Property control of sophorolipids: influence of fatty acid substrate and blending.

Sophorolipids (SLs) were synthesized by fed-batch fermentation of Candida bombicola on glucose and either palmitic acid (SL-p), stearic acid (SL-s), oleic acid (SL-o) or linoleic acid (SL-l) and the structural distribution accurately determined by atmospheric pressure chemical ionization-mass spectrometry (APCI-MS). The surfactant properties, including critical micelle concentration (CMC), minimum surface tension (min.ST) and oil-water interfacial tension (IFT) were measured by tensiometry. Minimum STs of 35-36 mN/m were obtained regardless of the substrate while IFTs ranged from 3-5 mN/m with the exception of SL-l, which had an IFT of 7 mN/m. The largest disparity occurred in the CMC values, which ranged from 35 ppm for SL-s to 250 ppm for SL-l. By manually mixing these four SLs in different ratios, it was possible to better control the CMC values without affecting the min.ST or IFT, which will prove beneficial as new applications for SLs are established.

Synthesis and characterization of canola oil-stearic acid-based trans-free structured lipids for possible margarine application.

Incorporation of stearic acid into canola oil to produce trans-free structured lipid (SL) as a healthy alternative to partially hydrogenated fats for margarine formulation was investigated. Response surface methodology was used to study the effects of lipozyme RM IM from Rhizomucor miehei and Candida rugosa lipase isoform 1 (LIP1) and two acyl donors, stearic acid and ethyl stearate, on the incorporation. Lipozyme RM IM and ethyl stearate gave the best result. Gram quantities of SLs were synthesized using lipozyme RM IM, and the products were compared to SL made by chemical catalysis and fat from commercial margarines. After short-path distillation, the products were characterized by GC and RPHPLC-MS to obtain fatty acid and triacylglycerol profiles, 13C NMR spectrometry for regiospecific analysis, X-ray diffraction for crystal forms, and DSC for melting profile. Stearic acid was incorporated into canola oil, mainly at the sn-1,3 positions, for the lipase reaction, and no new trans fatty acids formed. Most SL products did not have adequate solid fat content or beta' crystal forms for tub margarine, although these may be suitable for light margarine formulation.

Catalytic synthesis of biodiesel from high free fatty acid-containing feedstocks.

Recyclable and reusable heterogeneous diarylammonium catalysts are highly effective in catalyzing the esterification of the free fatty acid (FFA) present in greases to methyl esters to reduce the FFA content from 12-40 wt% to 0.5-1 wt%; the resulting ester-glyceride mixture (pretreated grease) could then be readily converted to methyl esters by base-catalyzed transesterification.

Simplified soy molasses-based medium for reduced-cost production of sophorolipids by Candida bombicola.

A simplified medium containing only soy molasses and oleic acid as ingredients was developed for the production of sophorolipids (SLs) from Candida bombicola. We achieved a product yield of 53 +/- 3 g of purified sophorolipids per liter of starting culture volume, which is 71 +/- 4% of the yield obtained with growth medium that also additionally contains the costly yeast extract and urea as nitrogen source. The large majority of the SL components existed in the lactone form (87%), and the predominant component is SL containing (omega-1)-hydroxyoleic acid as the lipid moiety. The study demonstrated for the first time the usefulness of the low-value soy molasses as a combined nitrogen- and carbon-source for SL production at a reduced cost.

Conversion of agricultural feedstock and coproducts into poly(hydroxyalkanoates).

Aside from their importance to the survival and general welfare of mankind, agriculture and its related industries produce large quantities of feedstocks and coproducts that can be used as inexpensive substrates for fermentative processes. Successful adoption of these materials into commercial processes could further the realization of a biorefinery industry based on agriculturally derived feedstocks. One potential concept is the production of poly(hydroxyalkanoate) (PHA) polymers, a family of microbial biopolyesters with a myriad of possible monomeric compositions and performance properties. The economics for the fermentative production of PHA could benefit from the use of low-cost agricultural feedstocks and coproducts. This mini-review provides a brief survey of research performed in this area, with specific emphasis on studies describing the utilization of intact triacylglycerols (vegetable oils and animal fats), dairy whey, molasses, and meat-and-bone meal as substrates in the microbial synthesis of PHA polymers.

The use of fatty acid esters to enhance free acid sophorolipid synthesis.

