# Online Monitoring of Chip-Based Microscale Perfusion Fermentations

## TL;DR

A new platform for online monitoring of microscale fermentations allows efficient tracking of metabolites and productivity in small-volume bioreactors.

## Contribution

A compact, commercially available online analytical platform for microscale perfusion fermentations is developed and demonstrated.

## Key findings

- The platform enables uninterrupted monitoring of metabolites in parallel fermentations without blockages or cross-contamination.
- Fermentations of lactic-acid-producing Saccharomyces cerevisiae strains were continuously monitored over 5–24 hours.
- The system uses minimal cell culture media (<1.5 mL over 24 h) and is adaptable for various biotechnology products.

## 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.