Product formation growth-associated

Biocatalyst limitations Transport barriers - diffusion trough the cell membrane Permeability issues Highly complex cellular system Often product formation growth-associated Measurement and stability of enzyme activity Compartmentalization in some cases... 

Figure 6.7. Growth-associated (a) and non-growth-associated (b) product formation, growth, and productivity D-p inCSTR operation without Pinhibition. Ypjx = 0.5 and...

Product formation kinetics in mammalian cells has been studied extensively for hybridomas. Most monoclonal antibodies are produced at an enhanced rate during the Gq phase of the cell cycle (8—10). A model for antibody production based on this cell cycle dependence and traditional Monod kinetics for cell growth has been proposed (11). However, it is not clear if this cell cycle dependence carries over to recombinant CHO cells. In fact it has been reported that dihydrofolate reductase, the gene for which is co-amplified with the gene for the recombinant protein in CHO cells, synthesis is associated with the S phase of the cell cycle (12). Hence it is possible that the product formation kinetics in recombinant CHO cells is different from that of hybridomas. 

The values of S and e may be estimated by calculating the slope and intercept of the plot of the specific rate of product formation ( /X)(dP/dt) against //. Clearly, for a purely growth associated product e will tend to zero but will dominate the expression for a growth independent product. 

Product Formation It is important to check that the product formed is growth associated. Some products are formed after cell mass reaches the stationary phase of the growth cycle and, therefore, are not growth associated. The evolution of carbon dioxide can be measured and related stoichiometrically to the cell growth. One product that is quite general to many fermentations is the hydrogen ion. The amount of alkali added to the fermentation broth to maintain the pH may be proportional to the growth. 

Some bacterial and fungal strains are capable of differentiation (sporulation, filamenta-tion). This property can affect product formation and the physical properties of the fermentation broth. Some enzymes are synthesized as secondary products and their production does not appear to be growth associated. Production strains are distinguished on the basis of their fermentation behavior such as viscosity or recoverability. They can also be distinguished by patent or proprietary... 

There are a large number of different types of fermentation processes that are used commercially, which are selected based on several different factors.19 21 Depending on the strain to be used, the fermentation could be aerobic or anaerobic, and the desired product could be either the biomass itself or a metabolite or polymer produced by the biomass. The kinetics of product formation, whether growth associated or nongrowth associated, also influences the process. Often procedures downstream of the fermentation unit operation have a major control of the overall process and determine how the fermentation is conducted. 

Specific growth rates (/Xx), specific substrate rates (/xs), and ethanol production rates (/Xp) were calculated, and results are shown in Fig. 2 for 5o= 103.1 g L. Similar profiles were obtained for the other initial substrate concentration studied (data not shown). As can be observed, specific growth rates, substrate consumption, and product formation followed a typical pattern for ethanol fermentation [16]. The specific rate of substrate consumption (ps) and ethanol production (/Xp) present sunilar profiles, thus correlating it very well. The specific growth rate (/Xx) presents, approximately, the same course of the others two curves. Then, ethanol formation is associated with growth, consumption of substrate, and catabolism reaction, typical of a primary metabolite (Fig. 2). 

Another important factor often associated with product formation is the inhibition of growth by the product. This is prominent in one of the most important processes, alcohol formation via fermentation of sugar by yeast. Here the anaerobic growth of brewer s yeast is inhibited by alcohol. A time-tested model for this case replaces equation (3-75) with... 

Sato and Yoshizawa [119] modeled the production of ethanol, a catabolic end product, during SSF of Saccharomyces cerevisiae. The rate of ethanol production was assumed to be directly proportional to the rate of carbon dioxide formation, which had both growth and non-growth associated terms as shown in Eq. (18). The non-growth associated term represented maintenance metabolism and the non-growth associated rate constant was assiuned to decrease exponentially as the ethanol concentration increased, in order to describe the inhibitory effect of ethanol on its own production. 

In the case of mcZ-PHAs, a Swiss group reports the continuous, growth-associated production of poly(3-hydroxyalkanoate-co-3-hydroxyalkenoates) in one-stage chemostat cultures of Pseudomonas putida ATCC 29147 in a single CSTR. The applied substrates encompassed 5-phenylvalerate, octanoate and 10-undecenoate. Multiple nutrient limited growth conditions were chosen at a dilution rate of D = 0.1 h . Different mixtures of the substrates in the feed resulted in the formation of copolyesters with varying compositions and different amounts of aromatic and unsaturated side chains that make the products accessible for further modification. Based on the results, the authors conclude that the steady state conditions in a continuous culture provide a strategy especially suited for the production of tailored PHA copolymers [117]. 

Ester formation is associated with yeast growth in the early phase of fermentation. Acetate esters are produced via the reaction between an alcohol and acetyl Co-A, which is catalysed by the enzyme alcohol acetyl transferases (ATFl and ATF2). Ethanol, branched-chain alcohols and 2-phenylethanol are the common moieties of acetate esters. Ethyl esters of medium-chain fatty adds are formed through the reaction between ethanol and respective fatty acyl Co-A, which is catalysed by the enzyme alcohol acyl transferases. Saccharomyces cerevisiae strains also produce esterases that hydrolyse esters, and thus the final concentration of esters in beers is the net balance between ester synthesis and hydrolysis. Strains of brewing yeasts produce predominantly ethyl esters of fatty acids, particularly ethyl octanoate, with relatively little formation of acetate esters. Ester production in beer is regulated by a number of factors such as yeast strain, temperature, hydrostatic pressure, wort composition, sugar type and concentration, type and amount of yeast-assimilable nitrogen, aeration, and unsaturated fatty acids (Hiralal, Olaniran, PiUay, 2014 Pires et al., 2014). 

Figure 5.41. Concentration/time plot of the three basic types of microbial product formation (I) growth association, (II) mixed growth association, and (III) nongrowth association (Gaden, 1955).

Product formation associated with growth and nongrowth la and lb... 

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