Introduction to Part 1

Jean-Marc ENGASSER and Mohamed GHOUL

Laboratoire Réaction et Génie des Procédés (LRGP), CNRS, Université de Lorraine, Nancy, France

In order to ensure proper and optimal extrapolation of a bioreactor process, the engineer must consider a number of technological choices. These choices include the sequence of upstream and downstream unit operations, the selection of the necessary equipment and the operating modes associated with this equipment. In terms of the biotransformation operation itself under the effect of different microorganisms, technological extrapolation decisions involve the composition of the medium, which varies according to the microorganism used, the bioreactor technology and its implementation.

With regard to the composition of the medium, which must meet the nutritional requirements of the strain used, the choice involves selecting the nutrients and determining their appropriate concentrations. Indeed, the composition of the medium has a significant influence on the course of the process, as well as on the final concentrations of the obtained products. Moreover, it can also have an impact on other process steps, including sterilization, extraction and purification. The composition of the medium can also have a significant impact on the cost of the process. In the case of large-scale metabolite production, for example, the cost of the medium can account for more than half the total cost of production.

The elements to be optimized in bioreactor culture media are the carbon source, typically sugars or mineral carbon for microalgae, the nitrogen source, which can be ammonia, nitrates, amino acids and peptides, the phosphorus source, usually phosphates, growth factors or micronutrients such as vitamins, iron, zinc, copper and manganese, as well as the source and brightness in the case of microalgae cultivation.

For bacterial, yeast and fungal cultures, most industrial fermentation media are complex media containing elements such as yeast extract, corn steep or peptones, the composition of which is not precisely known. However, media in which the concentrations of all of the elements are specified are also used. Although these media are generally more expensive, they have the advantage of facilitating product purification and ensuring reproducibility.

Microalgae can be grown under autotrophic conditions, using sunlight or artificial light as an energy source and CO2 or bicarbonate as a carbon source. In heterotrophic cultures, microalgae use simple sugars as a carbon and energy source. The culture medium must also provide sufficient quantities of mineral ions, and possibly growth factors or vitamins.

With regard to bioreactor technology, there is a wide range of bioreactors available for microbial fermentations or microalgae cultivation. The most common technology revolves around using cells in suspension in mixed tanks. In the laboratory, microorganisms are cultivated in containers that are agitated and in small mixed bioreactors, with capacities ranging from a few liters to several tens of liters. These cultures are then extrapolated to tanks with volumes of up to several thousand cubic meters. Other, less frequently used technologies include membrane bioreactors, in which the cells are retained by microfiltration membranes, and fixed-bed or fluidized-bed bioreactors, in which the cultured cells are located on the surface or inside solid supports.

An essential feature of a bioreactor is its agitation and/or aeration system. For anaerobic microbial fermentations, tanks may be equipped with moderate agitation systems, or may not require agitation at all. In such cases, agitation of the medium can be achieved by the release of CO2, like in the cylindrical-conical tanks used in the brewing industry. For aerobic fermentations, bioreactors are equipped with more powerful agitation and aeration systems. The mechanically aerated and agitated bioreactor, fitted with various types of agitator, is the most commonly chosen option for bacterial and yeast culture. Other, less shearing systems are used for microalgae and fungi. Other aeration configurations include bubble columns, air lifts and external loop bioreactors, where the medium is continuously stirred by rising air bubbles. The choice of agitation and aeration technology depends on the oxygen demand of the process under study, the rheological properties of the medium and the sensitivity of the cells to shear forces.

In terms of the bioreactor operating mode, aerobic or anaerobic bioreactors with suspension cells can be operated in three different modes: batch, fed-batch and continuous. In batch mode, the entire culture medium is initially introduced into the reactor. Fed-batch mode involves the initial introduction of part of the culture medium, followed by the continuous addition of the rest of the medium over time. The continuous mode is characterized by the continuous supply of fresh medium and the simultaneous removal of used medium. Continuous culture can also be implemented using several bioreactors that are arranged in a series.

The continuous bioreactor can also be combined with cell recovery and recycling. This operation can be carried out using a centrifuge, a microfiltration membrane module or simply by decantation in the case of flocculating cells.

For a specified operating mode, the bioprocess engineer must also optimize its operational variables. The predominant operational variable is the composition of the medium, which is characterized by the concentrations of substrates and nutrients added, notably sugar concentration for bacteria and minerals, and CO2 for microalgae. Concentrations of other nutrients, such as sources of nitrogen or phosphorus, are also adjusted. The substrate concentrations to be optimized are the concentrations in the initially added medium and, in the case of continuous or fed-batch operation, the substrate concentrations in the supply medium.

The second category of operational variables concerns bioreactor aeration. It includes air flow rate and agitation power of the medium in the case of mechanically agitated biotransformation. For yeast and bacterial cultures, it is also possible to increase the mole fraction of oxygen in the inlet air in order to overcome any oxygen limitations.

Temperature and pH are other operational parameters to consider. In most cases, temperature and pH are kept constant. However, they can also be modified over time, for example, to promote metabolite excretion.

The choice of technology and operating mode for a bioreactor depends on a number of criteria. First, it is influenced by the nature of the transformation, depending on the microorganism used, whether cell or metabolite production is involved, as well as its aerobic or anaerobic nature. Second, the kinetics of the microorganism used play a crucial role, particularly with regard to inhibitions of cell growth at high concentrations of substrates and metabolites. It is also important to consider whether or not metabolite production is linked to cell growth, as well as the morphology of cultured cells and their sensitivity to shear stress in bioreactors. Additional criteria to be considered include the quality of the products obtained, the sterility requirements of the process and the economic aspects related to the cultivation process, which depend on the species of microorganism used.

Given the wide range of possible transformations and the different types of cells used (bacteria, yeast, fungi and microalgae), there is no global optimal bioreactor process design. Bioreactor technology and implementation must be specifically adapted to the biotransformation reaction, the characteristics of the cells and the industrial context in which they are used. However, to speed up the implementation and optimization of bioreactors, regardless of the strain used, the engineer can benefit from the methodology of bioprocess engineering, which favors the use of simulation models. The aim of these simulators is to predict and compare the progress of a bioprocess under different batch, continuous or fed-batch operating conditions, and with different operational variables, such as substrate concentrations or the intensity and quality of light for microalgae.

This approach to digital extrapolation and optimization of biotransformation in a bioreactor uses a “digital twin”, enabling virtual biotransformations to be carried out, in order to determine the optimal operating mode and associated variables. The use of bioreactor simulators reduces the need for experimentation during the R&D phase. It also speeds up process extrapolation and reduces R&D costs.

The most effective simulators are those that rely on knowledge-based models, which are themselves based on a thorough understanding of the phenomena and factors that influence or control bioreactor operation under various operational conditions.

Thus, the first step in the methodology for building mechanistic or knowledge-based simulation models involves identifying the phenomena that influence bioreactor behavior or limit its operation. These phenomena include the transformation reaction by the microorganism used, material and heat transfers, as well as flows inside the bioreactor.

For a given microorganism culture operation in a bioreactor, it is the transformation reaction, that is specific to the microorganism used, that largely controls variations in substrate, cell and metabolite concentrations. It is therefore essential to understand the factors related to the composition of the medium that influence the rates of cell growth, substrate...