Fatty acid esters were prepared by transesterification of soy oil with methanol (methyl-soyate, Me-Soy), ethanol (ethyl-soyate, Et-Soy) and propanol (propyl-soyate, Pro-Soy) and used with glycerol as fermentation substrates to enhance production of free-acid sophorolipids (SLs). Fed-batch fermentations of Candida bombicola resulted in SL yields of 46 +/- 4 g/l, 42 +/- 7 g/l and 18 +/- 6 g/l from Me-Soy, Et-Soy, and Pro-Soy, respectively. Liquid chromatography with atmospheric pressure ionization mass spectrometry (LC/API-MS) showed that Me-Soy resulted in 71% open-chain SLs with 59% of those molecules remaining esterified at the carboxyl end of the fatty acids. Et-Soy and Pro-Soy resulted in 43% and 80% open-chain free-acid SLs, respectively (containing linoleic acid and oleic acid as the principal fatty acid species linked to the sophorose sugar at the omega-1 position), with no evidence of residual esterification.

Biosynthesis of medium-chain-length poly(hydroxyalkanoates) from soy molasses.

Pseudomonas corrugata was selected from a screening process for the bioconversion of inexpensive soy molasses into medium-chain-length poly(hydroxyalkanoates) (mcl-PHA). We obtained yields of 1.5 g cell dry weight (CDW)/l culture with growth medium supplemented with 2% (w/v) soy molasses, and of an average of 3.4 g CDW/l with 5% (w/v) soy molasses. Crude PHAs were obtained at 5-17% of CDW. The most prominent repeat-unit monomers in the PHAs were 3-hydroxydodecanoate, 3-hydroxyoctanoate, 3-hydroxydodecanoate, and 3-hydroxytetradecenoate. This work represents the first description of fermentative mcl-PHA production from the soy molasses.

A process model to estimate biodiesel production costs.

'Biodiesel' is the name given to a renewable diesel fuel that is produced from fats and oils. It consists of the simple alkyl esters of fatty acids, most typically the methyl esters. We have developed a computer model to estimate the capital and operating costs of a moderately-sized industrial biodiesel production facility. The major process operations in the plant were continuous-process vegetable oil transesterification, and ester and glycerol recovery. The model was designed using contemporary process simulation software, and current reagent, equipment and supply costs, following current production practices. Crude, degummed soybean oil was specified as the feedstock. Annual production capacity of the plant was set at 37,854,118 l (10 x 10(6)gal). Facility construction costs were calculated to be US dollar 11.3 million. The largest contributors to the equipment cost, accounting for nearly one third of expenditures, were storage tanks to contain a 25 day capacity of feedstock and product. At a value of US dollar 0.52/kg (dollar 0.236/lb) for feedstock soybean oil, a biodiesel production cost of US dollar 0.53/l (dollar 2.00/gal) was predicted. The single greatest contributor to this value was the cost of the oil feedstock, which accounted for 88% of total estimated production costs. An analysis of the dependence of production costs on the cost of the feedstock indicated a direct linear relationship between the two, with a change of US dollar 0.020/l (dollar 0.075/gal) in product cost per US dollar 0.022/kg (dollar 0.01/lb) change in oil cost. Process economics included the recovery of coproduct glycerol generated during biodiesel production, and its sale into the commercial glycerol market as an 80% w/w aqueous solution, which reduced production costs by approximately 6%. The production cost of biodiesel was found to vary inversely and linearly with variations in the market value of glycerol, increasing by US dollar 0.0022/l (dollar 0.0085/gal) for every US dollar 0.022/kg (dollar 0.01/lb) reduction in glycerol value. The model is flexible in that it can be modified to calculate the effects on capital and production costs of changes in feedstock cost, changes in the type of feedstock employed, changes in the value of the glycerol coproduct, and changes in process chemistry and technology.

Synthesis of short-/medium-chain-length poly(hydroxyalkanoate) blends by mixed culture fermentation of glycerol.

