Solvent-free enzymatic synthesis of structured lipids containing CLA from coconut oil and tricaprylin

Lipase-catalyzed acidolysis of different TAG with CLA was performed to produce structured lipids (SL) containing CLA. An immobilized lipase from Mucor miehei (Lipozyme IM, Novo Nordisk Inc., Franklinton, NC) was used as the biocatalyst in a solvent-free system. Conconut oil and tricaprylin, which are sources of medium-chain FA, were the starting substrates, and a mixture of FFA (MFFA) containing 73% CLA was the donor of the acyl groups. For each TAG, four different ratios of TAG/MFFA were blended to prepare about 500 g of mixture containing 10, 20, 30, and 40% CLA (w/w). Each blend was reacted with 5% lipase at 65°C for 48 h under nitrogen. Over the range of TAG/MFFA ratios examined, CLA was incorporated effectively by the enzyme. Lipozyme IM exhibited no special preference for any particular FA, since the incorporation of FA was proportional to their concentration in the system. FFA, PV, p-anisidine value (p-AV), iodine value (IV), and saponification number (SN) were evaluated for all the SL. FFA, PV, and p-AV depended on the purification process and showed no significant deterioration of SL with respect to the original TAG, whereas IV and SN depended on the composition of the SL, mainly the CLA content.     

The effects of emulsified polydimethylsiloxane FG‐10 on the oxygen transfer coefficient (kLa) and lipase production by Staphylococcus warneri EX17

BACKGROUND: In this study the effects of the addition of emulsified polydimethylsiloxane (PMDS) FG‐10 on the oxygen transfer coefficient (k_La) of submerged cultures of Staphylococcus warneri EX17 and its lipase production is described. FG‐10 is an emulsified silicone capable of dissolving 50 times more oxygen than water. The combined effects of FG‐10 concentration and different conditions of agitation were optimized in bioreactors using statistical design tools, and the cultures were run using raw glycerol from biodiesel synthesis as the sole carbon source. RESULTS: The optimal conditions found to improve lipase production were FG‐10 concentration of 11.2% (v/v) and speed agitation of 527 rpm, respectively, producing around 861 U L^?1 of lipolytic activity, a maximal cell concentration of 8.4 g L^?1, and a k_La of 99 h^?1, values that are approximately 3 times higher than cultures without FG‐10. CONCLUSIONS: This is the first report in the literature on the use of this class of chemicals as oxygen carriers in microbial cultures and its effect on k_La and lipase production, demonstrating the potential use of FG‐10 in microbial cultures. Copyright © 2012 Society of Chemical Industry     

Enhanced microbial oil production by activated sludge microorganisms via co‐fermentation of glucose and xylose

The co‐fermentation of glucose and xylose by activated sludge microorganisms for the production of microbial oils for use as biodiesel feedstock was investigated. Various carbon sources at initial concentration of 60 g/L and C:N ratio 70:1 were investigated: xylose, glucose, and 2:1 and 1:2 (by mass) glucose/xylose mixtures. Oil accumulation ranged between 12 to 22% CDW, the highest of which was obtained when xylose was the sole substrate used. Kinetic modeling of the fermentation data showed that specific growth and oil accumulation rates were similar in all substrate types and the lipid coefficient ranged from 0.02 to 0.06 g/g of sugar consumed. The fatty acid methyl ester yield and composition of the lipids showed their suitability for conversion to biodiesel. Based on the results, lignocellulose sugars could be used as fermentation substrates by activated sludge microorganisms for enhancing the oil content of sewage sludge for its use as a sustainable biofuel feedstock source. © 2013 American Institute of Chemical Engineers AIChE J, 59: 4036–4044, 2013     

Utilization of molasses for biosurfactant production by two Bacillus strains at thermophilic conditions

Traditionally, biosurfactants have been produced from hydrocarbons. Some possible substitutes for microbial growth and biosurfactant production include urban wastes, peat hydrolysate, and agro-industrial by-products. Molasses, a nonconventional substrate (agro-industrial by-product) can also be used for biosurfactant production. It has been utilized by two strains of Bacillus subtilis (MTCC 2423 and MTCC1427) for biosurfactant production and growth at 45°C. As a result of biosurfactant accumulation, the surface tension of the medium was lowered to 29 and 31 dynes/cm by the two strains, respectively. This is the first report of biosurfactant production by strains of B. subtilis at 45°C. Potential application of the biosurfactant in microbial enhanced oil recovery is also presented.     

