Widespread applications of shake flask off-gas analysis across biotechnology

Introduction: the need for advanced monitoring in shake flasks

Shake flasks have been a cornerstone of biotechnological research for nearly a century and remain one of the most commonly used cultivation systems for early-stage bioprocess development today. Despite the emergence of more sophisticated bioreactor systems, shake flasks continue to play a crucial role in microbial, fungal and mammalian cell culture research, as they offer cost efficiency, ease of handling and high experimental throughput. A recent review highlighted that the number of scientific publications mentioning shake flasks has been steadily increasing, with more than 20,000 papers per year referencing their use in screening and process development studies (Brauneck et al, 2025).

However, while shake flasks are widely used, early-stage process development at this scale often suffers from a lack of process insights. Many processes are still operated as black boxes, where key parameters such as oxygen transfer, carbon dioxide evolution and metabolic activity remain unknown due to limited monitoring capabilities. This can lead to unforeseen challenges in later-scale bioreactor experiments, where process conditions change dramatically. For instance, oxygen limitation in shake flasks — caused by inadequate gas exchange or incorrect shaking conditions — can falsely select for strains or conditions that perform well under oxygen stress but fail under well-aerated industrial conditions (Brauneck et al, 2025).

To ensure successful bioprocess development, it is essential to obtain quantitative process knowledge even at the small scale. Understanding oxygen transfer rates (OTR), carbon dioxide transfer rates (CTR) and respiration quotient (RQ) can provide critical insights into cellular metabolism, helping researchers identify optimal growth conditions, metabolic shifts and potential bottlenecks early in the development pipeline.

This review provides an overview of shake flask off-gas analysis and focuses on applications across biotechnology.

Technical principle

One approach for off-gas analysis for shake flask scale is the Kohner TOM system. It utilises a bypass-based design, which allows continuous measurement of gas exchange without interfering with the shaking motion of the culture flasks. Unlike direct sensor placement inside or on top of the cultivation vessel, this design ensures stable measurement conditions while maintaining the natural gas transfer dynamics of shake flask systems (Schulte et al, 2025).

Applications and case studies

Mammalian cell culture and biopharmaceutical applications

1. CHO cell cultivation and scale-down modelling

CHO cells are widely used for biopharmaceutical production, and process conditions at small scales must match those at production scale. Ihling et al (2023) used shake flask off-gas analysis to refine a scale-down model where OTR-based monitoring ensured oxygenation conditions in microtitre plates resembled those in larger shake flasks, improving process comparability. Additionally, Neuss et al (2024) investigated the impact of hydromechanical stress on CHO metabolism, using shake flask off-gas analysis to correlate oxygen uptake with productivity, helping define optimal agitation conditions for cell growth and monoclonal antibody production (Ihling et al, 2023; Neuss et al, 2024).

2. Assessing metabolic load in monoclonal antibody production using shake flask off-gas analysis

Monoclonal antibody (mAb) production in CHO cells imposes a metabolic burden, often reducing growth rates and productivity. Reyes et al (2025) used shake flask off-gas analysis to monitor metabolic responses during recombinant protein expression. Induced CHO cells exhibited slower growth, likely due to the energy demand of protein synthesis. Off-gas analysis detected metabolic shifts early, particularly the transition to the stationary phase, helping optimise induction timing. Delaying induction and adjusting temperature led to a twofold increase in viable cell density and a 30% higher mAb yield. This study demonstrates how real-time respiration monitoring can refine process control, minimise metabolic stress and enhance antibody production efficiency.

3. Predicting key culture parameters (glucose, lactate, VCD) in mammalian cell cultures

Monitoring glucose consumption, lactate accumulation and viable cell concentration in CHO cell cultures traditionally requires frequent sampling, which is time-consuming and provides only intermittent data. Ihling et al (2022) demonstrated that by tracking oxygen transfer rate (OTR) in real time, these key parameters can be predicted without manual intervention. This non-invasive approach reduces the need for offline measurements while providing continuous insights into metabolic activity. It allows for early detection of metabolic shifts, improving process control and reproducibility in mammalian cell cultures — an essential step for biopharmaceutical production and scale-up.