Glycerol was used as a substrate in the bio-production of poly(hydroxyalkanoates) (PHAs) in an effort to establish an alternative outlet for glycerol and produce value-added products. Pseudomonas oleovorans NRRL B-14682 and Pseudomonas corrugata 388 grew and synthesized poly(3-hydroxybutyrate) (P3HB) and medium-chain-length PHA (mcl-PHA) consisting primarily of 3-hydroxydecanoic acid (C(10:0); 44 +/- 2 mol %) and 3-hydroxydodecenoic acid (C(12:1); 31 +/- 2 mol %), respectively, from glycerol at concentrations up to 5% (v/v). Cellular productivity maximized at 40% for P. oleovorans in 5% (v/v) glycerol and 20% for P. corrugata in 2% (v/v) glycerol after 72 h. Increasing the glycerol media concentration from 1% to 5% (v/v) caused a 61% and 72% reduction in the molar mass (M(n)) of the P3HB and mcl-PHA polymers, respectively. Proton-NMR analysis of the glycerol-derived P3HB revealed that the M(n) decrease was the result of esterification of glycerol onto the polymer in a chain terminating position. However, no evidence of glycerol-based chain termination was present in the mcl-PHA. The growth patterns of P. oleovorans and P. corrugata on glycerol permitted their use as mixed cultures to produce natural blends of P3HB and mcl-PHA. By incorporating a staggered inoculation pattern and varying the duration of the fermentations, P3HB/mcl-PHA ratios were achieved that varied from 34:66 to 96:4.

Production of sophorolipids by Candida bombicola grown on soy molasses as substrate.

Sophorolipids (SLs) were produced from Candida bombicola using soy molasses and oleic acid as co-substrates. The purified SLs were obtained at 21 g l(-1) and were 97% in lactone form. The major SL constituent (81% relative abundance) of the product mixture contains an oleoyl chain. The surface properties of the SLs obtained from the soy molasses/oleic acid fermentation had minimum surface-tension values of 37 mN m(-1) (pH 6) and 38 mN m(-1) (pH 9), and critical micelle concentration values of 6 mg l(-1) (pH 6) and 13 mg l(-1) (pH 9).

Transesterification activity of lipases immobilized in a phyllosilicate sol-gel matrix.

Lipases from Pseudomonas cepacia (P.c.) and Thermomyces lanuginosa (T.l.) were immobilized in a phyllosilicate sol-gel matrix and studied for their ability to catalyze the alcoholysis of fats and oils to simple alkyl esters. At 50 degrees C and 48 h reaction immobilized T.l. lipase gave higher alkyl ester yields (70 to 100%) from fats and oils regardless of chain length or degree of unsaturation of the acyl groups in the triacylglycerols than did immobilized P.c. lipase (20-90%), which preferred unsaturated oils. Both immobilized lipases catalyzed ester formation (80-90%) from greases containing a range of free fatty acids (2.6 to 36%). Molecular sieves had no effect on ester yields in the immobilized T.l. lipase-catalyzed alcoholysis of greases but did improve yields (5-10%) in the immobilized P.c. lipase-catalyzed reactions.

LC/MS analysis and lipase modification of the sophorolipids produced by Rhodotorula bogoriensis.

The extracellular glycolipids produced by the yeast, Rhodotorula bogoriensis (formerly Candida bogoriensis), were analyzed using an LC/API-MS method. The analysis confirmed that the predominant form the sophorolipid structure contained a C22 hydroxy carboxylic acid. A minor amount (<10%) of a C24 hydroxy carboxylic acid in the sophorolipid was also found, which had not been reported previously. The sophorolipid product, which contained varying degrees of acetylation at the primary hydroxy groups of the sophorose sugar, was deacetylated with sodium methoxide. The des-acetylated sophorolipid was esterified using an immobilized lipase as catalyst in tetrahydrofuran and the product analyzed by mass spectrometric techniques. The product was screened for dimer or polymer formation but only a monomeric lactonized sophorolipid structure was detected.

Optimization of alkyl ester production from grease using a phyllosilicate sol-gel immobilized lipase.

Simple alkyl ester derivatives of restaurant grease were prepared using a lipase from Pseudomonoas cepacia immobilized within a phyllosilicate sol-gel matrix as biocatalyst. Alcoholysis reactions of grease were carried out in solvent-free media using a one-step addition of alcohol to the reaction mixture. The immobilized lipase was active from 40 to 70 degrees C. Ester yields (60-97%) were highest when using a ratio of reactants of 2 mmol grease to 8 mmol alcohol and the biocatalyst was 10% (w/w) of grease in the presence of molecular sieves.

