Adrie J. J. Straathof

The production of fine chemicals by biotransformations

Today, biocatalysis is a standard technology for the production of chemicals. An analysis of 134 industrial biotransformations reveals that hydrolases (44%) and redox biocatalysts (30%) are the most prominent categories. Most products are chiral (89%) and are used as fine chemicals. In the chemical industry, successful product developments involve on average a yield of 78%, a volumetric productivity of 15.5 g/(L.h) and a final product concentration of 108 g/L. By contrast, the pharmaceutical industry focuses on time-to-market. The implications of this for future research and development on biocatalysis are discussed.

Fumaric acid production by fermentation

The potential of fumaric acid as a raw material in the polymer industry and the increment of cost of petroleum-based fumaric acid raises interest in fermentation processes for production of this compound from renewable resources. Although the chemical process yields 112% w/w fumaric acid from maleic anhydride and the fermentation process yields only 85% w/w from glucose, the latter raw material is three times cheaper. Besides, the fermentation fixes CO2. Production of fumaric acid by Rhizopus species and the involved metabolic pathways are reviewed. Submerged fermentation systems coupled with product recovery techniques seem to have achieved economically attractive yields and productivities. Future prospects for improvement of fumaric acid production include metabolic engineering approaches to achieve low pH fermentations.

The enantiomeric ratio : origin, determination and prediction

Abstract The enantiomeric ratio E =(kcatR/KmR)/(kcatS/KmS) offers a concise representation of the enantioselective properties of an enzyme in reactions that involve chiral compounds. Both as a measure of the intrinsic selectivity of the catalyst, and as a parameter to model the performance of enzymatic processes for the production of enantiopure fine-chemicals, its merits have been well-recognized. Several methods for the determination of E exist. The scope and limitations of these methods are evaluated in terms of accuracy and feasibility. There appears to be no single method that is both reliable and readily applicable in all cases. Complementary methods, however, are available. The outstanding characteristics of the enantiomeric ratio as a quantitative measure of the effects of physical and chemical conditions on the intrinsic enantioselectivity of enzymes are presented in terms of the difference in Gibbs energies of the diastereomeric enzyme-substrate transition states. The prospects of molecular modeling strategies for the prediction of E are discussed.

A simple method to determine the enantiomeric ratio in enantioselective biocatalysis

The enantiomeric ratio (E) is commonly used to characterize the enantioselectivity in enzyme-catalyzed kinetic resolution. In this paper this parameter is directly derived from the enantiomeric excess of substrate and product. This is formally more correct than using Chens equation after calculating the degree of conversion from both ee values using the relation of Sih and Wu. New expressions and useful graphs have been generated for reversible and irreversible uni-uni reactions. The theoretical predictions have been verified experimentally for various reactions. Values for E and the thermodynamic equilibrium constant, KEQ, were obtained for a (DL)-dehalogenase-catalyzed dehalogenation, a hydrolysis reaction by porcine pancreatic lipase, and for C. Cylindracea lipase-catalyzed esterification and transesterification. In view of the current developments in the field of chiral analysis, this method is an easily available tool in the quantitative treatment of enzyme-catalyzed resolution of enantiomers.

Recovery of carboxylic acids produced by fermentation

Carboxylic acids such as citric, lactic, succinic and itaconic acids are useful products and are obtained on large scale by fermentation. This review describes the options for recovering these and other fermentative carboxylic acids. After cell removal, often a primary recovery step is performed, using liquid-liquid extraction, adsorption, precipitation or conventional electrodialysis. If the carboxylate is formed rather than the carboxylic acid, the recovery process involves a step for removing the cation of the formed carboxylate. Then, bipolar electrodialysis and thermal methods for salt splitting can prevent that waste inorganic salts are co-produced. Final carboxylic acid purification requires either distillation or crystallization, usually involving evaporation of water. Process steps can often be combined synergistically. In-situ removal of carboxylic acid by extraction during fermentation is the most popular approach. Recovery of the extractant can easily lead to waste inorganic salt formation, which counteracts the advantage of the in-situ removal. For industrial production, various recovery principles and configurations are used, because the fermentation conditions and physical properties of specific carboxylic acids differ.

