Substrate-feeding

Based on the model described in the previous section, an optimization strategy has been developed for substrate feeding with maximum PAC production as its objective fimction. A molar ratio of pyruvate benzaldehyde of 1.2 1 was maintained in the feed as some of the pyruvate is converted also to by-products acetaldehyde and acetoin. 

Pirt [16] described a diffusion capsule for the addition of a solute at a constant rate to a liquid medium. The diffusion capsule consists of a small cylindrical container that can be completely filled with solution and then sealed with a small semipermeable membrane at one end. The device was designed for obtaining the low substrate feed rates required for multiple cultures of microbes. 

A key consideration in development of all multi-step bioprocesses is the type of bioreactor it may be necessary to accommodate a range of conditions including compartmentalization of the enzymes, cofactor recycle, adequate oxygen supply, variable temperature and pH requirements, and differential substrate feed rates. Examples described below include a range of different reactors, of which membrane bioreactors are clearly often particularly useful. 

Figure 9.1 2 -Deoxyribonucleoside production from glucose, acetaldehyde and a nucleobase in a one-pot (substrate-feeding) reaction.

Since the presumed cytosolic pathway interfaces directly with the network of secondary metabolism, the observed induction of DS-Co and CM-2 isozymes in response to wounding was expected. However, the even greater response of plastidic isozymes was unexpected. Perhaps the increased pull on carbohydrate metabolism in the cytosol affects the balance of substrates feeding into the aromatic pathway of the plastid. If so, a tendency to starvation for pathway endproducts may trigger derepression of the plastidic-pathway isozymes. 

Professor Microbe has submitted a paper for publication in which he studied the growth of a new strain of bug in a mixed flow fermenter Vm = 46.4) using a pure substrate feed (C o = 150, Crq = Qo = 0). His raw data is as follows... 

In such systems, biotransformations are generally carried out in a reaction medium composed of an aqueous phase containing the biocatalyst and a water-immiscible organic solvent which may be the substrate itself to be converted [21] or may serve as a reservoir for substrates and products [22] (Fig. 24.2). In these conditions, a constant substrate feeding in the aqueous phase is obtained owing to the partition coefficient. The substrate is used by the biocatalyst to be converted into the product of interest, which is then continuously extracted into the organic phase. 

The magnitude of current and substrate feed rate determine the degree of hydrogen replacement per pass, typically 20 - 50 % per pass, but partially fluorinated materials may be recycled to give higher fluorinated products. 

In Eq. (8.11), Sf is the substrate feed rate, and FSf consequently is the molar flow into the fermenter (there is no flow out of the fermenter). Yx/s and YpyS are the two empirical yield coefficients of cell X or product P on substrate S [g (g substrate)-1] and qp is the specific production formation rate [g product (g cells)-1 h-1] (qp-X = rp, the product formation rate [g product h 11). 

This article presents the design and implementation of a software sensor for the continuous determination of substrate concentration based on a simple model of a fed-batch fermentation process and the available signals of two other sensors—one for on-line biomass determination (7) and the other for on-line ethanol determination (8)—developed in previous works. The software sensor proposed provides a continuous signal that can be used in a control loop to manipulate the substrate feed flow in order to maintain almost constant substrate concentration and obtain an excellent level of productivity and yield during all of the process, as shown in experimental control strategy studies in previous works (9). 

At the 4th day of the fermentation 2.5 g compactin substrate is added in sterile filtered aqueous solution. Calculated for the volume of the broth 0.5-1.0% glucose was added into the culture depending on the pH in the form of 50% solution sterilized at 121°C for 25 min in parallel with the substrate feeding. After 24 hours the compactin substrate is consumed from the culture (is detected by HPLC) and was converted to pravastatin. By lyophilization of the aqueous residue 1.3 g pravastatin was obtained. The chromatographically pure product was crystallized from a mixture of ethanol and ethyl acetate. Melting point 170-173°C (decomp.). 

Hassell T, Butler M (1990), Adaptation to non-ammoniagenic media and selective substrate feeding lead to enhanced yields in animal cell culture, J. Cell Sci. 96 501-508. 

My third example of chemical engineering challenges in biotechnology is a problem in monitoring and control. In virtually all practical fermentations where the medium contains solids and/or where the cell concentrations are greater than 10 g/liter, it is impossible to monitor directly the cell concentration and hence cell activity. It would be most helpful to know the instantaneous cell concentration and activity in order to control the substrate feed rate, particularly in a fed-batch operation. 

Substrate Conversion11 (%) Turnover rate (sec" ) Substrate feed rate (mol/hr) Ionization potential (eV)... 

In order to keep the amount of substrate (or virtual level) constant in a three phase system, the whole reactor was placed with flexible tubing on a Mettler scale with a special resolution of 0.1 g. This signal was used as the input to the weight (level) controller. The substrate feed was kept at a constant value by controlling the feed pump. 

Figure 173 (a) Time course of reaction rate in UF-membrane bioreactor at different temperature. Appropriate substrate feed solution (benzonitrile or benzamide) lOmM, resting cell load 2mgocw, flow-rate 12mlh. Filled symbols for nitrile hydratase activity ... 

The space velocity effect was investigated and data analysis allowed evaluation of the steady-state conversion of the reactor loaded with 10 mg and a substrate feed of 4mM in buffer (50mM of sodium phosphate buffer at pH 7.0) solution. These data are illustrated in Figure 17.6b. An increase in T gives a higher product conversion but the reactor capacity is lower. 

Figure 17.7 % Conversion at steady state ( ) and reactor capacity (o) as a function of cell loading in UF-membrane reactors operated at 50°C with substrate feed lOOmM Nicotinamide in buffered solution.

A quantitative jump was finally brought by a two-in-one resin-based in situ substrate feeding product removal method (SFPR) [104]. An adsorbent polymeric resin acts simultaneously as a reservoir for the substrate and as a trap for the product (Figure 21.5). The substrate, preloaded onto the resin, slowly diffuses into the broth and is hansformed by cells while the formed product is readsorbed onto the soUd. The system is conveniently tuned by a simple choice of substrate resin ratio. Thus, both the substrate and product concentrations are controlled in order to maintain them below their inhibitory or toxic levels and allow a higher productivity. Additional benefits are also to be considered for prachcal large-scale applica-... 

To date, only a few examples of laboratory preparative-scale processes based on purified enzyme have been reported. Several studies have focused on the small-scale implementation of processes associating a new co-factor regenerating system, enzyme immobilization, membrane reactor, continuous substrate feeding, or resin-based SFPR with various results [110], Using the outstanding stabihty of PAMO, a 200 ml biotransformation of 5g/l phenyl cyclohexanone by an engineered mutant under two-Hquid phase conditions using methyl tert-butyl ether as solvent was described [102]. 

G. Catapano, G. lorio, E. Drioli, and M. Filosa, Experimental analysis of a cross-flow membrane bioreactor with entrapped whole cells Influence of trans-membrane pressure and substrate feed concentration on reactor performance, J. Membrane Sci 55 325 (1988). 

The concentration of the growth-limiting substrate in the feed medium should be set to that it becomes limiting while other nutrients are still present in relative excess. Small-scale batch culture experiments in which the cell yield is determined at different initial substrate concentrations will give some indication of a suitable substrate feed concentration (5 r). Once at steady state, it can be confirmed that the cells are limited by the chosen substrate because a change in the substrate feed concentration should give a proportionate change in the cell concentration. 

Substrate utilization (neglect maintenance energy and substrate removal) Substrate accumulation equals Substrate Feed minus Growth minus Enzyme Production... 

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