酿酒酵母工程菌发酵产青蒿酸的工艺优化

通过酿酒酵母工程菌(Saccharomyces cerevisiae 1211)发酵制备青蒿酸,并通过单因素实验和响应面优化,考察了发酵温度、pH、半乳糖质量浓度、发酵碳源及发酵氮源等对青蒿酸发酵产量的影响。结果表明:在发酵温度30℃,发酵培养基初始pH=5.5,发酵培养基中蔗糖质量浓度91.8 g/L,半乳糖质量浓度10.1 g/L,硫酸铵质量浓度10.3 g/L,磷酸二氢钾质量浓度8.7 g/L的条件下,青蒿酸发酵产量可达(1529.7±12.6)mg/L,与未优化时的发酵产量相比,提升了67.1%。     

Scale-Up studies of non-aerated fed-batch fermentation of dextransucrase and the industrial synthesis of dextran using the enzymatic route

High activities of the enzyme dextransucrase were repeatedly produced using slowly agitated non-aerated fed-batch fermentations of Leuconostoc mesenteroides B-512(F). Activities in excess of 24.0 U cm^?3 were obtained consistently in a 16 dm³ laboratory fermenter using a 6 dm³ initial work volume. Yeast extract type was identified to be one of the important factors influencing the enzyme yield. Studies on aerating the medium with different gases indicated that the presence of carbon dioxide in the medium favoured high enzyme production. Agitation rates did not appear to have significant effects on either cell growth or enzyme production. One type of antifoam (silicone antifoam) was observed to affect enzyme production but not the cell growth. Scale-up of the non-aerated process was carried out up to a 1000 dm³ scale with enzyme broths containing up to 21.0 U cm^?3 being produced. Two batches of the enzyme that were produced at the large scale were used for the first time to synthesize dextran at a 50000 dm³ industrial scale. The dextran yields were up to 95.5% of the conventional industrial yields and were achieved in much shorter reaction time intervals.     

Medium optimization for lipid production through co‐fermentation of glucose and xylose by the oleaginous yeast Lipomyces starkeyi

Co‐fermentation of lignocellulose‐based carbohydrates is a potential solution to improve the economics of microbial lipid production. In the present paper, experiments were performed to optimize the media composition for lipid production by the oleaginous yeast Lipomyces starkeyi AS 2.1560 through co‐fermentation of glucose and xylose (2 : 1 wt/wt). Statistical screening of nine media variables was performed by a Plackett–Burman design. Three factors, namely mixed sugar, yeast extract and FeSO₄, were found as significant components influencing cellular lipid accumulation. Further optimization was carried out using a Box–Behnken factorial design to study the effects of these three variables on lipid production. A mathematical model with the R² value at 96.66% was developed to show the effect of each medium composition and their interactions on the lipid production. The model estimated that a maximal lipid content of 61.0 wt‐% could be obtained when the concentrations of mixed sugar, yeast extract and FeSO₄ were at 73.3 g/L (glucose 48.9 g/L, xylose 24.4 g/L), 7.9 g/L and 4.0 mg/L, respectively. The predicted value was in good accordance with the experimental data of 61.5%. Compared with the initial media, the optimized media gave 1.59‐fold and 2.03‐fold increases for lipid content and lipid productivity, respectively.     

