Chemostat process

Suspension systems can be operated in different modes batch, fed-batch, chemostat, and perfusion (Fig. 1). These operation modes differ basically in the way nutrient supply and metabolite removal are accomplished, which in turn determines cell concentration, product titer and volumetric productivity that can be achieved [8]. The intrinsic limitation of batch processes, where cells are exposed to a constantly changing environment, limits full expression of growth and metabolic potentials. This aspect is partially overcome in fed-batch cultures, where a special feeding strategy prolonges the culture and allows an increase in cell concentration to be achieved. In perfusion and chemostat processes nutrients are continuously fed to the bioreactor, while the same amount of spent medium is withdrawn. However, in perfusion cultures the cells are retained within the bioreactor, as opposed to continuous-flow culture (chemostat), which washes cells out with the withdrawn medium [9]. 

Fermentation and alternative production techniques, such as roller bottles, can be carried out in four different ways. They are (1) batch process, (2) fed-batch process, (3) chemostat process, and (4) perfusion process. Batch and fed-batch processes require termination of cell growth while chemostat and perfusion processes allow continuous cell cultivation. 

Jung, K., Hazenberg, W., Prieto, M.A. and Witholt, B. (2001) Two stage chemostat process development for the effective production of medium-chain-length poly(3-hydroxyalkanoates). Biotechnology and Bioengineering, 72,19-24. 

We can see that for type 1 processes, high growth rate is obligately linked to a high rate of product formation. Indeed, this is the case for all products produced by a fermentative mode of metabolism, eg ethanol, lactic add, acetone. Chemostat studies have shown that for most aerobic processes when growth is limited by some nutrient other than the carbon source, the yield of product decreases with increase in spedfic growth rate (p or D p = dilution rate (D) in chemostat culture). Conversely, both the biomass yield and the spedfic rate of substrate utilisation (qs g substrate g biomass-1 h-1) increase with spedfic growth rate. 

Reaction times of fermentation range from a few hours to several days. Batch processes are common, but continuous stirred tanks also are used either singly or in stages. A continuous stirred tank reactor (CSTR) also is called a chemostat. Figure 8.4 is a schematic of a fermentor with representative dimensions from the literature. 

Batch, fed-batch and perfusion processes are commonly used in large-scale cultivation of mammalian cells. Chemostat cultures are a valuable tool for cell physiology studies but not a serious contender as a production process [3]. 

Fig. 1. Schematic view of the different operation modes of suspended-cell processes batch, fed-batch, chemostat and perfusion...

Many reviews and several books [61,62] have appeared on the theoretical and experimental aspects of the continuous, stirred tank reactor - the so-called chemostat. Properties of the chemostat are not discussed here. The concentrations of the reagents and products can not be calculated by the algebraic equations obtained for steady-state conditions, when ji = D (the left-hand sides of Eqs. 27-29 are equal to zero), because of the double-substrate-limitation model (Eq. 26) used. These values were obtained from the time course of the concentrations obtained by simulation of the fermentation. It was assumed that the dispersed organic phase remains in the reactor and the dispersed phase holdup does not change during the process. The inlet liquid phase does not contain either organic phase or biomass. 

Continuous fermentation processes are primarily used in the research and development stage. However, more chemostat operations are being used at the production level as the understanding of this reactor increases. Examples include ethanol fermentation for the production of fuel grade ethanol and single-cell protein production from methanol substrates. 

The chemostat is an arrangement for continuous fermentation in which the properties of the system are regulated by a controlled supply of some limiting nutrient. It is a powerful research tool in microbial physiology and for evaluating process parameters. The chemostat... 

The following cultivation modes have been described for production of MAbs in in vitro large-scale systems batch, fed-batch, chemostat and perfusion (Reuveny Lazar, 1989). Batch and fed-batch processes are the most common methods. More demanding are chemostat and perfusion modes, which allow a continuous production of MAbs. All methods are applicable in homogeneous and, with the exception of chemostat, non-homogeneous systems. 

A second type of culture described by Monod kinetics is the continuous culture, in which a chemical is constantly fed into a vessel and both microbial cells and the chemical are constantly lost from the vessel at a given rate. This culture is often called a chemostat when operated under steady-state conditions. Like the batch culture, a continuous culture may be a useful model of certain environmental systems, such as lakes receiving continuous discharges of pollutants. Continuous cultures are common in industrial processes as... 

A similar process can be accomplished without access to a chemostat. Samples from a culture of an initial concentration of xenobiotic can be placed in other containers with successively higher concentrations of the toxicant, achieving the same selective pressures as found in the chemostat (Figure 10.12). Again, it may take long periods for evolution of a degradative organism or community to arise. 

One of the drawbacks in the current commercial fermentation process is that the predominant form of the product is the deprotonated lactate rather than lactic acid, requiring more expensive and wasteful product purification steps. This is because the Lactobacillus fermentation operates at a minimum pH of 5.0-5.5 which is above the pA a of lactic acid (3.87). To overcome this limitation, a powerful strain improvement method, genome shuffling, was used to improve the acid tolerance of a poorly characterized industrial strain of LactobacillusA population of strains with subtle improvement in pH tolerance was isolated using classical strain improvement methods such as chemostats, and were then shuffled by recursive pool-wise protoplast fusion to create mutant strains that grow at substantially lower pH than does the wild-type strain. 

Fig. 2 Reactor types used for fermentation processes (A) batch reactor, (B) continuous reactor or chemostat, and (C) fed-batch reactors with either a feed stream or a product withdrawal stream.

In the pharmaceutical industry, the reactors are closed to maintain aseptic conditions, and agitation and aeration are provided as needed. The classic example is the Monod chemostat, often found in laboratories. A representative chemostat, fermentation vessel, or bioreactor, is shown in Fig. 2 with an air sparger, agitator, and feed or exit streams. The continuous flow system is usually replaced by a batch process for production in the pharmaceutical and biotechnology industries. The general relationships for a constant volume system are developed in terms of cell concentration (A), often the component of interest, and substrate concentration (5), such as a sugar or oxygen. 

The continuous operations of Table 2 are elaborated in Table 3 as three types of operations. In a chemostat without feedback control, the feed medium containing all the nutrients is continuously fed at a constant rate (dilution rate) and the cultured broth is simultaneously removed from the fermenter at the same rate. A typical chemostat is shown in Fig. 1. The chemostat is quite useful in the optimization of media formulation and to investigate the physiological state of the microorganism. A turbidostat with feedback control is a continuous process to maintain the cell concentration at a constant level by controlling the medium feeding rate. A nutristat with feedback control is a cultivation technique to maintain a nutrient concentration at a constant level. A phauxostat is an extended nutristat which maintains the pH value of the medium in the fermenter at a preset value. 

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