Optimisation of media and cultivation conditions for L(+)(S)-lactic acid production by Lactobacillus casei NRRL B-441.

Process variables and concentration of carbon in media were optimised for lactic acid production by Lactobacillus casei NRRL B-441. Lactic acid yield was inversely proportional to initial glucose concentration within the experimental area (80-160 g l(-1)). The highest lactic acid concentration in batch fermentation, 118.6 g l(-1), was obtained with 160 g 1(-1) glucose. The maximum volumetric productivity, 4.4 g 1(-1) h(-1) at 15 h, was achieved at an initial glucose concentration of 100 g l(-1). Similar lactic acid concentrations were reached with a fedbatch approach using growing cells, in which case the fermentation time was much shorter. Statistical experimental design and response surface methodology were used for optimising the process variables. The temperature and pH optima for lactic acid production were 35 degrees C, pH 6.3. Malt sprout extract supplemented with yeast extract (4 g l(-1)) appeared to be an economical alternative to yeast extract alone (22 g l(-1)) although the fermentation time was a little longer. The results demonstrated both the separation of the growth and lactic acid production phases and lactic acid production by non-growing cells without any nutrient supplements. Resting L. casei cells converted 120 g l(-1) glucose to lactic acid with 100% yield and a maximum volumetric productivity of 3.5 g l(-1) h(-1).

Refolding of denatured/reduced lysozyme at high concentration with diafiltration.

Refolding of reduced and denatured protein in vitro has been an important issue for both basic research and applied biotechnology. Refolding at low protein concentration requires large volumes of refolding buffer. Among various refolding methods, diafiltration is very useful to control the denaturant and red/ox reagents in a refolding solution. We constructed a refolding procedure of high lysozyme concentration (0.5-10 mg/ml) based on the linear reduction of the urea concentration during diafiltration under oxygen pressure. When the urea concentration in the refolding vessel was decreased from 4 M with a rate of 0.167 M/h, the refolding yields were 85% and 63% at protein concentrations, 5 mg/ml and 10 mg/ml, respectively, after 11 h. This method gave a high productivity of 40.1,microM/h of the refolding lysozyme. The change in refolding yields during the diafiltration could be simulated using the model of Hevehan and Clark.

Kinetic Analysis of the Autoxidation of Ethyl Eicosapentaenoate at Different Oxygen Levels.

The autoxidation of such n-3 polyunsaturated fatty acid (PUFA) ethyl esters as ethyl eicosapentaenoate and ethyl docosahexaenoate was investigated at various temperatures. Extensive studies of the oxidative reaction for ethyl eicosapentaenoate were carried out at different oxygen levels. At the same oxygen level and temperature, the autocatalytic reaction rate, by which oxidation progressed in the first half period, was about 1.5-2.5 times larger than the first-order reaction rate which governed the oxidation in the second half period. The reaction rate constants for ethyl eicosapentaenoate at different oxygen levels correlated well with the Langmuir-type equation of the oxygen concentration, in which the Langmuir parameter was independent of temperature.

Kinetic Analysis of Temperature-Programmed Autoxidation of Ethyl Eicosapentaenoate and Ethyl Docosahexaenoate.

On the basis of an autocatalytic and a first order reaction kinetics, a nonisothermal oxidation reaction model was developed for n-3 polyunsaturated fatty acid (PUFA) under temperature-programmed linear heating conditions. With this model, the activation energy of oxidative reaction can easily be obtained with at least three linear heating conditions. The temperature-programmed oxidation experiments of ethyl eicosapentaenoate and ethyl docosahexaenoate were done under linear heating conditions of 4 to 12 K/h. The activation energies and the frequency factors obtained were in good agreement with those by the isothermal oxidation experiments reported previously.

Biodegradable products by lipase biocatalysis.

