Yu-Yen Linko

Effect of temperature and various nitrogen sources on L(+)-lactic acid production by Lactobacillus casei

Abstract Two homofermentative strains, Lactobacillus casei NRRL B-441 and Lactobacillus casei subsp. rhamnosus NRRL B-445 were selected for further study from 17 lactic acid bacterial strains screened for lactic acid production. The effect of temperature on lactic acid production with the selected strains was investigated by adapting both strains to four different temperatures. The production of L(+)-lactic acid by both strains was most efficient at 37°C, although with L. casei the highest lactic acid concentration was obtained at 41°C. The maximal volumetric productivity with L. casei was 4.1 g l-1 h-1 and with L. casei subsp. rhamnosus 3.5 g l-1 h-1. The composition of the medium was studied in order to replace the costly yeast extract with less expensive sources of nitrogen and amino acids. From 11 different nitrogen sources investigated at 37°C, barley malt sprouts (88 g l-1 lactic acid in 66 h) and grass extract (74 g l-1 lactic acid in 73 h) were the best economic alternatives. The effect of different combinations of yeast extract, peptone and malt sprouts was further studied by using statistical experimental design, and an empirical second-order polynomial model was constructed on the basis of the results. With the right combination most of the yeast extract could be substituted by barley malt sprouts for efficient lactic acid production. A method for extraction of nutrients and growth factors from malt sprouts is also described.

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.

An investigation of crude lipases for hydrolysis, esterification, and transesterification

Abstract Nine commerically available powdered lipases were investigated for their catalytic ability to hydrolyze olive oil and synthesize 1-butyl oleate by direct esterification and 2-ethyl-1-hexyl ester of rapeseed oil by transesterification. Under the experimental conditions used, a lipase from Candida rugosa exhibited the highest hydrolytic activity at 88 U mg−1 enzyme. Lipase from Pseudomonas fluorescens gave the highest conversion of oleic acid at about 95% in 24 h. Furthermore, lipases from C. rugosa and Rhizopus sp. resulted in the highest conversion of rapeseed oil at nearly 100%. Porcine pancreatic lipase showed the lowest hydrolytic activity at 1.5 U mg−1 enzyme and also the lowest synthetic activity with the conversions of oleic acid and rapeseed oil at only 50% and 19% respectively. For the lipase from Rhizomucor miehei, only esterification and transesterification activities were related. Finally, for the lipase from Chromobacterium viscosum, no relationship between the hydrolytic and synthetic activities was observed. The low multiple correlation coefficients in the order of R = 1 0.35–0.40 obtained from the regression analysis for the hydrolytic and synthetic activities for all lipases studied suggested little relationship between the hydrolytic and synthetic activities; however, the high multiple correlation coefficient of R = 0.97∗∗∗ for the conversion of oleic acid by esterification and rapeseed oil by transesterification by eight of the nine lipases studied suggested that there was a close relationship between esterification and transesterification. According to the results, the hydrolytic lipase activity may be of little value in predicting the synthetic activity, and in extreme cases, a lipase may exhibit no synthetic activity while possessing a high hydrolytic activity.

Simultaneous liquefaction, saccharification, and lactic acid fermentation on barley starch

Abstract It has been demonstrated that a l (+)-lactic acid concentration as high as 162 g l −1 can be obtained from barley starch in a relatively short processing time of less than 48 h with a balanced simultaneous liquefaction, saccharification, and fermentation. Yields of 98 and 87% were obtained from 130 and 170 g l −1 starch, respectively. A 10% inoculum was determined to be optimal under the experimental conditions investigated.

Biotechnology of bread baking

Biotechnology includes the application of a wide variety of biological, biochemical, bioengineering, genetic, microbiological and control techniques. The baking of yeast-leavened and sourdough breads represents one of the oldest biotechnical processes, together with the brewing of beer, sake and wine, and the production of yoghurt and cheese, etc. A modern baking process may take advantage of biotechnology in its widest sense, from the improvement of cereal grains and starter cultures by recombinant DNA technology, through the use of enzymes as processing aids, to application of the most advanced batch and continuous fermentation technologies.

Industrial Applications of Immobilized Cells

Although the application of the natural attraction of many microorganisms to surfaces has been applied in vinegar production since the early 1980s, and has long been utilized in waste water purification, the development of microbial cell immobilization techniques for special applications dates back only to the early 1960s. The immobilization may involve whole cells, cell fragments, or lysed cells. Whole cells may retain their metabolic activity with their complex multienzyme systems and cofactor regeneration mechanisms intact, or they may be killed in the process with only a few desired enzymes remaining active in the final biocatalyst. Cells may also be coimmobilized with an enzyme to carry out special reactions. Although relatively few industrial scale applications exist today, some are of very large scale. Current applications vary from relatively small scale steroid conversions to amino acid production and high fructose syrup manufacture. A vast number of potential applications are already known, and one of the most interesting applications may be in continuous fermentation such as ethanol production by immobilized living microorganisms. 373 references.

