微水相超声波协同固定化脂肪酶催化酯交换过程优化

超声波协同固定化脂肪酶催化制备生物柴油的最佳工艺条件为：超声波功率70W、叔丁醇为反应介质、叔丁醇用量3％（v／v）、醇油比3：1且甲醇分三批加入、反应温度40℃、水含量为2％（v／v）。副产物甘油对固定化脂肪酶使用寿命影响最大,使用后的固定化脂肪酶用丙酮洗去表面的甘油,进行酯交换反应,酶的稳定性大为提高,可连续使用16批次。     

荧光假单胞菌脂肪酶固定化及催化制备生物柴油的工艺研究

研究了不同因素对制备固定化荧光假单胞菌脂肪酶的影响及固定化酶的酶学性质,并初步探讨了利用该固定化酶制备生物柴油的工艺。以海藻酸钠明胶为复合载体,采用包埋法制备固定化荧光假单胞菌脂肪酶,考察了载酶量、颗粒直径等因子对固定化效果的影响,并用制备的固定化酶进行了酶促酯交换合成生物柴油的工艺研究,考察了反应条件如酶量、反应温度、甲醇流加方式、醇油比等因素对甲酯得率的影响。试验结果表明,制备固定化荧光假单胞菌脂肪酶的最优条件为:每克载体给酶量为300 IU,选用6号注射器针头(内径为0.5 mm);通过酯交换,催化大豆油合成生物柴油的最佳反应工艺参数为:固定化酶25%,醇油比4:1,含水量6%,反应温度40℃;此条件下反应35 h后,甲酯的最高得率可达82%。     

复合脂肪酶协同催化制备生物柴油的研究

【目的】探讨复合酶协同催化体系在含水量较高的体系中催化油脂制备生物柴油的工艺条件。【方法】通过基因工程手段在毕赤酵母中分别高效分泌表达南极假丝酵母脂肪酶(CALB)和米根霉脂肪酶(ROL),构建CALB和ROL复合酶协同催化体系制备生物柴油,利用单因素实验优化工艺条件,以甲酯化得率作为复合酶协同催化体系效能的评价标准。【结果】优化工艺条件为:CALB?ROL最佳复合酶配比为7?3,每克大豆油中加入16 U的复合脂肪酶,甲醇与大豆油摩尔比为4?1,并按0 h时2?1醇油摩尔比,12 h和24 h时以1?1醇油摩尔比分批加入甲醇,含水量为30%-60%之间,40°C反应29-34 h,甲酯得率达到93%。【结论】该复合酶协同催化体系对环境友好,与常规酶法制备生物柴油工艺相比对酶的使用量和催化时间减少幅度都在50%以上,本复合酶协同催化体系能有效降低生物柴油制备成本,具有较好的工业化应用前景。     

脂肪酶催化合成生物柴油的瓶颈问题及其对策研究进展

生物柴油,一种新型的清洁能源燃料,具有可再生、可生物降解、环境友好等优良的品性,可部分或全部替代石化柴油。碱催化法、脂肪酶催化法及超临界法是合成生物柴油的主要工艺,其中脂肪酶催化法是一种节能型、环保型工艺,在节能和环保方面,有着碱催化法无可比拟的优越性,具有良好的工业应用前景。但目前在实现产业化的进程中仍存在如酶成本高、稳定性较差、甲醇对酶的失活效应及反应时间长等瓶颈问题。通过固定化技术和全细胞催化剂的采用、甲醇流加方式的改进、溶剂工程的改善及酰基受体和耐醇酶的开发等技术手段,结合固定床生物反应器,较好地解决了这些瓶颈问题,从而推进了酶催化法合成生物柴油的工业化进程。本文主要对酶法合成生物柴油工艺存在的主要问题及相应对策研究进展进行概括介绍,并对其工业化发展前景进行讨论。     

固定化脂肪酶催化花椒籽皮油转酯化反应研究

采用固定化脂肪酶催化花椒籽皮油制备生物柴油,研究了该转酯化反应的工艺条件.结果表明:在脂肪酶用量25%(质量分数).含水量10%(质量分数),正己烷用量15%(质量分数).醇油比3:1.分三次添加甲醇,于反应温度45℃下反应时间24 h,固定化脂肪酶使花椒籽皮油的棕榈酸甲酯的转化率达到82.5%.     

固定化脂肪酶催化制备生物柴油条件优化

本文探讨了以固定化脂肪酶为催化剂催化制备生物柴油中醇油比、水含量、游离脂肪酸酸值和催化剂使用寿命对菜子油酯交换反应的影响,并与以NaOH、固体碱纳米水滑石为催化剂生物柴油的制备条件相比较.研究表明:固定化脂肪酶为催化剂所需最佳醇油比最低,仅为4:1,游离脂肪酸含量对酯交换反应影响甚微,且有较强的抗水性,固定化脂肪酶催化剂可可重复使用6次;NaOH为催化剂酯交换反应抗水性最强,随游离脂肪酸的增加,酯交换转化率显著降低;纳米水滑石为催化剂可重复使用5次,酯交换产物易分离,所得产品完全符合德国生物柴油标准.     