Enzymatic synthesis of a galactopyranose sophorolipid fatty acid-ester.

Sophorolipid lactones produced by Candida bombicola were deacetylated and ring opened with sodium methoxide to their corresponding methyl esters. The methyl esters, after re-acetylation with vinyl acetate using an immobilized lipase, were transesterified with 1,2-3,4-di-O-isopropylidene-D-galactopyranose in tetrahydrofuran using the same lipase catalyst. The di-O-isopropylidene sophorolipid sugar esters were hydrolyzed to give the galactopyranose sophorolipid esters as the final products.

Regioselective enzyme-catalyzed synthesis of sophorolipid esters, amides, and multifunctional monomers.

Novel enzyme-mediated synthetic routes were developed to provide a new family of sophorolipid derivatives and glycolopid-based amphiphilic monomers. These compounds are of great interest for their potential use in immunoregulation, as well as for other biological properties. In the present work, an efficient lipase-catalyzed conversion of sophorolipid ethyl ester to (a) the 6'-monoacylated derivatives using Novozym 435, (b) 6' '-monoacylated derivatives using Lipase PS-C, (c) secondary amide derivatives using Novozym 435, and (d) 6',6' '-diacylated amide derivatives using Novozym 435 in an one-pot reaction and (e) the regioselective monoacylation of an amide derivative at the 6'- and 6' '-positions using Novozym 435 and Lipase PS-C, respectively, are described. The ethyl ester produced by esterification of the sophorolipid mixture with sodium ethoxide was subjected to acylation catalyzed by Novozym 435 in dry tetrahedrofuran (THF) with vinyl acetate and vinyl methacrylate to produce 6'-monoacylated derivatives. In contrast, Lipase PS-C catalyzed acylations of sophorolipid ethyl ester in dry THF with vinyl acetate and vinyl methacrylate to give the corresponding 6'-monoacylated derivatives. Novozym 435 mediated amidation of sophorolipid ethyl ester in dry THF with phenethylamine, tyramine, p-methoxyphenethylamine, 2-(p-tolyl)ethylamine, and p-fluorophenethylamine generated the corresponding secondary amides but not tertiary amides. The formation of diacyl derivatives of amides was achieved by their treatment with vinyl acetate and vinyl methacrylate in dry THF using Novozym 435 as catalyst. The conversion of sophorolipid ethyl ester to the same diacyl derivatives of amide (i.e., both amidation and acylation) in high yield was also demonstrated in dry THF by a one-pot reaction using Novozym 435. Furthermore, regioselective monoacylation of a sophorolipid amide at 6' and 6' ' in dry THF with vinyl acetate and vinyl methacrylate using Novozym 435 and Lipase PS-C was also demonstrated.

Isolation of unsaturated diols after oxidation of conjugated linoleic acid with peroxygenase.

Oat seeds are a rich source of peroxygenase, an iron heme enzyme that participates in oxylipin metabolism in plants. An isomer of CLA, 9(Z),11 (E)-octadecadienoic acid (1), believed to have anticarcinogenic activity, was used as a substrate for peroxygenase in an aqueous medium using t-butyl hydroperoxide as the oxidant. After acidification of the reaction medium, the products were extracted with ethyl ether, converted to their methyl esters, and characterized using HPLC. Major products after reaction for 24 h showed resonances from 1H NMR spectroscopy that were further downfield than the expected epoxides and were thought to be diol hydrolysis products. However, analyses by HPLC with atmospheric pressure chemical ionization MS (APCI-MS) of the putative allylic diols or their bis-trimethylsilyl ether derivatives gave incorrect M.W. The M.W. of the diols could be obtained by APCI-MS after removal of unsaturation by hydrogenation or by EI-MS after conversion of the allylic 1,2-diols to cyclic methyl boronic esters. Data from MS in conjunction with analyses using 1H and 13C NMR showed that the methylated products from 1 were methyl 9,10(threo)-dihydroxy-11 (E)-octadecenoate, methyl 9,10(erythro)-dihydroxy-11(E)-octadecenoate, methyl 9,12(erythro)-dihydroxy-10(E)-octadecenoate, and methyl 9,12(threo)-dihydroxy-10(E)-octadecenoate. Solid-phase extraction without prior acidification and conversion of the products to methyl esters allowed identification of the following epoxides: methyl 9,10(Z)-epoxy-11 (E)-octadecenoate (6M), methyl 9,10(E)-epoxy-11 (E)-octadecenoate, and methyl 11,12(E)-epoxy-9(Z)-octadecenoate. At times of up to at least 6 h, 6M accounted for approximately 90% of the epoxide product. Product analysis after the hydrolysis of isolated epoxide 6M showed that hydrolysis of epoxide 6 could largely account for the diol products obtained from the acidified reaction mixtures.

