Kaili Nie

Immobilized lipase Candida sp. 99-125 catalyzed methanolysis of glycerol trioleate: solvent effect.

The immobilized lipase Candida sp. 99-125 catalyzed methanolysis of glycerol trioleate was studied in twelve different solvents in order to deduce the solvent effect through an attempt to correlate the highest yield with such solvent properties as hydrophobicity (log P), dielectric constant (epsilon), and Hildebrand solubility parameter (delta). The results showed that the conversion of glycerol trioleate and yield of oleic acid methyl ester were quite dependent on the solvent. The catalyst lipase in various solvents also needed different optimum amount of water to keep its maximum activity, and generally this lipase in more hydrophobic solvents required more water. The correlation between the highest yield and log P value was found to be reasonable except deviation of data points of certain solvents, while no obvious correlation existed between the other two parameters, dielectric constant (epsilon) and Hildebrand solubility parameter (delta), and the enzyme activity. The study revealed that more hydrophobic solvents such as n-hexane or cyclohexane were more suitable solvents for Candida sp. 99-125 catalyzed transesterification of glycerol trioleate to oleic acid methyl ester.

Immobilization of lipase on macroporous resin and its application in synthesis of biodiesel in low aqueous media

Lipase from Candida sp. 99-125 was immobilized by physical adsorption onto macroporous resins. The results showed that the nonpolar resin NKA was the best carrier used in low aqueous media. 98.98% of degree of immobilization can be achieved when the adsorption procedure was performed in the presence of heptane. The hydrolytic activity and the apparent activity recovery of lipase adsorbed on resin in heptane was 4.07 and 3.43 times higher than that of lipase adsorbed in sodium phosphate buffer, respectively. The catalytic properties of immobilized lipase for production of biodiesel in low aqueous media were studied. Immobilized lipase displayed the highest activity when the crude enzyme/resin weight ratio was 1.92:1 and the water content(water/oil weight ratio) was 15% at 40 degrees C under pH 7.4. As lipase was adsorbed on NKA in heptane to produce biodiesel, the batch conversion rate can reach 97.3% when a three-step methanolysis protocol was used. After 19 consecutive batches, the conversion rate remained 70.2%.

Production of biodiesel by immobilized Candida sp. lipase at high water content

A new process for enzymatic synthesis of biodiesel at high water content (10–20%) with 96% conversion by lipase from Candida sp. 99–125 was studied. The lipase, a no-position-specific lipase, was immobilized by a cheap cotton membrane and the membrane-immobilized lipase could be used at least six times with high conversion. The immobilized lipase could be used for different oil conversion and preferred unsaturated fatty acids such as oleic acid to staturated fatty acids such as palmitic acid. The changes in concentration of fatty acids, diglycerides, and methyl esters in the reaction were studied and a mechanism of synthesis of biodiesel was suggested: the triglycerides are first enzymatically hydrolyzed into fatty acids, and then these fatty acids are further converted into methyl esters.

Synthesis of Wax Esters by Lipase-catalyzed Esterification with Immobilized Lipase from Candida sp. 99–125

Abstract Wax esters were synthesized in a solvent free system catalyzed by immobilized lipase from Candida sp. 99-125, with oleic acid and cetyl alcohol. The effects of substrate molar ratio, lipase dosage and water removal were investigated in a 50 ml flask incubated in a thermostatic cultivation cabinet. The optimized conditions were: temperature 40°C, shaking at 170 r·min−1, acid/alcohol molar ratio 1:0.9, lipase dosage in 10% (by mass) of oleic acid, and open reaction for water removal. As a result, the conversion rate reached 98% for reaction of 8 h. The volume of reactor was scaled up to 1 L three-neck flask. The optimized parameters were: 200 r·min−1 agitation speed, 2.5% (by mass) lipase dosage, others were the same as the parameters described above. The conversion rate reached 95% for reaction of 24 h. The lipase retained 46% conversion rate after reuse for 6, 7 batches. The products were purified by removing remained cetyl alcohol and fatty acids with ethanol and saturated sodium carbonate solution, respectively. The purity of the wax ester, cetyl oleate, was 96%. The physical and chemical properties of cetyl oleate were tested and compared with those of jojoba oil. The results show that the product cetyl oleate has great potential to use as the substitute of natural jojoba oil.

Production of fumaric acid from biodiesel-derived crude glycerol by Rhizopus arrhizus.