Feasibility of acrylic acid production by fermentation

Acrylic acid might become an important target for fermentative production from sugars on bulk industrial scale, as an alternative to its current production from petrochemicals. Metabolic engineering approaches will be required to develop a host microorganism that may enable such a fermentation process. Hypothetical metabolic pathways for insertion into a host organism are discussed. The pathway should have plausible mass and redox balances, plausible biochemistry, and plausible energetics, while giving the theoretically maximum yield of acrylate on glucose without the use of aeration or added electron acceptors. Candidate metabolic pathways that might lead to the theoretically maximum yield proceed via β-alanine, methylcitrate, or methylmalonate-CoA. The energetics and enzymology of these pathways, including product excretion, should be studied in more detail to confirm this. Expression of the selected pathway in a host organism will require extensive genetic engineering. A 100,000-tons/year fermentation process for acrylic acid production, including product recovery, was conceptually designed based on the supposition that an efficient host organism for acrylic acid production can indeed be developed. The designed process is economically competitive when compared to the current petrochemical process for acrylic acid. Although the designed process is highly speculative, it provides a clear incentive for development of the required microbial host, especially considering the environmental sustainability of the designed process.

Influence of fermentation by-products on the purification of ethanol from water using pervaporation

Pervaporation is claimed to be a promising separation technique for the purification of ethanol from fermentation broths during bio-ethanol production. In this study, influence of fermentation by-products on the purification of ethanol from water during hydrophobic pervaporation was investigated. Sugars and salts were found to increase the membrane performance. Reason for this was a change in vapor/liquid equilibrium. 2,3-butanediol decreased the ethanol flux and selectivity factor, while glycerol exhibited no effect. This was explained by a strong sorption of butanediol into PDMS and no sorption of glycerol. Due to the presence of carboxylic acids, hydrophobicity degree of the Pervap 4060 membrane decreased, which resulted in an irreversible increase in water flux and decrease in separation performance. These observations suggested the presence of silicalite-based fillers in the membrane. When the pH was raised to a value above the dissociation constant, no changes in hydrophobicity degree and membrane performance were found.

Reductions of 3-oxo esters by baker's yeast : Current status

The objective of the current review is to present a mechanism and process engineering approach of stereospecific reductions of 3-oxo esters by bakers yeast. The stereospecific outcome of a reduction by bakers yeast depends on the kind of 3-oxo ester reductases involved and their specific activity. Various competing 3-oxo ester reductases are present in a yeast cell. An important aspect for efficient biotransformations with whole cells is the regeneration of NADH and NADPH cofactors. Use of different electron donors leads to the involvement of different metabolic routes influencing the reduction process. Optimization of the process conditions such as aeration, immobilization of cells, use of additives, or use of two phases, will enhance re-use of bakers yeast, yield, stereospecific outcome and scale up. Since the genome of bakers yeast is known, genetic engineering will soon increase the possibilities of stereoselective reductions.

Development of a low pH fermentation strategy for fumaric acid production by Rhizopus oryzae

Dicarboxylic acids that are produced from renewable resources are becoming attractive building blocks for the polymers industry. In this respect, fumaric acid is very interesting. Its low aqueous solubility facilitates product recovery. To avoid excessive waste salt production during downstream processing, a low pH for fumaric acid fermentation will be beneficial. Studying the influence of pH, working volume and shaking frequency on cell cultivation helped us to identify the best conditions to obtain appropriate pellet morphologies of a wild type strain of Rhizopus oryzae. Using these pellets, the effects of pH and CO(2) addition were studied to determine the best conditions to produce fumaric acid in batch fermentations under nitrogen-limited conditions with glucose as carbon source. Decreasing either the fermentation pH below 5 or increasing the CO(2) content of the inlet air above 10% was unfavourable for the cell-specific productivity, fumaric acid yield, and fumaric acid titer. However, switching off the pH control late in the batch phase did not affect these performance parameters and allowed achieving pH of 3.6. A concentration of 20 gL(-1) of fumaric acid was obtained at pH 3.6 while the average cell mass specific productivity and fumaric acid yield were the same as at pH 5.0. Consequently, relatively modest amounts of inorganic base were required for pH control, while recovery of the acid should be relatively easy at pH 3.6.

Fundamental bottlenecks in the application of continuous bioprocesses

Continuous culture is applied mainly as a research tool and much less as a production process. Fundamental bottlenecks in continuous culture are discussed to help shed light on this apparent contradiction. Based on a discussion of technical, process related, and economic/market bottlenecks it is concluded that the often mentioned productivity argument in favor of continuous processing is much too simple. The optimal choice of a process mode is determined by a full understanding of the equipment and production plant factors and of the economic/market factors. Very often the resulting choice will be the fed batch and/or the cell retention process mode which is characterized by low growth rates. Therefore more research towards product formation at low growth rates (less than 0.05 h-1) is needed.