Production methods

The entire medium is charged to the vessel at the start. From inoculation to harvest, only gas and the acid, base and antifoam for pH and foam control are added. The culture passes through the lag, exponential and stationary phases. Batch operation suits process development, inoculum preparation and productions that do not show substrate inhibition.

On Talamus systems

Temperature, pH and dissolved oxygen are held at set point by PID loops. On pilot systems, the sterilisation, cooling and cultivation phases run automatically; the defined process variables are logged with time stamps.

Limits

A high initial substrate concentration can cause substrate inhibition and overflow metabolism; fed-batch operation is then preferred.

The culture is grown by controlled feeding of the limiting substrate, and the working volume increases over the batch. The feed rate sets the specific growth rate and keeps the substrate concentration below the thresholds for inhibition, catabolite repression and overflow metabolism. In exponential feeding the feed rate is F(t) = μ × X₀V₀ × exp(μt) / (Yx/s × S_F). Here X₀V₀ is the biomass at the start of feeding, Yx/s the biomass yield and S_F the substrate concentration in the feed solution; substrate consumed for cell maintenance is neglected. High cell density and high product concentration are achieved with this mode.

On Talamus systems

The feed profile is entered on the control unit. On laboratory and pilot systems it is applied with peristaltic pumps; on laboratory systems, feed rate measurement is added as an option. On industrial lines, the feed pump and flow measurement are selected within the project. Feeding linked to the dissolved oxygen or pH signal (DO-stat, pH-stat) and feed correction with the respiratory quotient (RQ) calculated from exhaust gas analysis are configured within the project.

Limits

Vessel volume is selected for the maximum working volume at the end of feeding. At high cell density, oxygen transfer and heat removal capacity limit the feed rate.

At the end of a batch, part of the culture is harvested; the remainder is left in the vessel as the inoculum for the next batch and fresh medium is added. Because the vessel is not re-sterilised and no new inoculum is prepared between batches, dead time is shortened. The mode is applied to genetically stable strains and to processes in which contamination risk can be kept low.

On Talamus systems

Partial harvest and refilling follow a valve and pump sequence linked to level measurement. This sequence is defined within the project, together with harvest line sterility and the sterile addition of fresh medium.

Limits

Strain stability and contamination status are monitored in every cycle; the number of cycles is limited according to these data.

Fresh medium is fed continuously and culture is withdrawn at the same rate; the working volume stays constant. In a chemostat the feed rate is constant, and at steady state the specific growth rate equals the dilution rate (D = F/V). In a turbidostat, feeding is adjusted to the optical density or biomass signal to keep the cell concentration constant. Continuous culture is used for physiology studies, strain characterisation and productions in which volumetric productivity matters; commercial mycoprotein production is a known example.

On Talamus systems

Feed and harvest rates are balanced against level measurement; this arrangement is configured within the project. Turbidostat operation requires in-line optical density or biomass measurement; these measurements are added to Talamus systems as PAT options.

Limits

When the dilution rate approaches the maximum specific growth rate (μmax), cells are washed out of the reactor; a chemostat is therefore operated at D < μmax. In long-term operation, contamination and strain stability are the main risks. We size the sterile feed tank for the run length and design the sampling arrangement to monitor these risks.

The oxygen requirement of the organism determines the gas strategy. In aerobic processes, dissolved oxygen is kept above the critical value. Microaerophilic processes require a low and controlled oxygen concentration. In anaerobic processes, oxygen is removed from the medium with nitrogen or carbon dioxide and the vessel headspace is swept with inert gas.

On Talamus systems

Dissolved oxygen is held by cascade control through agitation speed and mass-flow-controlled air flow. Oxygen enrichment and vessel pressure control and, for anaerobic and microaerophilic operation, a nitrogen or carbon dioxide line and redox potential (ORP) measurement are defined within the project.

The inoculum for the production reactor is prepared in reactors of increasing volume. Stage volumes, the inoculation ratio (usually 1–10 percent of the working volume of the vessel being inoculated) and the transfer time between stages determine the production schedule and the contamination risk. Transfer is made while the seed culture is in the exponential growth phase, usually towards its end.

On Talamus systems

On the TLM120, transfer from the seed reactor to the pilot reactor runs through a sterile line. On industrial lines, the seed stages and transfer lines are defined in the Piping and Instrumentation Diagram (P&ID); sterilisation of the transfer lines is part of this design.

Microalgae and cyanobacteria grow on light energy and carbon dioxide. Yield depends on light intensity, the distribution of light within the culture, CO₂ supply and pH control. As the culture becomes denser, the depth to which light penetrates decreases; reactor geometry is therefore one of the determinants of yield. As the culture consumes CO₂, pH rises; CO₂ supply is both the carbon source and a means of lowering pH. Oxygen produced by photosynthesis inhibits photosynthesis if it accumulates in the culture; the gas flow also strips this oxygen.

On Talamus systems

In BioSIS Airlift systems, mixing and gas transfer are provided by the air flow. pH is held at set point with acid and base pumps; dissolved oxygen, which rises with photosynthesis, is monitored.