Extractive bioconversion systems

A prehminary study of the use of larch AGs in aqueous two-phase systems [394] revealed that this polysaccharide provides a low-cost alternative to fractionated dextrans for use in aqueous two-phase, two-polymer systems with polyethylene glycol (PEG). The narrow molecular-weight distribution (Mw/Mn of 1-2) and low viscosity at high concentration of AG can be exploited for reproducible separations of proteins under a variety of conditions. The AG/PEG systems were used with success for batch extractive bioconversions of cornstarch to cyclodextrin and glucose. 

Straightforward. We have therefore employed XAD-4 to combine biocatalytic synthesis with simultaneous product extraction. The system (Figure 15.8) comprises a continuously stirred tank reactor, a starting material feed pump, a product recovery loop with a (semi-) fluidized bed of XAD-4, and a pump to circulate the entire reaction mixture through the loop." ° Preliminary studies indicated that XAD-4 had no detrimental effects on E. coli JMlOl (pHBP461), hence, separation of biomass and reaction liquid prior to catechol extraction was not required. The biocatalytic reaction was carried out at very low concentrations of the toxic substrate and product. This was achieved by feeding the substrate at a rate lower than the potential bioconversion rate in the reactor. 

Other applications besides purification of biomolecules are affinity partitioning (Labrou, 1994), extractive bioconversions (Andersson, 1990 Kaul, 1991 Zijlstra, 1998), liquid-liquid partition chromatography, and analytical assays. Although industrial applications of aqueous two-phase systems have not gained widespread use to date, practical and economic feasibility has been proved (Tjemeld, 1990 Cunha, 2002). 

R. KAurand B. Mattiasson, Extractive bioconversions in aqueous two-phase systems, Bioproc. Technol. 1991, 11,... 

Monitoring Enzymes That Degrade Macromolecular Substrates by Extractive Bioconversion in Aqueous TVo-Phase Systems... 

Hahn-Hagerdal, B. Mattiasson, B. Albertsson, P.-A. "Extractive Bioconversion in Aqueous Two-Phase Systems. A Model Study on the Conversion of Cellulose to Ethanol" Biotech. Letters. 1981, 3, 2, pp 53-58. 

Although CO2 is inhibitory to microbes, compressed hydrocarbon solvents may be appropriate for extractive bioconversions and extractions in biphasic (aqueous-compressed solvent) systems. Our laboratory investigated the metabolic activity of the anaerobic, thermophilic bacteria Clostridium ther-mocellum as a model system (45). Thermophilic bacteria have a distinct advantage over conventional yeasts for ethanol production in their ability to use a variety of inexpensive biomass feedstocks. Extractive fermentation using compressed solvents is an approach to address the end-product toxicity of these bacteria to ethanol and improve the economic viability of biofuel production by thermophilic organisms. 

Aqueous two-phase systems are generated by mixing aqueous solutions of two water-soluble polymers, or a polymer and a salt. These systems offer extremely mild conditions for separation of cells, organelles, proteins and other biomolecules, in biochemical processes. Considerable attention has been directed towards the use of the two-phase systems in several areas of biotechnology. The present paper summarizes the state of the art concerning extractive bioconversions for production of small as well as macromolecules, and protein purification using aqueous two-phase system. 

Figure 1. Principle for extractive bioconversion in aqueous two-phase systems, where the biocatalyst is temporarily immobilized in the droplets of one of the phases. Reproduced with permission from Ref. 7. Copyright 1984. Society of Chemical Industry.

One of the common side effects observed during extractive bioconversion is the accumulation of unwanted by-products in the system which may affect the productivity during continuous operation (14). The build up of glycerol and other non-volatile products was shown to decrease the ethanol yields during repeated fermentations in a two-phase system (12). The problem was, however, solved by dialysing the fermentation broth and also adding more yeast cells. It appears that the combination of ultrafiltration with the phase system may circumvent the problem of by-product inhibition in most of the cases. 

These studies, therefore, set the basis for a continuous system for extractive bioconversion of steroids. The interesting point that comes to notice is that the combination of two techniques such as aqueous two-phase separation, and adsorption, which are generally used individually for performing extractive bioconversions, could be advantageous for efficient product recovery in case of fine chemicals as well. 

The versatility and the potential of aqueous two-phase systems in future biotechnology has been amply demonstrated. The applications described here deal with extractive bioconversions, isolation and purification of proteins. Biochemical analyses in terms of binding assays have also been successfully applied in the two-phase systems... 

In membrane-based bioconversion systems, mass transfer resistance due to diffusion through the membrane and partition of the solutes over the membrane have to be added to the film resistances in the two Hquid phases. An overall mass transfer coefficient (K) can thus be defined for each solute, based on the individual resistances. The concentration profile of a solute being extracted through a membrane is a function of some physical characteristics of the membrane, namely its wettabiHty character, its geometry, porosity, and tortuosity, of the location of the aqueous and organic phases, and of the free diffusion coefficient of the solute, as evidenced in a detailed study performed by Prasad and Sirkar [189]. 

Membrane-assisted extractive bioconversions have been mainly developed for systems leading to toxic end-products, mainly short chain alcohols and acids. In... 

The effect of detoxification of the medium by removal of toxic compounds with UF membranes was demonstrated by Boyaval et al. [36] in the fermentation of propionic acid. UF runs led to an eightfold increase in volumetric productivity relative to fed batch experiments. The effectiveness of membrane bioreactors in the lowering of toxicity of the compounds involved in the bioconversion system was demonstrated by Edwards and co-workers [159]. An eightfold increase in the removal of phenoHc compounds from effluents was observed when polyphenoloxidase was immobilized in a capillary poly(ether)sulfone membrane as compared to the use of the free enzyme. Butanol recovery from the fermentation medium with organic solvent extraction or membrane solvent extraction led to similar results, both processes leading to decreased product inhibition. Due to the low toxicity of the extractive solvent used (isopropyl myristate) on Clostridium beyerinckii cells, no protective effect of the membrane was observed. However, precipitates observed in two-Hquid phase extraction were not observed... 

ILIX Li, X., Lian, Z., Dong, B., Xu, Y., Yong, Q., and Yu, S., Extractive bioconversion of xylan for production of xylobiose and xylotriose using a PEG 6000/sodium citrate aqueous two-phase system, Korean J. Chem. Eng., 28,1897, 2011. 

Li, M., Kim, J.-W., Peeples, T. L. (2002). Amylase partitioning and extractive bioconversion of starch using thermoseparating aqueous two-phase systems. Journal of Biotechnology, 93, 15-26. 

Biocatalysis. Biocatalysis, also termed biotransformation and bioconversion, makes use of natural or modified isolated enzymes, enzyme extracts, or whole-cell systems for the production of small molecules. A starting material is converted by the biocatalyst in the desired product. Enzymes are differentiated from chemical catalysts particularly with regard to stereoselectivity. 

The aqueous two-phase system can be applied to plant cell suspension cultures as an in situ extraction method to enhance the production of hydrophobic secondary metabolites or extracellular proteins such as enzymes. Selective removal of the product during bioconversion using plant cells may also be possible in ATPS. Prior to obtaining phy to chemicals with high yields using ATPS, however, reliable cultivation of plant cells in ATPS should be proven. 

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