High cell density fermentation via a metabolically engineered Escherichia coli for the enhanced production of succinic acid

BACKGROUND: Succinic acid is a valuable four‐carbon organic chemical with applications in many fields. It was found that cell mass was an important factor in succinic acid production by metabolically engineered Escherichia coli strains. In this work, high cell density fermentation was investigated for succinic acid production by a metabolically engineered strain SD121 with ldhA, pflB, ptsG mutation and heterogenous cyanobacterial ppc overexpression. RESULTS: Under two‐stage cultivation, the controlled DO feeding strategy during the aerobic growth phase facilitated biomass up to a dry cell weight of 19.6 g L^?1, and enhanced succinic acid production in the following anaerobic fermentation phase to a concentration of 116.2 g L^?1. A near theoretical maximum succinic acid yield of 1.73 mol mol^?1 glucose was achieved with an average productivity of 1.55 g L^?1 h^?1. CONCLUSION: The results indicated the potential advantage of high cell density fermentation for improvement of succinic acid production by E. coli. Copyright © 2010 Society of Chemical Industry     

发酵法生产甘油的技术经济评述

介绍了甘油的国内外市场状况及发酵法生产甘油的技术进展，对１０００ｔ／ａ的发酵法甘油装置进行了经济评价。     

One-step methodology for the synthesis of FA picolinyl esters from intact lipids

Picolinyl derivatives are used for structural determination of FA by GC-MS. Although they provide reliable diagnostic fragments, the usual multistep methodologies applied for their preparation require TAG hydrolysis or acid chloride formation prior to picolinyl synthesis. These reaction conditions may result in the presence of artifact molecules in the samples and thus compromise analytical quality and accuracy. To address these problems, a rapid, simple and quantitative methodology for the synthesis of FA picolinyl esters from intact lipids was developed. It involves their transesterification under basecatalyzed conditions using 3-potassiooxamethylpyridine in methylene chloride. The catalyst was prepared by proton exchange between potassium tert-butoxide and anhydrous 3-hydroxymethylpyridine. Mild reaction conditions allowed complete derivatization of TAG and phospholipids in 2 min at room temperature, and of FAME in 15 min at 45°C. The proposed procedure, which can be used on a routine basis, was applied to Ipomoae imperialis seed lipids and used to confirm occurrence of γ-linoleic acid at a level of 0.9%.     

Studies on the contamination of feed molasses for continuous ethanol production in an immobilised yeast cell reactor

With the present world problem of fossil fuel limitations, alcohol production from renewable resources such as sugar cane molasses, particularly by continuous processes, has become important. In continuous processing, feed contamination problems could become critical. A useful inexpensive technique, which can minimise these problems when molasses is used as the substrate, is to ensure that the time period elapsing after dilution of molasses and before feeding to the reactor is minimal. Experiments were conducted to determine how long the clarified molasses feed solution could be kept under non-sterile conditions without affecting the performance of an immobilised whole cell reactor for alcohol production. For this purpose, the fastest growing contaminant (under the prevailing conditions) present in molasses feed solution was isolated by plating technique. From the initial total number of contaminant microorganisms in the feed solution, and from the experimentally determined generation time of the fastest growing contaminant cells, the safe period of storage of diluted molasses was calculated. This value was in reasonable agreement with the experimental observations.     

Effects of selected substrate forms on the synthesis of structured lipids by two immobilized lipases

Two immobilized lipases, IM 60 from Rhizomucor miehei and SP 435 from Candida antarctica, were used to synthesize structured lipids (SL). Tricaprin and trilinolein were interesterified to produce SL that contained one linoleic acid per triacylglycerol molecule (SL1) and SL with two linoleic acids (SL2). SL1 and SL2 were separated by silver nitrate thin-layer chromatography according to their unsaturation, and the fatty acid at the sn-2 position was determined after pancreatic lipasecatalyzed hydrolysis of SL1 and SL2. With IM 60, 57.7 mol% capric acid and 42.3 mol% linoleic acid were found at the sn-2 position of SL1, while 43.3 mol% capric acid and 56.7 mol% linoleic acid were at the sn-2 position of SL2. The fatty acid at the sn-2 position of SL1 with SP 435 as biocatalyst was 43.6 mol% capric acid and 56.4 mol% linoleic acid, while SL2 contained 56.6 mol% capric acid and 43.4 mol% linoleic acid. Different structural forms of the capric acid-containing substrate (triacylglycerol vs. ethyl ester) and different chainlengths of triacylglycerol were selected to study the substrate selectivity of lipases. Results indicated that SP 435 had some degree of preference for the triacylglycerol form (tricaprin), and IM 60 produced SL more rapidly and reached steady state faster with tricaprin as substrate than with capric acid ethyl ester. For chainlength selectivity, mol% of synthesized SL from tricaprin + trilinolein and tristearin + trilinolein were compared. SP 435 exhibited no apparent preference for either tricaprin or tristearin. However, IM 60 showed a more rapid reaction with tricaprin than with tristearin.     

