Smart fermentation for aquaculture: How a VOC sensor could revolutionize the production of innovative tilapia vaccines

Dr. Ansgar Stratmann (W42 Industrial Biotechnology) with the fermSYS - CUBE in the laboratory

Global aquaculture is growing rapidly – and with it the challenge of effectively protecting fish stocks from viral and bacterial diseases. In tilapia farming in particular, pathogenic viruses and bacteria cause significant economic losses. One promising solution is the development of novel, orally administered vaccines based on recombinant yeast.

In this development project by W42 Industrial Biotechnology, Komagataella phaffii (formerly Pichia pastoris) is being used as a production platform for a multivalent vaccine against two major viral fish pathogens. What makes this project unique is that the yeast cells themselves are used as a functional component of fish feed following fermentation. This means that the yeast serves simultaneously as the production cell, the packaging, and a natural immune booster.

But how can such a process be monitored efficiently? This is where real-time measurement of volatile organic compounds (VOCs) comes into play.

Vaccine production in yeast: a “whole-cell-vaccine” approach

The aim of the project is to produce artificially designed proteins that combine several immunologically relevant epitopes from two viral pathogens affecting tilapia. The epitopes are selected using rational design strategies and AI-assisted analyses.

The resulting multi-epitope protein is expressed in Komagataella phaffii. After cultivation, the yeast cells are harvested, dried and mixed directly into the fish feed.

Advantages of this approach

• No complex protein purification required

• Can be given orally

• The yeast cell wall acts as a natural immune booster and helps to build a strong protective response.

• Lower production costs

• Scalable for industrial applications

The fermentation process

Production follows an established high-cell-density process and comprises the following phases:

1. Glucose batch: Rapid cell growth and initial biomass accumulation.

2. Glycerol fed-batch: Controlled, respiratory metabolism to achieve very high cell densities.

3. Transition phase: Start of methanol feeding and adaptation of the cells to MeOH metabolism. Activation of the Pw42-2 promoter in the time window from minute 1500 to 1750.

4. Methanol induction:

   1. Switching the cells to a ‘mixed feed’ of glycerol and methanol, gradually increasing the MeOH concentration and producing the recombinant vaccine protein. From minute 3000 onwards, the final glycerol/MeOH mixture is reached, enabling optimal production.

   2. Cell densities of OD₆₀₀ > 400 are a realistic possibility – and have already been achieved in our process.

VOC monitoring: A real-time insight into metabolic processes

Volatile organic compounds are produced as by-products of cellular metabolism and can be detected online in the exhaust air stream.

A VOC sensor thus provides additional information about the physiological state of the culture:

• Excess substrate

• Overflow metabolism

• Adaptation phases

• Stress responses

• Steady-state production phases

Interpretation of the VOC signal in the process

VOC measurement curve for a fermentation process using Komagataella phaffii (formerly Pichia pastoris) as a production platform for a multivalent vaccine

High VOC level in the glucose batch (Phase 1)

A clear signal was observed at the start. This suggests the formation of volatile metabolites such as alcohols or organic acids – a typical sign of overflow metabolism.

Decline in the glycerol fed-batch (Phase 2)

The signal dropped significantly following the switch to glycerol. This is characteristic of respiratory metabolism with low by-product formation.

Rise during methanol adaptation (Phase 3)

When methanol supply began, the signal rose again, presumably due to intermediate metabolites, whilst the cells adapted to methanol metabolism.

Stable phase with methanol (Phase 4)

Following adaptation, a constant signal level was observed until the end of the process – a strong indication of metabolic stability and efficient methanol utilisation.

Why a VOC sensor sees more than DO and pH

Traditional process parameters such as pH, DO or temperature only provide an indirect indication of what is happening inside the cells.

A VOC sensor, on the other hand, directly measures the gaseous metabolic products and thus provides:

• Early warning signs of metabolic stress

• Information on substrate accumulation

• Indications of toxic intermediates

• A real-time window into cellular metabolism

Next step: VOC-controlled methanol feeding

Methanol is a powerful but critical inducer. An overdose can lead to the accumulation of toxic intermediates such as formaldehyde.

The planned VOC-based control strategy for the methanol phase:

• VOC signal rises: Reduce methanol feed

• VOC signal drops too sharply: Increase methanol feed

• VOC signal remains stable: Optimal production conditions

This would mean that methanol feeding would be directly tailored to the metabolic state of the cells – rather than simply to indirect parameters such as DO or others.

Advantages of VOC-controlled regulation

• Prevention of methanol accumulation

• Reduced cellular stress

• More stable protein expression

• Higher product yields

• Faster process development

• Potential for scale-up and automation

Particularly valuable for whole-cell vaccines

As the entire yeast is used as a vaccine in this project, it is not only the amount of protein that is crucial, but also:

1. high biomass

2. intact cell structure

3. stable physiology

4. reproducible antigen production

A VOC sensor helps to monitor precisely these factors in real time.

What’s more – real-time data at a glance – anytime, anywhere

Anyone who works with high-cell-density fermentations will know the feeling: even before you’ve finished your first cup of coffee, your thoughts are already turning to the bioreactor.

• Is the methanol induction stable?

• Is the VOC signal normal?

• Are there any signs of stress or feeding problems?

• Do I need to go to the lab straight away?

The fermHUB data platform can significantly reduce this very uncertainty. The platform consolidates all relevant process data into a central dashboard. Whether you’re in the office, working from home or on the move – you can check the current status of the fermentation process at any time.

Conclusion

The combination of recombinant yeast, AI-based antigen design and real-time VOC monitoring opens new possibilities for the cost-effective production of oral fish vaccines.

Our fermentation process clearly demonstrates that the VOC sensor reliably identifies metabolic transitions and stable production phases.

The next logical step is VOC-controlled methanol feeding, which keeps the cells precisely in the state in which they perform at their best.

In this way, a sensor signal becomes an intelligent tool for bioprocess control – and yeast cells become an innovative vaccine for sustainable aquaculture.

• Try it now – Monitoring Fermentation Processes is Easy with the fermSYS - Cube Solution

• Read more about aquaculture and W42:  https://w42biotechnology.de/Aquaculture 

Literature:

Devereux Taylor, R. (2019, 13. Dezember). Sea lice could be eradicated by new treatment at salmon farms. The Herald.

Dindial, A., Monaghan, S., Haywood, J., McLean, K., Androscuk, D., Thompson, K., Roy, W., & Bron, J. (2025). Investigation of proteins identified in the secretory and excretory products (SEPs) of the infectious copepodid stage of the salmon louse Lepeophtheirus salmonis. Veterinary Parasitology, 340, 110608. https://doi.org/10.1016/j.vetpar.2025.110608