This work investigated the capability of Rhizopus arrhizus to assimilate biodiesel-derived crude glycerol and convert it into fumaric acid. After optimizing the initial glycerol concentration, spore inoculum and yeast extract concentration, smaller pellets (0.7 mm) and higher biomass (3.11 g/L) were obtained when R. arrhizus grew on crude glycerol. It was found that crude glycerol was more suitable than glucose for smaller R. arrhizus pellet forming. When 80 g/L crude glycerol was used as carbon source, the fumaric acid production of 4.37 g/L was obtained at 192 h. With a highest concentration of 22.81 g/L achieved in the co-fermentation of crude glycerol (40 g/L) and glucose (40 g/L) at 144 h, the fumaric acid production was enhanced by 553.6%, compared to the fermentation using glycerol (80 g/L) as sole carbon source. Moreover, the production cost of fumaric acid in co-fermentation was reduced by approximately 14% compared to glucose fermentation.

Synthesis of medium chain length fatty acid ethyl esters in engineered Escherichia coli using endogenously produced medium chain fatty acids

Microbial biosynthesis of fatty acid-derived biofuels from renewable carbon sources has attracted significant attention in recent years. Free fatty acids (FFAs) can be used as precursors for the production of micro-diesel. The expression of codon optimized two plants (Umbellularia californica and Cinnamomum camphora) medium-chain acyl-acyl carrier protein (ACP) thioesterase genes (ucFatB and ccFatB) in Escherichia coli resulted in a very high level of extractable medium-chain-specific hydrolytic activity and caused large accumulation of medium-chain free fatty acids. By heterologous co-expression of acyl-coenzyme A:diacylglycerol acyltransferase from Acinetobacter baylyi ADP1, specific plant thioesterases in E. coli, with supplementation of exogenous ethanol, resulted in drastic changes in fatty acid ethyl esters (FAEEs) composition ranging from 12:0 to 18:1. Through an optimized microbial shake-flask fermentation of two modified E. coli strains, yielded FFAs and FAEEs in the concentration of approximately 500 mg L(-1)/250 mg L(-1) and 2.01 mg g(-1)/1.99 mg g(-1), respectively. The optimal ethanol level for FAEEs yield in the two recombinant strains was reached at the 3% ethanol concentration, which was about 5.4-fold and 1.93-fold higher than that of 1% ethanol concentration.

Biodiesel production by combined fatty acids separation and subsequently enzymatic esterification to improve the low temperature properties.

The poor low-temperature properties of biodiesel, which provokes easy crystallization at low temperature, can cause fuel line plugging and limits its blending amount with petro-diesel. This work aimed to study the production of biodiesel with a new process of improving the low temperature performance of biodiesel. Waste cooking oil was first hydrolyzed into fatty acids (FAs) by 60g immobilized lipase and 240g RO water in 15h. Then, urea complexation was used to divide the FAs into saturated and unsaturated components. The conditions for complexation were: FA-to-urea ratio 1:2 (w/w), methanol to FA ratio 5:1 (v/v), duration 2h. The saturated and unsaturated FAs were then converted to iso-propyl and methyl esters by lipase, respectively. Finally, the esters were mixed together. The CFPP of this mixture was decreased from 5°C to -3°C. Hydrolysis, urea complexation and enzymic catalyzed esterification processes are discussed in this paper.

Biosynthesis of medium chain length alkanes for bio-aviation fuel by metabolic engineered Escherichia coli

The aim of this work was to study the synthesis of medium-chain length alkanes (MCLA), as bio-aviation product. To control the chain length of alkanes and increase the production of MCLA, Escherichia coli cells were engineered by incorporating (i) a chain length specific thioesterase from Umbellularia californica (UC), (ii) a plant origin acyl carrier protein (ACP) gene and (iii) the whole fatty acid synthesis system (FASs) from Jatropha curcas (JC). The genetic combination was designed to control the product spectrum towards optimum MCLA. Decanoic, lauric and myristic acid were produced at concentrations of 0.011, 0.093 and 1.657mg/g, respectively. The concentration of final products nonane, undecane and tridecane were 0.00062mg/g, 0.0052mg/g, and 0.249mg/g respectively. Thioesterase from UC controlled the fatty acid chain length in a range of 10-14 carbons and the ACP gene with whole FASs from JC significantly increased the production of MCLA.