Assessment of lipase- and chemically catalyzed lipid modification strategies for the production of structured lipids

The purpose of the present study was to devise a two-step reaction to produce partial glycerides, which would subsequently be used as substrates in both lipase-catalyzed and chemically catalyzed esterification reactions with caprylic acid. The yields and kinetics of these two-step reactions were compared to established lipase-catalyzed acidolysis and transesterification as well as to chemical transesterification reactions. Acyl migration did not occur during the hydrolysis or short-path distillation steps in the preparation of free fatty acid-free partial glycerides for esterification reactions. No significant differences in final yields (59.9% to 82.8% w/w of total triacylglycerols) of new structured lipids were detected among lipase-catalyzed (24 h) and chemically catalyzed (5 h) reactions; however, the yield of new structured triacylglycerols (TAG) after 2 h was lower for acidolysis than for the other lipase-catalyzed reactions (P≤0.05). Since no differences in final yields were detected among the reactions, chemical esterification using hydrolyzed oil could represent the best synthetic option, since it offers the advantage of positional distribution control associated with lipase-catalyzed reactions as well as rapid reaction times associated with chemically catalyzed reactions. Attempts to evaluate the positional distribution of caprylic acid using allyl magnesium bromide (Grignard reagent) were hampered by production of unknown species, which prevented accurate determination of the concentration of some key fatty acids.     

Fatty acid vinyl esters as acylating agents: A new method for the enzymatic synthesis of monoacylglycerols

Lipase-catalyzed synthesis of monoacylglycerols (MAG) was performed by transesterification reactions between fatty acid vinyl esters and either glycerol (1) or 1,2-O-isopropylidene-rac-glycerol (2), without solvents or in the presence ofn-pentane. Vinyl decanoate, vinyl laurate, vinyl stearate and vinyl palmitate have been converted to the corresponding monoacylglycerols. As expected for the reaction with1, a mixture of mono-, di- and triacylglycerols was synthesized. The highest concentrations of MAG were achieved with vinyl stearate (30% 2-MAG and 15% 1-MAG). The reactions of fatty acid vinyl esters with the protected glycerol (2) led to the corresponding protected 3-monoacylglycerols with 100% conversion after short reaction times. The subsequent cleavage of these acetonides was performed by four different methods. The fastest cleavage was found with trifluoroacetic acid as catalyst, whereas the highest concentration of MAG (100%) was obtained for the boric acid-catalyzed hydrolysis of the acetonides.     

Identification of naturally isolated Southern Louisiana's algal strains and the effect of higher CO2 content on fatty acid profiles for biodiesel production

BACKGROUND: Microalgae, with both high biomass productivity and oil content, are regarded as attractive candidates for the production of alternative biodiesel as well as for CO₂ biofixation. In the present study, four microalgal strains native to southeastern Louisiana's waters were isolated and identified to evaluate their potential for the production of biodiesel. Selected strains were identified through genomic DNA in sequencing of either 16S rRNA or 18S rRNA genes followed by lipid and fatty acid content characterization and quantification. RESULTS: High correlation was found with known nucleotide sequence identities at 98% with Sellaphora pupula, and 99% with Synechococcus sp., Chlorella sorokiniana, Scenedesmus abundans, and Chlorella vulgaris (control). The fatty acid profiles of these organisms changed when using 5% CO₂ aeration. Total fatty acids (TFA) decreased from 20.63 to 17.62, 54.83 to 24.4, and 29.82 to 23.99 g kg^?1 in Synechococcus sp., Sellaphora pupula and Chlorella sorokiniana, respectively. TFA increased from 14.14 to 31.49 and 15.14 to 47.52 g kg^?1 dry biomass in Scenedesmus abundans and Chlorella vulgaris (control), respectively. CONCLUSION: Chlorella sorokiniana, with a lower C18:3 and the highest biomass yield at 5% CO₂ aeration, was found to be the best candidate for biodiesel production. © 2012 Society of Chemical Industry