Immobilised cells

Freitas dos Santos, L.M., Pavasant, P., Pistikopoulos, E.N., Livingston, A.G., Growth of immobilised cells results and predictions for membrane-attached biofilms using a novel in situ biofilm thickness measurement technique. Immobilised Cells Basics and Applications, Ed. Wijfells, R.H., Buitelaar, R.M., Bucke, C., Tramper, J., Progess in Biotech. 11, pp.290-297, 1996. 

Conversions carried out by immobilised cells give higher yields than those carried out by growing and dividing cells. 

The alternative to batch mode operation is continuous operation. In the continuous mode there is a continuous flow of medium into the fermentor and of product stream out of the fermentor. Continuous bioprocesses often use homogenously mixed whole cell suspensions. However, immobilised cell or enzyme processes generally operate in continuous plug flow reactors, without mixing (see Figure 2.1, packed-bed reactors). 

Process B Genetic instability Poor enzyme stability Cofactor requirement Product (non-polar) inhibition Biocatalyst Free enzyme Free cells Immobilised enzyme Immobilised cells... 

Polysaccharides can also be used to immobilise cells or enzymes, permitting the re-use of the catalyst and continuous flow systems. Alginates have the advantage that gel formation occurs under mild conditions, therefore cells remain viable and enzymes are not denatured but calcium gradually leaches out and the gel dissolves. Gellan or other combinations may prove superior for this application. 

A rapid activity loss (a few days) was observed with whole cells. Immobilisation increased the stability and continuous production of L-phenylalanine was possible using alginate bead immobilised cells of P. fluorescerts for 60 days. However, to achieve this the cofactor pyridoxalphosphate had to be continuously added to the beads to correct for the dissociation of the cofactor and loss from the cells. 

Polyazetidine prepolymer may be cross-linked in aqueous solution by reaction with amine, thiol, hydroxyl, carboxylic add or other polyazetidine groups. Cross-linking occurs upon water removal, heating or by changing to a basic pH. The immobilised cell/polymer composition may be prepared in the form of membranes, fibres, tubes or beads. 

Table 9.6 Examples of hydroxylation of steroid nuclei by micro-organisms. (Data derived from Neidleman, SL "Industrial Chemicals Fermentation and Immobilised Cells" in "Biotechnology the Science and the Business", edited by Moses V and Cape RB, published by Harwood Academic, London 1991).

In fact, significant substrate concentration gradients may exist for cells immobilised in biofilm. Cells located close to the nutrient supply are likely to maintain higher quality and activity compared with cells located relatively further away, leading to differentiation in the quality or activity of the immobilised cell population. This differentiation is more pronounced if there are starvation regions. In practice, zero substrate concentration may exist inside the biofilm, because in these regions the cell physiology may be markedly different from that of the freely suspended cells. 

Since 1978, several papers have examined the potential of using immobilised cells in fuel production. Microbial cells are used advantageously for industrial purposes, such as Escherichia coli for the continuous production of L-aspartic acid from ammonium fur-marate.5,6 Enzymes from microorganisms are classified as extracellular and intracellular. If whole microbial cells can be immobilised directly, procedures for extraction and purification can be omitted and the loss of intracellular enzyme activity can be kept to a minimum. Whole cells are used as a solid catalyst when they are immobilised onto a solid support. 

Fig. 8.1. Alternatives methods used to immobilise cells, (a) Carrier binding (b) entrapment (c) cross-linking.3...

Thirdly, nutrient depletion and any inhibitory compounds do not, in general, have a large effect on the immobilised cells because the cells are fixed by immobilisation. In batch and CSTR fermentation, nutrient depletion, inhibition and accumulation of toxic by-products are major problems, but immobilised cells are usually unaffected by toxic by-products. 

However, there are disadvantages to using immobilised cells. The cell may contain numerous catalytically active enzymes, which may catalyse unwanted side reactions. Also, the cell membrane itself may serve as a diffusion barrier, and may reduce productivity. The matrix may sharply reduce productivity if the microorganism is sensitive to product inhibition. One of the disadvantages of immobilised cell reactors is that the physiological state of the microorganism cannot be controlled. 

The immobilised cell reactor (ICR) experiments were undertaken to determine the performance of immobilised Propionibacterium acidipropionici in a plug-flow tubular reactor. The... 

CASE STUDY ETHANOL FERMENTATION IN AN IMMOBILISED CELL REACTOR USING SACCHAROMYCES CEREVISIAE... 

Fermentation of sugar by Saccharomyces cerevisiae, for production of ethanol in an immobilised cell reactor (ICR), was successfully carried out to improve the performance of the... 

Keywords immobilised cell reactor (ICR) Saccharomyces cerevisiae ethanol fermentation encapsulated beads calcium alginate... 

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... 

Fig. 8.9. Percentage, growth of immobilised cells with various initial beads loading. Reprinted from Najafpour et al. (2004).18 Copyright with permission from Elsevier.

In preparing immobilised cells, 1.5, 2, 3 and 6% alginate was used. By pressing them manually, the hardness and rigidity of the beads were tested. The physical criteria of the... 

The economics of an immobilised cell process depend on the lifetime of the microorganism and a continued level of clean product delivered by the fixed cells. It is important to eliminate the free cells from the downstream product without the use of any units such as centrifuge or filtration processes. Since the cells are retained in the ICR, the activity of intracellular enzymes may play a major role. It is assumed that the deactivation of the enzyme at constant temperature follows a first-order equation as shown below 17... 

Effect of High Concentration of Substrate on Immobilised cells... 

By using the unsteady-state model, the maximum oxygen penetration depth for highly packed immobilised cells has been reported to be in the range of 50-200 xm. 

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