Immobilized lipase-catalysed production of alkyl esters of restaurant grease as biodiesel.

Simple alkyl ester derivatives of restaurant grease were prepared using immobilized lipases as biocatalysts. The lipases studied included those of Thermomyces lanuginosa and Candida antarctica supported on granulated silica (gran- T.l. and gran- C.a., respectively), C. antarctica supported on a macroporous acrylic resin (SP435) and Pseudomonas cepacia immobilized within a phyllosilicate sol-gel matrix (IM PS-30). All alcoholysis reactions were carried out in solvent-free media employing a one-step addition of the alcohol to the reaction system. Of the lipases studied, IM PS-30 was found to be the most effective in catalysing the methanolysis and ethanolysis of grease. The processes catalysed by gran- T.l. and gran- C.a. lipases gave poor conversions to esters, and the SP435-catalysed reactions gave intermediate yields of ethyl and methyl esters. Water activity (a(w)) was an important factor in the methanolysis reactions; reaction media with a(w)<0.5 resulted in the highest conversions to methyl esters. Molecular sieves also improved methyl ester yields by as much as 20% in transesterification reactions catalysed by IM PS-30. The immobilized lipases also were evaluated for their ability to produce alkyl esters of grease with several additional normal and branched-chain alcohols.

Physiological characterization and genetic engineering of Pseudomonas corrugata for medium-chain-length polyhydroxyalkanoates synthesis from triacylglycerols.

Pseudomonas belonging to the rRNA-DNA homology group I produce medium-chain-length (mcl)-polyhydroxyalkanoates (PHA). We show that P. corrugata, a member of this group, accumulates 0.5-1.0 g of mcl-PHA/L of culture when grown on glucose (Gl) or oleic acid (Ol). The predominant monomers of Gl-PHA and Ol-PHA are beta-hydroxydecanoate and beta-hydroxyoctanoate, respectively. The molecular masses and polydispersity of P. corrugata PHAs are higher than those typically found with other Pseudomonas. We electrotransformed P. corrugata with a plasmid pCN51lip-1 carrying Pseudomonas lipase genes to generate strain III111-1. The recombinant strain grew on intact triacylglycerols (TAGs) to 1.9-2.7 g of cell-dry-weight/L of culture. The yields and the predominant repeat-units of PHAs obtained from the lard- and tallow-grown III111-1 were similar to those of Ol-PHA from wild-type cells. In contrast to other Pseudomonas species, P. corrugata III111-1 grown on TAGs at temperatures up to 36 degrees C was not significantly affected with regard to cell yields, amounts of PHA produced, and the repeat unit compositions of the polymer.

Purification of lipoxygenase from Chlorella: production of 9- and 13-hydroperoxide derivatives of linoleic acid.

Oxygenation of linoleic acid by the enzyme lipoxygenase (LOX) that is present in the microalga Chlorella pyrenoidosa is known to produce the corresponding 9- and 13-hydroperoxide derivatives of linoleic acid (9- and 13-HPOD, respectively). Previous work with this microalga indicated that partially purified LOX, present in the 30-45 and 45-80% saturated (NH4)2SO4 precipitate fractions, produced both HPOD isomers but in different ratios. It was not clear, however, if the observed activity in the two isolates represented the presence of one or more isozymes. In the present work, LOX isolated from the intracellular fraction of Chlorella by (NH4)2SO4 precipitation (35-80% saturated) was purified by ion exchange and hydrophobic interaction chromatography to apparent homogeneity. Analysis of the purified protein by SDS-PAGE and subsequent native size exclusion chromatography demonstrated that LOX in Chlorella is a single monomeric protein with a molecular mass of approximately 47 kDa. The purified LOX produced both the 9-HPOD and 13-HPOD isomers from linoleic acid in equal amounts, and the isomer ratio was not altered over the pH range of 6 to 9. Optimal activity of LOX was at pH 7.5.