Cell recycle fermentors

Y. L. Lee, H. N. Chang (1990) High cell density culture of a recombinant Escherichia coli producing penicillin acylase in a membrane cell recycle fermentor. Biotechnol. Bioeng., 36 330-337. 

The final system, shown in Figure 30.4D, is the continuous system with a partial (PRF) or complete (RF) cell recycle. It is similar to the continuous system, but cells are returned to the fermentor by means of a biomass separation device. Cross-filtration units, centrifuges, and settling tanks have all been used for biomass separation.22 In the partial cell recycle fermentor, a steady state is achieved as in the continuous system. This process is typically used to increase the productivity of the system and is used commonly in wastewater treatment and ethanol production type applications. 

Because (1 + r — rC) < 1, it is possible to operate the system at dilution rates greater than the maximum growth rate. It is this stability imparted by the cell recycle fermentor system that makes it useful, especially in waste treatment applications. 

Cell recycle fermentors consist of two main units a vessel where the biomass is allowed to grow, and a membrane separation unit (as in Figure 7.40). Vessels are usually designed to insure a uniform concentration of nutrients and pH throughout the whole volume. Due to complete mixing, process control and stability of the microbial slurry are not difficult to achieve.88 After anaerobic stabilization, when the biomass is well developed, the reactor biomass is pumped to the UF unit where solid-liquid separation occurs. The sludge is flushed back to the reactor. In most cases, the flow rate of nutrient feed is kept equal to the permeate flow rate thus keeping a constant liquid level in the anaerobic reactor. 

Higher cell concentrations are usually achieved in cell recycle fermentors than in the usual fermentor. Steady state mass balances on viable cells and on the limiting growth substrates for a continuous fermentor (Figure 7.43) can be written as 117... 

A steady state mass balance on a continuous cell recycle fermentor, over the vessel alone is as follows (see Figure 7.40) ... 

The cell concentration within the vessel of a cell recycle fermentor is then greater by a factor of 1/(1 + a - Ca ). 

Biomass recycle as sketched in Figure 30.4D is frequently used in fermentors as a way of increasing the biomass productivity. The increased biomass productivity obtained with a recycle fermentor is a function of the recycle ratio, r, and the cell concentration factor, C = XJX, achieved in the concentrator. Equations expressing the recycle system behavior are derived from material balances around the reactor. For the cell biomass balance at steady state ... 

Figure 7.44 shows the typical dependence at steady state of substrate and product concentrations, and of productivity on permeate flow rate, i.e., dilution rate, for lactose fermentation to ethanol by Kluyveromices fragilis in a cell recycle membrane fermentor.89 As dilution rate increases fermentor productivity increases, attains a maximum value and then decreases. Product and substrate concentrations in the permeate, instead, steadily decrease and increase, respectively, as dilution rate increases. A compromise generally has to be made between production rate and product concentration in the effluent. When the absence of substrate in the permeate is required, it obviously limits fermentor productivity, as in the case of wastewater treatment in the dairy industry. On the other hand, low substrate concentrations in the permeate keep recovery... 

Figure 7.44 Fermentation kinetics of a membrane recycle fermentor. Feed concentration = 150 g/C lactose. Cell concentration = 90 g/C.89...

Kluyveromices fragilis. The behavior of the fermentor is similar to that of a cell recycle reactor with the same fermentation system. Higher productivity and yields than with a batch fermentation are obtained (Tables 7.1, 7.2). The long term cell stability in an HFF (Figure 7.46) is also better than in a batch fermentor. However, even with HFF, reactor productivity is increased at the expense of low substrate conversions, at least at low dilution rates. 

With regard to the fermenters, the most common configurations are membrane recycle fermentor (MRF) and hollow fibre fermentor (HFF). In the MRF the membrane module forms a semi-closed loop with a conventional fermentation vessel the MRF gives much better performance than the HFF, where the microbial cells are loaded onto the shell-side and the feed is pumped through the lumen side. Further advantages of this system are a cell/particulate-free product stream and the reduction of capital costs. Furthermore, in these systems cell growth is a caitical point. 

For example, different fermentation schemes have been developed for the production of ethanol. Conventional batch, continuous, cell recycle and immobilized cell processes, as well as membrane, extraction and vacuum processes, which selectively remove ethanol from the fermentation medium as it is formed, were compared on identical bases using a consistent model for yeast metabolism (Maiorella et al., 1984). The continuous flow stirred tank reactor (CSTR) with cell recycle, tower and plug flow reactors all showed similar cost savings of about 10% compared to batch fermentation. Cell recycle increases cell density inside the fermentor, which is important in reducing fermentation cost. 

FIGURE 12.6 Continuous fermentor with recycle of live cells. 

It may also be economical to remove the inhibitory product directly from the ongoing fermentation by extraction, membranes, or sorption. The use of sorption with simultaneous fermentation and separation for succinic acid has not been investigated. Separation has been used to enhance other organic acid fermentations through in situ separation or separation from a recycled side stream. Solid sorbents have been added directly to batch fermentations (18,19). Seevarantnam et al. (20) tested a sorbent in the solvent phase to enhance recovery of lactic acid from free cell batch culture. A sorption column was also used to remove lactate from a recycled side stream in a free-cell continuously stirred tank reactor (21). Continuous sorption for in situ separation in a biparticle fermentor was successful in enhancing the production of lactic acid (16,22). Recovery in this system was tested with hot water (16). 

What has been previously said about the advantages of continuous membrane fermentors also applies to such complex systems. UF membranes can in fact be used to recycle both enzymes and microbial cells thus increasing overall system productivity.98... 

A cross-flow nanofillration module (SEPA CFII, GE Osmonics, Miime lis, MN) was used for this process with a maximum operating pressure of 7.0 MPa. The sur ce area of the membrane is 140 cm. The holdup volume of the membrane unit is 70 mL. The fermentation broth was placed in a 5-L fermentation vessel to control the temperature, agitation, and pH. A bench-top pump (M03-S, Hydra cell, MinneapoUs, MN) was used to pump the fermentation broth through the cross-flow membrane separation unit and recycle back to the fermentor (Fig. 2). The permeate was collected on a digital balance attached to a laptop computer with a RS-COM version 2.40 system (A D, Milpitas, CA) that recorded the amount of permeate collected every 0.5 min. The fermentation brofli was kept at constant temperature (37 °C), pH (5.5), and agitation (200 rpm). Transmembrane pressures of 1.4, 2.1, and 2.8 MPa were used in the nanofiltration tests. Each condition was tested twice, and each test lasted for 2 h. Samples of the original broth (before separation), permeate, and letentate were collected for analysis. 

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