4. Cytotoxicity assessment in mammalian bioprocesses

Traditional cytotoxicity assays rely on endpoint measurements, missing real-time metabolic changes. Ihling et al (2022) used shake flask off-gas analysis to continuously track OTR in CHO cells exposed to toxic compounds, enabling early detection of metabolic shifts and determination of IC50 values based on respiration activity. This approach provided a faster and more reliable alternative to conventional assays (Ihling et al, 2022).

Microbial and fungal biotechnology for bio-based compounds

5. Optimising itaconic acid production with microbial co-cultures

Itaconic acid is an important bio-based platform chemical used in polymer production and biofuels. A study by Schlembach et al (2020) used shake flask off-gas analysis to monitor a synthetic microbial consortium of Trichoderma reesei and Ustilago maydis, where T. reesei secreted cellulases to break down cellulose, providing sugars for U. maydis to produce itaconic acid. By analysing oxygen uptake and carbon dioxide evolution, shake flask off-gas analysis helped optimise metabolic interactions between the two species, leading to a titre of 34 g/L under optimised conditions (Schlembach et al, 2020).

6. Improving mycofactocin production in Mycobacterium smegmatis

Mycofactocins are redox cofactors with industrial applications. Peña et al (2020) used shake flask off-gas analysis to monitor oxygen uptake rates during fermentation, identifying optimal aeration conditions that maximised mycofactocin biosynthesis while avoiding oxidative stress. The study demonstrated that oxygen transfer efficiency directly influenced yield, showing the value of off-gas analysis for optimising metabolic pathways (Peña et al, 2020).

7. Enhancing psilocybin production in fungal cultures

Psilocybin, a promising compound for treating psychiatric disorders, is traditionally extracted from wild mushrooms, but biotechnological production using engineered fungi offers a scalable alternative. Janevska et al (2024) used shake flask off-gas analysis to monitor oxygen uptake in Aspergillus nidulans, a host engineered for high-yield psilocybin biosynthesis. By tracking respiration activity in real time, researchers were able to adjust aeration strategies to maintain optimal metabolic conditions, preventing oxygen limitations that could reduce yield. These optimisations led to a psilocybin concentration of 267 mg/L, demonstrating the potential of shake flask off-gas analysis for enhancing secondary metabolite production in fungal fermentations.

8. Optimising jagaricin production using shake flask off-gas analysis

Jagaricin, an antifungal cyclic lipopeptide, is produced by Janthinobacterium agaricidamnosum and has potential applications in medicine and agriculture. Schlosser et al (2021) used shake flask off-gas analysis to monitor oxygen uptake and carbon dioxide evolution during shake flask cultivations, optimising conditions for higher jagaricin yields. By tracking oxygen transfer rates (OTR), the study revealed that temporary oxygen limitation enhanced production, while adjusting carbon sources and phosphate levels led to a 458% increase in yield. Shake flask off-gas analysis also supported process scale-up, ensuring consistent metabolic activity from shake flasks to stirred-tank bioreactors.

Synthetic biology and metabolic engineering

9. Cannabinoid precursor production in amoebae

Scaling up alternative hosts for bioproduct synthesis is a major challenge. Kufs et al (2022) demonstrated how shake flask off-gas analysis can assist in scaling up Dictyostelium discoideum fermentations for olivetolic acid production, a precursor for cannabinoids. By monitoring hydromechanical stress and oxygen supply, researchers successfully scaled the process from shake flasks to a 300 L stirred tank reactor without yield reduction from shear stress (Kufs et al, 2022).

10. Controlling metabolic fluxes with optogenetics

Optogenetics is emerging as a tool to dynamically regulate metabolic pathways. Chen et al (2021) engineered a photo-switchable isocitrate dehydrogenase (IDH) in Saccharomyces cerevisiae, allowing researchers to modulate the citric acid cycle with light. Shake flask off-gas analysis was crucial for evaluating metabolic responses to optogenetic activation, providing insights into real-time metabolic control strategies (Chen et al, 2021).

