Microbial cell fermentation

Over half of all biopharmaceuticals thus far approved are produced in recombinant E. coli or S. cerevisiae. Industrial-scale bacterial and yeast fermentation systems share many common features, an overview of which is provided below. Most remaining biopharmaceuticals are produced using animal cell culture, mainly by recombinant BHK or CHO cells (or hybridoma cells in 

Simple organic molecules Ethanol S. cerevisiae Pachysolen tannophilus 

Enzymes Proteases Various bacilli, e.g. Bacillus licheniformis 

Typically, the manufacture of a batch of biopharmaceutical product entails filling the production vessel with the appropriate quantity of purified water. Heat-stable nutrients required for producer cell growth are then added and the resultant medium is sterilized in situ. This can be achieved by heat, and many fermenters have inbuilt heating elements or, alternatively, outer jackets through which steam can be passed in order to heat the vessel contents. Heat-labile ingredients can be sterilized by filtration and added to the fermenter after the heat step. Media composition can vary 

---

We can also use microbial cells (fermentation) containing the desired catalytic activity without isolating the enzymes responsible. 

Figure 5.9 Design of a generalized microbial cell fermentation vessel (a) and an animal cell bioreactor (b). Animal cell bioreactors display several structural differences compared with microbial fermentation vessels. Note in particular (i) the use of a marine-type impeller (some animal cell bioreactors-air lift fermenters-are devoid of impellers and use sparging of air-gas as the only means of media agitation) (ii) the absence of baffles (iii) curved internal surfaces at the bioreactor base. These modifications aim to minimize damage to the fragile animal cells during culture. Note that various additional bioreactor configurations are also commercially available. Reprinted with permission from Proteins Biochemistry and Biotechnology (2002), J. Wiley Sons...

Mammalian cell culture is more technically complex and more expensive than microbial cell fermentation. Therefore, it is usually only used in the manufacture of therapeutic proteins that show extensive and essential post-translational modifications. In practice, this usually refers to glycosylation, and the use of animal cell culture would be appropriate where the carbohydrate content and pattern are essential to the protein s biological activity, its stability or serum half-life. Therapeutic proteins falling into this category include EPO (Chapter 10), the gonadotrophins (Chapter 11), some cytokines (Chapters 8-10) and intact monoclonal antibodies (Chapter 13). 

FIG. 18-28 Usually, the gas-liquid mass-transfer coefficient, K, is reduced with increased viscosity. This shows the effect of increased concentration of microbial cells in a fermentation process. 

The resultant fermentation broth was centrifuged to harvest the microbial cells, and they were washed with water and centrifuged a second time, whereupon a living cell paste was obtained. (There was obtained an amount of cells equivalent to 54 parts on a dry basis, which contained 920 /Jg of ubiquinone-10 per gram of dry cells.)... 

Synthesis of industrial chemicals by microbial cells may be by fermentation (free, living cells), immobilised growing cells, immobilised resting cells or immobilised dead cells. 

Study the unlabelled block diagram, and then replace the question marks with the words and phrases to give a generalised scheme of an industrial fermentation. Assume in this example that the product is excreted from the microbial cells. 

There several DO probes available. Some well-known branded fermenters, like New Brunswick, Bioflo series and the B. Braun Biotstat B fermenters are equipped with a DO meter. This unit has a 2 litre fermentation vessel equipped with DO meter and pH probe, antifoam sensor and level controllers for harvesting culture. The concentration of DO in the media is a function of temperature. The higher operating temperature would decrease the level of DO. A micro-sparger is used to provide sufficient small air bubbles. The air bubbles are stabilized in the media and the liquid phase is saturated with air. The availability of oxygen is major parameter to be considered in effective microbial cell growth rate. 

A strain of Azotobacter vinelandii was cultured in a 15 m3 stirred fermenter for the production of alginate. Under current conditions the mass transfer coefficient, kLa, is 0.18 s. Oxygen solubility in the fermentation broth is approximately 8 X 10 3 kgm-3.9 The specific oxygen uptake rate is 12.5 mmol g 1 h. What is the maximum cell density in the broth If copper sulphate is accidentally added to the fermentation broth, which may reduce the oxygen uptake rate to 3 mmol g 1 h 1 and inhibit the microbial cell growth, what would be the maximum cell density in this condition ... 

Use of biofilm reactors for ethanol production has been investigated to improve the economics and performance of fermentation processes.8 Immobilisation of microbial cells for fermentation has been developed to eliminate inhibition caused by high concentrations of substrate and product, also to enhance productivity and yield of ethanol. Recent work on ethanol production in an immobilised cell reactor (ICR) showed that production of ethanol using Zymomonas mobilis was doubled.9 The immobilised recombinant Z. mobilis was also successfully used with high concentrations of sugar (12%-15%).10... 

The potential use of immobilised cells in fermentation processes for fuel production has been described previously. If intact microbial cells are directly immobilised, the removal of microorganisms from downstream product can be omitted and the loss of intracellular enzyme activity can be kept to a minimum level.11... 

A special CSTR fermentation known as a chemostat bioreactor is used for microbial cell growth. The rate of biomass generation is given by ... 

Normally, fermentation processes can be classified depending on the objective of study. For example, in terms of products fermentation is divided into 4 types, namely, microbial cell, microbial enzyme, microbial metabolite and transformation process. If considering due to its contaminating conditions, it will be classified into 3 types septic, semi-septic and aseptic fermentation. However, in general, the fermentation processed are classified into 3 types as follows. 

In some cases it is more attractive to use whole microbial cells, rather than isolated enzymes, as biocatalysts. This is the case in many oxidative biotransformations where cofactor regeneration is required and/or the enzyme has low stability outside the cell. By performing the reaction as a fermentation, i.e. with growing microbial cells, the cofactor is continuously regenerated from the energy source, e.g. glucose. 

The immobilization of microbial cells under conditions where an activity or set of enzymic activities remains intact, but the normal metabolic processes cease, represents a novel technique for enzyme immobilization. Moreover, immobilized cells might enable the standard fermentation methods to be replaced by immobilized -cell -based continuous processes. 

Advantages Cells grow rapidly on relatively inexpensive media the fermentation technology for growing microbial cells is well established. 

In some cases, microbial cell fragments may arise and can be particularly problematic. For example, the disc centrifuges often used to remove yeast after brewery fermentations are known to produce shearing forces that break off yeast cell wall fragments (Siebert et ah, 1987). Agitation of yeast by other means is also problematic (Lewis and Poerwantaro, 1991 Stoupis et ah, 2003). In beer, the resulting particles resist sedimentation and impair filtration. 

---