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1.4 Fermentation Technology
ОглавлениеThe main challenge facing bioprocess specialists is the technological transfer from the laboratory to the industrial scale. Indeed, it is usually quite simple to cultivate a microorganism in an Erlenmeyer flask containing a few hundred milliliters of culture medium. However, the same operation is much more complicated in industry, where high culture volumes are processed. Nonetheless, whether in the laboratory or the industry, certain steps must be taken to cultivate a microorganism and make it produce a molecule of commercial interest.
In this regard, the fermentation industry provides a basic model in which a sequence of steps is commonly followed in the majority of bioprocesses. Whatever the strain used and the product sought, there are six major points to take into consideration when developing an industrial fermentation:
1 The formulation of an adequate culture medium that will promote the growth of the strain used and the production of the molecule of interest;
2 Sterilization of the culture medium, the bioreactor, and accessory components to prevent another microorganism developing there and contaminating the fermentation;
3 The development of an inoculum that will be used to produce a pure culture of the selected strain in sufficiently high concentration and volume to be able to adequately inoculate the bioreactor;
4 The growth of the strain within the bioreactor, under optimal and controlled production conditions;
5 Recovery and purification of the product from the culture (i.e. downstream processing steps); and
6 The elimination of effluents and other residues from the process.
The inoculum represents the biomass (e.g. cells, spores, mycelium, etc.) required for seeding the culture in a bioreactor. On one hand, it must provide a sufficient quantity of cells in homogeneous suspension to achieve rapid growth in the bioreactor, while, on the other hand, it must be free from any microbial contamination. To achieve this, an industrial lyophilized or frozen strain from a working cell bank (Figure 1.7) is used and grown in a liquid medium stirred in an Erlenmeyer flask to obtain a significant volume of culture. This volume will be then transferred aseptically to a small bioreactor, commonly called pre‐culture, which has the function of developing a very high and metabolically active cellular biomass concentration. These stages may require several days of work and, in the case of fermentation, are carried out in very large volumes; it is often even a real fermentation before the main fermentation. Generally, the volume of the inoculum represents between 5 and 10% of the volume of the main bioreactor where the fermentation will take place. Seeding steps at an industrial scale are presented in Figure 1.8.
Figure 1.8 Factory seed culture equipment. AF = air filter; FIC = flow indication control; STM = steam; TIC = temperature indication control; PIC = pressure indication control; ATM = atmosphere.
Source: Oka (1999).
In the field of industrial fermentation bioprocesses, developing a profitable process requires the consideration of some criteria for the culture medium:
1 The environment must be as inexpensive as possible (i.e. the cost of acquiring and storing raw materials must be affordable).
2 Raw materials should be available on an annual basis and ideally in the local market.
3 The quality of the raw materials must be constant, which allows obtaining similar results in terms of yield and productivity between the different batches.
4 The substrate must demonstrate good physicochemical stability during storage.
5 The medium must be easy to sterilize by usual techniques since certain media could be viscous or very heavily loaded with solid matter that affect the sterilization efficiency.
6 The medium must have an acceptable viscosity, since a too viscous medium is unfavorable for the aeration and homogenization of the substrate, in addition to causing a higher energy demand for agitation and a greater risk of formation of unwanted foam.
7 The raw materials must be able to guarantee the quality of the finished product (i.e. be free of toxic substances) and have the lowest possible content of impurities to facilitate the recovery and purification stages at the end of the process.
Any industrial fermentation aims to produce a molecule of interest in the highest quantity, in the shortest time, and at the lowest possible cost. For this, a microorganism must be cultivated under controlled physicochemical conditions within a large volume enclosure specially designed for this purpose: the bioreactor or fermenter. One of the most used bioreactors at the industrial scale is the stirred tank type, schematized in Figure 1.9. Other types of bioreactors exist and are described in Chapter 2.
Mainly, two types of bioreactors exist. The first one allows performing nonaseptic cultures such as brewing and effluent treatment, and the second one requires aseptic conditions for successful product formation, such as antibiotics, vitamins, polysaccharides, and recombinant proteins. Aseptic bioreactors can be sterilized repeatedly and can process a large volume of culture. Several peripheral systems are coupled to the tank and allow controlling automatically the parameters affecting the microbial growth such as the temperature, the pH, and the dissolved O2. Within the bioreactor, the microorganisms are suspended in the aqueous nutrient medium containing the necessary substrates for microbial growth and product synthesis. Changing the culture conditions has a great impact on the bioprocess performances, as discussed in Chapter 3.
Figure 1.9 Fermenter vessel schematic and terminology.
Source: Charles (1999).
In a bioreactor, fermentation occurs in a multiphase state: a gas phase where CO2, O2, and N2 can be exchanged, a liquid phase mainly composed of the aqueous medium, and a solid phase mainly composed of microorganisms and solid substrates. These phases must be perfectly mixed to ensure the most efficient and homogenous heat and mass transfer. To ensure a perfect mixing of the suspension, the shaft of the bioreactor is usually equipped with several impellers. Although a wide range of impeller types exist in the market, the six flat‐bladed (Rushton) turbine impellers are used in the majority of bioreactors. Usually, three to five impellers are mounted and spaced at intervals equivalent to one tank diameter along the shaft to avoid a swirling type of liquid movement.
An optimal production system could be achieved by taking a few requirements that include:
1 Avoiding the contamination of the culture, which can be achieved by designing a bioreactor where the inputs and outputs of the fermenter are controlled;
2 No leakage or water evaporation from the medium can occur;
3 Agitation of the medium should be perfect to allow a homogenous distribution of O2 in the medium and the maintenance of O2 concentration above the critical value; and
4 The fermentation parameters that include the pH, temperature, etc. must be automatically controlled.
A successful process occurs when all the parameters required for the growth and production of a molecule of interest are brought together. Yield and productivity constitute the two main aspects to be quantified during fermentation. They indicate the conversion efficiency of the substrate into cells and products. The following equations are given for culture in batch mode.
The yield in biomass YX/S is given by Eq. (1.3) and corresponds to the mass (g) of dry cell biomass produced per gram of substrate consumed (g g−1).
where X0 and X correspond, respectively, to the initial and final biomass concentrations (g l−1). S0 and S correspond, respectively, to the initial and final carbon substrate concentrations (g l−1).
The yield in product YP/S is given by Eq. (1.4) and corresponds to the weight in grams of product produced per gram of substrate consumed (g g−1).
where P0 and P correspond, respectively, to the initial and final product concentrations (g l−1). S0 and S correspond, respectively, to the initial and final carbon substrate concentrations (g l−1).
The productivity in biomass PX is given by Eq. (1.5) and corresponds to the weight in grams of biomass produced per liter of culture per hour (g l−1 h−1).
where X0 and Xt correspond, respectively, to the initial biomass concentration and that at time t (g l−1). t0 and t correspond, respectively, to the initial and final time (h).
The productivity in product Pp is given by Eq. (1.6) and corresponds to the weight in grams of product produced per liter of culture per hour (g l−1 h−1).
where P0 and Pt correspond, respectively, to the initial product concentration and that at time t (g l−1). t0 and t correspond, respectively, to the initial and final time (h).