The interest in the applications of biocatalysis in organic syntheses has rapidly increased. In this context, lipases have recently become one of the most studied groups of enzymes. We have demonstrated that lipases can be used as biocatalyst in the production of useful biodegradable compounds. A number of examples are given. 1-Butyl oleate was produced by direct esterification of butanol and oleic acid to decrease the viscosity of biodiesel in winter use. Enzymic alcoholysis of vegetable oils without additional organic solvent has been little investigated. We have shown that a mixture of 2-ethyl-1-hexyl esters can be obtained in a good yield by enzymic transesterification from rapeseed oil fatty acids for use as a solvent. Trimethylolpropane esters were also similarly synthesized as lubricants. Finally, the discovery that lipases can also catalyze ester syntheses and transesterification reactions in organic solvent systems has opened up the possibility of enzyme catalyzed production of biodegradable polyesters. In direct polyesterification of 1,4-butanediol and sebacic acid, polyesters with a mass average molar mass of the order of 56,000 g mol-1 or higher, and a maximum molar mass of about 130,000 g mol-1 were also obtained by using lipase as biocatalyst. Finally, we have demonstrated that also aromatic polyesters can be synthesized by lipase biocatalysis, a higher than 50,000 g mol-1 mass average molar mass of poly(1,6-hexanediyl isophthalate) as an example.

The Increased Effect of Kneading on the Formation of Inclusion Complexes between d-Limonene and ß-Cyclodextrin at Low Water Content.

The molecular inclusion powder of d-limonene in ß-cyclodextrin was prepared by using a twin-screw kneader at a low water content. The influence of water and alcohol content on the formation of the inclusion complex was studied in comparison with the inclusion complex obtained by the micro-aqueous method. The inclusion fraction of d-limonene in the complex powder in ß-cyclodextrin was much higher than that made by the micro-aqueous method. The marked differences in the inclusion fraction were observed particularly at the molar water ratio to ß-cyclodextrin of less than 10. The inclusion fraction increased sharply with the increase in the kneading torque. This means that the energy of kneading increased the inclusion of d-limonene. A mathematical model for estimating the inclusion fraction was derived by combining the effects of water and the kneading energy on the formation of the inclusion complex.

Formation of Inclusion Complexes of Cycldextrin with Ethanol under Anhydrous Conditions.

Complex formation of poorly water soluble organic compounds with cyclodextrin (CD) is quite difficult in an aqueous cyclodextrin system. Formation of the inclusion complex of d-limonene, phenyl ethanol, acetophenone, or menthol was investigated in a slurry form of α-, ß-, or γ-CD in organic solvents or alcohol under anhydrous conditions. Ethanol and methanol were found to be good solvents for this method. The use of ethanol as the solvent was investigated in greater detail. There existed an optimal amount of ethanol for the maximum inclusion of d-limonene as the guest compound. However, an excess of ethanol inhibited the inclusion. An adsorption model of alcohol on CD, analogous to the substrate inhibition model of enzyme kinetics, could correlate the inclusion ratio with the amount of alcohol added to CD.

Continuous itaconic acid production by immobilized biocatalysts.

The continuous itaconic acid production from sucrose with Aspergillus terreus TKK 200-5-3 mycelium immobilized on polyurethane foam cubes was optimized in column bioreactors using statistical experimental design and empirical modelling. The highest itaconic acid product concentration calculated on the basis of the obtained model was 15.8 g l-1 in the investigated experimental area, when sucrose concentration was 13.5%, aeration rate 150 ml min-1 and residence time 178 h. From sucrose with immobilized A. terreus TKK 200-5-3 mycelium itaconic acid production was stable for at least 4.5 months in continuous column bioreactors. In comparison, using glucose as substrate and immobilized A. terreus TKK 200-5-1 mycelium as biocatalyst similar stability was obtained with higher product concentration. The omission of copper sulphate from the production medium gave the highest itaconic acid product concentration (26 g l-1) from 9% glucose with 0.25% ammonium nitrate and 0.095% magnesium sulphate.

Citric acid production with alginate bead entrappedAspergillus niger ATCC 9142.

Aspergillus niger ATCC 9142 mycelium was entrapped in calcium alginate beads and employed in an air-lift completely stirred reactor for continuous production of citric acid. Maximum yield obtained from 10% (w/v) sucrose was 12 g dm(-3) with about 40% fermentation efficiency. Maximum rate of production 70 mg g(-1) h(-1) was about five times that obtained in classical batch fermentation.