Wasted culture medium again? How to accurately determine the fermentation endpoint?

In industrial fermentation production, determining the fermentation endpoint is directly tied to the final product quality and economic efficiency. If the endpoint is judged too early, substrates may not be fully utilized, resulting in insufficient product yield. If judged too late, cell autolysis, product degradation, and increased downstream extraction burdens may occur. Therefore, scientifically defining this critical point is a key topic in process optimization.

A successful fermentation endpoint determination must follow an integrated logic: accurately monitoring key signals, balancing decisions around production goals, and considering overall economic efficiency throughout the process. The following sections systematically outline the essential criteria and practical methods for making this determination.

 Clarify process objectives, establish the decision direction

Different fermentation products have different process priorities, and the key dimensions for endpoint determination should likewise vary.

• If raw material and fermentation costs are the dominant factors, the focus should be on improving yield and conversion rate, aiming for higher productivity per unit time.

• If downstream extraction is costly or the product has high value, then ensuring a sufficiently high final product concentration becomes the primary objective, while reasonably controlling the fermentation duration to reduce the difficulty and cost of subsequent purification.

Monitor key indicators, capture critical state shifts

In the late stage of fermentation, shifts in the metabolic state of the microorganisms are reflected in various measurable parameters. These serve as important bases for determining the fermentation endpoint:

• Abnormal increases in pH and dissolved oxygen (DO): During stable fermentation, pH and DO typically remain within a defined range. When they rise significantly, either simultaneously or sequentially, it often indicates that cell growth and product synthesis are stagnating, and the culture may be entering the decline or early autolysis phase. This signal is commonly regarded as the primary indicator for endpoint determination.

• Deterioration of mycelial morphology: Sampling observations may reveal that mycelia transition from thick and intact to sparse, broken, or fragmented. This visual change indicates a decline in cell viability and suggests that autolysis may soon begin.

• Residual sugar depletion and amino nitrogen rebound: When residual sugar drops to a low level while amino nitrogen begins to rise, it signifies that cells have shifted to endogenous metabolism due to the lack of external nutrients and have started degrading their own proteins. At this point, fermentation should be terminated promptly.

• Changes in the physical properties of the broth: Sudden increases or decreases in viscosity, darkening of color, or changes in foam characteristics can also serve as auxiliary indicators.

Conduct economic evaluation, pursue optimal overall efficiency

Endpoint determination should not focus solely on reaching the highest product concentration; instead, it should consider overall production efficiency. That is:

Total productivity = total product amount fermentation time + tank- cleaning preparation time total productivity = fermentation time + tank-cleaning preparation time total product amount

In some cases, ending the fermentation slightly earlier may result in a marginally lower yield per batch, but because the cycle time is shortened and equipment turnover improves, the total output over time can actually increase.

Reserve Operational Flexibility for Downstream Processes

The timing of harvest directly affects subsequent extraction and purification efficiency

Excess residual nutrients (such as sugars and proteins) increase the downstream processing load, whereas excessive autolysis of the cells can make the fermentation broth difficult to filter and destabilize the product. Therefore, before harvest, nutrient feeding must be carefully controlled, and the addition of exogenous substances such as sugars and antifoaming agents is typically stopped several hours in advance.

Differentiate Strategies Based on Process Maturity

• Mature processes: Operations can be carried out according to established workflows, but real-time monitoring is still required. In cases of mycelial abnormalities or other unexpected conditions, timely adjustments must be made to prevent further loss.

• New process development: Multiple batches of experimentation are needed, with systematic recording of product yield, cell viability, and key parameters at different time points. These data are used to gradually establish a reliable endpoint-determination model.

Ensure Reliable Equipment Support to Strengthen Decision-Making

In practical operations, stable and reliable fermentation equipment provides essential data support and ensures process consistency for endpoint determination. For example:

CytoLinX® GB 1-20 L Benchtop Glass Bioreactor

Figure 1. CytoLinX® GB 1-20 L Benchtop Glass Bioreactor

For process development and small-scale research, CytoLinX® GB 1-20 L Benchtop Glass Bioreactor provides robust support:

• Its high configurability and flexibility allow it to accommodate a wide range of working volumes tailored to specific needs in process development and characterization, while also supporting scale-up toward production.

• It serves a broad spectrum of application areas and can be customized according to specialized process requirements, such as those in ADC or mRNA production. The vessel is made of high-borosilicate glass, offering excellent resistance to high temperature and corrosion, while being easy to clean and highly durable.

• The system also provides strong operational stability, delivering precise and reliable cascade control of temperature, DO, and pH. In addition, it offers multiple aeration options, including macro-bubbles and micro-bubbles, to meet the individualized needs of both cell culture and microbial fermentation processes.

CytoLinX® BR 10–2000 L Single-use Bioreactor

Figure 2. CytoLinX® BR 10–2000 L Single-Use Bioreactor

• CytoLinX® BR 10–2000 L Single-use Bioreactor is equipped with a highly sensitive real-time pH and DO monitoring system that accurately captures metabolic transition points and reduces the risk of misjudgment caused by signal lag.

• Its flexible aeration and agitation design helps maintain stable mycelial morphology and facilitates observation and sampling for endpoint assessment.

• The system is available across multiple scales, supporting seamless scale-up from process development to large-scale production, ensuring that endpoint strategies defined at the lab scale can be effectively executed in full-scale manufacturing.

• Its control and data recording system complies with ISA-88 and 21 CFR Part 11, providing complete and reliable parameter profiles for every batch. This supports the establishment of a comprehensive process knowledge base and enhances endpoint decision-making.

In summary, determining the fermentation endpoint is an art of balance, requiring the operator to identify the optimal trade-off among product accumulation, time cost, equipment utilization, and downstream processing burden. By setting clear objectives, implementing systematic monitoring, conducting integrated assessments, and relying on stable and reliable process equipment, operators can progressively improve the accuracy and consistency of endpoint judgments, ultimately achieving comprehensive optimization of quality, efficiency, and overall performance.