Biopolymers and biosurfactants for industrial applications

11. Alginate production in Azotobacter vinelandii

Alginate, a biopolymer with applications in wound dressings and food additives, is sensitive to medium composition. A study by Sparviero et al (2023) used shake flask off-gas analysis to track respiration dynamics and oxygen uptake rates, revealing that different yeast extracts significantly influence alginate production efficiency. This highlights the importance of media selection and process control in biopolymer fermentations (Sparviero et al, 2023).

12. Foam-free biosurfactant production in Pseudomonas putida

Biosurfactant production, such as rhamnolipids, often suffers from excessive foaming, complicating bioreactor operations. Weiser et al (2022) used shake flask off-gas analysis to develop a pressurised, foam-free bioprocess for the production of 3-(3-hydroxyalkanoyloxy) alkanoic acid (HAA), a rhamnolipid precursor. The study showed that headspace aeration combined with overpressure allowed efficient oxygen transfer without excessive foam formation, making large-scale production more feasible (Weiser et al, 2022).

13. Biosurfactant production in marine bacteria

Biosurfactants play a role in bioremediation and oil spill clean-up. Cui et al (2022) investigated Alcanivorax borkumensis, a marine bacterium that produces glycine-glucolipid biosurfactants to facilitate alkane degradation. Using shake flask off-gas analysis, they tracked oxygen consumption and metabolic shifts between different carbon sources, identifying optimal conditions for biosurfactant production and hydrocarbon breakdown (Cui et al, 2022).

Environmental and sustainable bioprocesses

14. Biocalcification and carbon sequestration studies

Microbial-induced calcite precipitation (MICP) is an innovative biotechnology with applications in carbon sequestration, self-healing concrete and soil stabilisation. Lapierre et al (2020) used shake flask off-gas analysis to monitor oxygen uptake in Sporosarcina pasteurii, a bacterium known for its ability to precipitate calcium carbonate. By tracking real-time respiration activity, researchers were able to fine-tune nutrient formulations and aeration conditions, leading to improved bacterial growth and enhanced calcite precipitation. These optimisations helped increase process efficiency, making MICP a more viable and scalable solution for improving construction materials.

Conclusion

A key advantage of shake flask off-gas is its ability to provide continuous insights into respiration dynamics, reducing the need for frequent manual sampling and allowing for early detection of metabolic shifts. It has proven to be a versatile and valuable tool across multiple fields of biotechnology, enabling precise real-time monitoring of metabolic activity in both mammalian and microbial systems.

The broad range of applications covered in this article — from biopharmaceutical development and synthetic biology to biopolymer production and environmental sustainability — demonstrates the adaptability of shake flask off-gas for process optimisation in diverse biotechnological workflows. To learn more, contact Capella Science on (02) 9575 7512 or sales@capellascience.com.au.

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_{12. Kufs, J. E., Reimer, C., Steyer, E., Valiante, V., Hillmann, F., & Regestein, L. (2022). Scale-up of an amoeba-based process for the production of the cannabinoid precursor olivetolic acid. Microbial Cell Factories, 21(217).}

_{13. Chen, H., Mulder, L., Wijma, H. J., Wabeke, R., Vila Cha Losa, J. P., Rovetta, M., & Heinemann, M. (2021). A photo-switchable yeast isocitrate dehydrogenase to control metabolic flux through the citric acid cycle. bioRxiv}

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_{16. Cui, J., Hölzl, G., Karmainski, T., Tiso, T., Kubicki, S., Thies, S., Blank, L. M., Jaeger, K.-E., & Dörmann, P. (2022). The glycine-glucolipid of Alcanivorax borkumensis is resident to the bacterial cell wall. Applied and Environmental Microbiology, 88(16), e01126-22.}

_{17. Lapierre, F. M., Schmid, J., Ederer, B., Ihling, N., Büchs, J., & Huber, R. (2020). Revealing nutritional requirements of MICP-relevant Sporosarcina pasteurii DSM33 for growth improvement in chemically defined and complex media. Scientific Reports, 10:22448.}

Image ©Kuhner Shaker