Production of l-lactic acid with immobilized Lactobacillus delbrueckii

SummaryLiving Lactobacillus delbrueckii cells were entrapped in calcium alginate gel beads and employed both in recycle batch and continuous column reactors to produce l-lactic acid from glucose. The substrate contained l% (w/v) yeast extract as nutrient and 4.8% (w/v) solid calcium carbonate as buffer. The maxiumum lactic acid yield obtained was 97%, of which more than 90% was l-lactic acid. The biocatalyst activity half-life in continuous operation was about 100 d, and only about 10% of the activity was lost during intermittent storage of the bioreactor at +7°C for about 5 months.

Lipase-catalyzed linear aliphatic polyester synthesis in organic solvent

Abstract Enzymatic polymerization of bis(2,2,2-trifluoroethyl) sebacate and aliphatic diols was investigated with the purpose of obtaining a polyester of as high as possible average molecular weight. The reaction was carried out at 37°C, and vacuum was applied to shift the equilibrium forward by removal of the 2,2,2-trifluoroethanol formed. The results showed that the elimination of alcohol formed was critical for a high degree of polymerization. Among the four lipases studied, Mucor miehei lipase was the best biocatalyst for polytransesterification and diphenyl ether was the best solvent for the polymerization of bis(2,2,2-trifluoroethyl) sebacate with 1,4-butanediol. The highest average molecular weight ( M w ) of 46,400 g mol−1 (degree of polymerization = 184) was obtained when 0.51 m (187 g l−1) bis(2,2,2-trifluoroethyl) sebacate was polymerized with 0.51 m (46 g l−1) 1,4-butanediol in diphenyl ether for 168 h (7 days) with 36.5% M. miehei lipase powder.

Lipase-catalyzed transesterification of rapeseed oil and 2-ethyl-1-hexanol

Lipase-catalyzed transesterification (alcoholysis) of lowerucic acid rapeseed oil and 2-ethyl-1-hexanol without an additional organic solvent was studied in stirred batch reactors. Of a number of commercially available enzymes investigated, the best results were obtained with aCandida rugosa lipase. The optimal transesterification conditions were an oil/alcohol molar ratio of 1∶2.8, a minimum of 1.0% (w/w) added water, and with a temperature of 37–55°C. Under the optimal conditions, a nearly complete conversion was obtained in one hour with 14.6% (w/w) lipase, whereas 0.3% (w/w) lipase required 10 h for similar results. The enzyme was inactivated at 60°C.

Lipase biocatalysis in the production of esters

Lipase biocatalysis was investigated as a tool for the production of butyl oleate and rapeseed oil 2-ethyl-1-hexyl ester by esterification and transesterification, respectively. We screened 25 commercially available lipases and found that butyl oleate was produced at high yields from oleic acid and 1-butanol by lipases fromCandida rugosa, Chromobacterium viscosum, Rhizomucor miehei, and Pseudomonas fluorescens. The initial water content of the system, lipase quantity, and the molar ratio of 1-butanol to oleic acid were important factors in influencing the ester yield. In general, no ester was formed without the addition of water. The exception wasCh. viscosum lipase, which yielded 98% of ester in 12 h with 1-butanol excess without additional water. The addition of 3.2% water increased the initial rate of reaction. With an oleic acid excess and only 0.3% lipase,C. rugosa andR. miehei lipases yielded 94 and 100% esters with initial water contents of 3.2 and 14%, respectively. Lipase-catalyzed alcoholysis of low-erucic acid rapeseed oil and 2-ethyl-1-hexanol without additional organic solvent also was studied in stirred batch reactors. In this case,C. rugosa lipase was the best biocatalyst with an optimal 2-ethyl-1-hexanol to rapeseed oil molar ratio of 2.8, a minimum of 1.0% added water, and 37°C. An increase in temperature up to 55°C increased the rate of reaction but did not affect the final ester yield. The enzyme was inactivated at 60°C. Under optimal conditions, the ester yield increased from 88% in 7 h to nearly complete conversion in 1 h when the lipase content was increased from 0.3 to 14.6%. In a 2-kg small pilot scale, up to 90% conversion (97% of theoretical) was obtained in 8 h at 37°C with 3.4% lipase in the presence of Amberlite XAD-7 resin with 3% added water.