake flasks) whereby conventional offline sampling can only provide limited feedback on fermentation bioprocess parameters including strain productivity. To address these limitations, a new sensitive and selective online analytical platform consisting entirely of commercially available components with a small footprint (valves, 2DLC hardware, LC separation, and online tandem mass spectrometry) was developed for online monitoring of chip-based microbioreactors. Fermentations of microbial cell factories (Saccharomyces cerevisiae) were cultivated in 20 μL bioreactors, requiring perfusion of cell culture media at low μL/min rates delivered by syringe pump modules, operated in a multiplexed configuration with a flow-through stream selection valve, and monitored with a 2DLC-MS/MS system adapted for microscale operation. This allows uninterrupted multiplexed microperfusions to be monitored with online measurements of metabolites from parallel fermentations without the occurrence of blockages or cross-contamination between independent fermentations. Fermentations of lactic-acid-producing strains ofS. cerevisiaewere continuously monitored over 5-24 h, demonstrating the suitability of the platform for online monitoring of product quantity and key metabolites for fermentation biotechnology. Offering minimal consumption of biological material and using <1.5 mL of cell culture media over 24 h per experiment, this new platform can be used for monitoring a broad range of biomolecules, rapid strain selection, and screening of microenvironmental factors and is adaptable for targeting other key biotechnology products.

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Figures

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1
Representation of the sampling time required to park one cut (i.e., collect one fraction in a sample loop) for (A) the range of 5–20% sample loop filling according to microperfusion flow rate delivered by the 1D pump. Both 40 μL (formula image) and 10 μL sample loops (formula image) were considered. The upper boundaries represent 20% loop filling (formula image for 40 μL loops and formula image for 10 μL loops) and the lower boundaries are for 5% loop filling (formula image for 40 μL loops and formula image for 10 μL loops). Typical microfluidic perfusion rates of P = 1, 0.5, 0.25 h–1 are indicated by dashed lines. (B) Zoomed in region of relevant perfusion rates for the present work. The formula image line represents the practical limit imposed on the system due to the contribution of transfer capillary volume.
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Schematic representation of the full 2DLC-MS/MS analytical μ-platform. The multiplexed perfusion fermentations in microbioreactors are performed at 37 °C inside a microchip incubator with a syringe pump delivering cell medium at 1 μL/min. An additional syringe pump is used to deliver the wash flow at 10 μL/min to flush the flow path after each measurement. The 10-port, 5-position flow-through stream selection (FSS) valve is used to switch between independent fermentations and wash without interruption of flow in any microchip chambers. The 2DLC hardware (ASM valve and Deck A sampling loops) is used to collect sequential fractions from each perfusion fermentation being monitored. Collected fractions are transferred onto the LC column from the sample loop on Deck A using a 100 μL/min flow rate from the 2D pump and analyzed by MS/MS. The effluent from fermentations not actively being transferred to the ASM valve (formula image) can be transferred to other analytical modules or discarded to waste. See Figure S3 for detailed schematics of all valve operations. (Instrumentation images reproduced with permission courtesy of Agilent Technologies, Inc.).
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Results for metabolites monitored over 5 h fermentations ofS. cerevisiae. The peak area for each metabolite was measured every hour for lactate (A), isoleucine/leucine (B), methionine (C), phenylalanine (D), and tyrosine (E) during concurrent 20 μL perfusion fermentations with 1a (▲), theS. cerevisiaestrain with lower production of lactate, and 1e (●), the higher-producing strain. For each time point, both fermentations were measured in duplicate. The shaded areas represent the standard deviation of the duplicates using a correction factor to compensate for the small sample size, as proposed by Roesslein et al. .
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Comparison of lactate production in the 1a (low-producing) and 1e (high-producing) strains at different pH. The cell media was buffered at pH 3 or 5 for the 8 h perfusion fermentations with an analysis rate of five sequential cuts every hour. Lactate peak area for each strain was compared across 5 h (two biological replicates per strain measured at 4 time points during the fermentation) at pH 3 and 5 after stabilization in lactate production. Statistical analysis comparing the two strains at pH 3 and 5 was performed with a two-sided Welch Two Sample t test with unequal variance. **** represents a significance level of p < 0.0001.
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