海滨锦葵油制备生物柴油工艺条件优化

以海滨锦葵油为原料制备生物柴油。通过单因素试验及正交试验研究了反应温度、催化剂用量、醇油摩尔比、反应时间、搅拌强度等因素对酯交换率的影响。结果表明,在试验范围内各影响因素对酯交换率作用的大小依次为：搅拌强度＞催化剂用量＞醇油摩尔比＞反应时间＞反应温度。海滨锦葵油制备生物柴油的最佳工艺参数为：搅拌强度为1800r.min-1,催化剂KOH用量为海滨锦葵油质量的1%,醇油摩尔比6/1,反应时间60min,反应温度65℃,在该工艺条件下,酯交换反应三次,酯交换率达到97.8%。     

脂肪酶协同催化猪油合成生物柴油工艺研究

探讨了以乙酸甲酯为酰基受体两种脂肪酶协同催化猪油转酯合成生物柴油的工艺条件。首先利用单因子试验确定2种固定化脂肪酶Novozym435、Lipozyme TLIM单独作为催化剂时的最佳酶用量为40％,反应温度为50℃,乙酸甲酯用量为14（相对于油的摩尔比）。在此基础上,采用3因素5水平和3个中心点的中心组分旋转设计法研究了上述2种脂肪酶协同使用时脂肪酶用量（g/g）、混合酶的配比（%/%）以及乙酸甲酯用量诸因素共同作用对转酯反应转化率的影响。优化后的反应条件为：总酶用量为40％,混合酶配比为50/50,乙酸甲酯用量为14,在该条件下甲酯得率可达97.6％,比同质量的Novozym435、Lipozyme TLIM的催化活性分别高出7.6％、22.3％。表明脂肪酶协同催化猪油合成生物柴油工艺可以较好地提高甲酯得率,并且节约生产成本。     

大孔树脂共吸附固定葡聚糖-脂肪酶催化合成香茅酯

以大孔树脂为载体对脂肪酶和葡聚糖进行共吸附固定,考察葡聚糖的共吸附对脂肪酶固定化效果的影响,并应用所得固定化酶在无溶剂体系催化合成月桂酸香茅酯。结果表明:在固定化过程中添加终质量浓度为0.75mg/m L的葡聚糖可提高固定化酶酶活回收率,使用该固定化酶在无溶剂体系催化月桂酸与香茅醇酯化,酶的催化效率及操作稳定性均有提高。在底物月桂酸与香茅醇物质的量的比为1∶1,加入1 U的固定化脂肪酶,在50℃时无溶剂体系中反应10 h,反应的酯化率达95.3%。添加终质量浓度为0.75 mg/m L的T-20及T-40(葡聚糖相对分子质量为2×10~4和4×10~4)制备的固定化酶可将到达95%酯化率的反应时间缩短至6 h,其中添加T-40的固定化酶经10次连续催化后,仍保持75%以上的催化活性。     

固定化脂肪酶催化毛棉籽油制备生物柴油

研究了固定化脂肪酶Lipozyme TL IM和Novozym435催化毛棉籽油和乙酸甲酯制备生物柴油的过程。通过向反应体系中添加甲醇,可减少乙酸的抑制,明显提高生物柴油得率,确定最佳反应条件为:正己烷作溶剂,乙酸甲酯与油摩尔比9:1,添加油重3%的甲醇、油重10%的LipozymeTLIM和5%的Novozym435复合使用,温度55°C,反应8h,生物柴油得率达到91.83%。最后探索了酶催化毛棉籽油合成生物柴油的动力学,得到动力学方程。     

Biodiesel production—current state of the art and challenges

Biodiesel is a clean-burning fuel produced from grease, vegetable oils, or animal fats. Biodiesel is produced by transesterification of oils with short-chain alcohols or by the esterification of fatty acids. The transesterification reaction consists of transforming triglycerides into fatty acid alkyl esters, in the presence of an alcohol, such as methanol or ethanol, and a catalyst, such as an alkali or acid, with glycerol as a byproduct. Because of diminishing petroleum reserves and the deleterious environmental consequences of exhaust gases from petroleum diesel, biodiesel has attracted attention during the past few years as a renewable and environmentally friendly fuel. Since biodiesel is made entirely from vegetable oil or animal fats, it is renewable and biodegradable. The majority of biodiesel today is produced by alkali-catalyzed transesterification with methanol, which results in a relatively short reaction time. However, the vegetable oil and alcohol must be substantially anhydrous and have a low free fatty acid content, because the presence of water or free fatty acid or both promotes soap formation. In this article, we examine different biodiesel sources (edible and nonedible), virgin oil versus waste oil, algae-based biodiesel that is gaining increasing importance, role of different catalysts including enzyme catalysts, and the current state-of-the-art in biodiesel production. JIMB 2008: BioEnergy—special issue.     

An effective acid catalyst for biodiesel production from impure raw feedstocks

Biodiesel consists of fatty acids short chain alkyl esters produced through transesterification and esterification of fats and oils. Production of biodiesel is strongly affected by the purity of raw lipids, and catalysts play important role in these processes. Although direct utilization of impure feedstocks is more economical, their use necessitates development of effective catalysts to overcome hindering influences of impurities. In this study, sulfuryl chloride, thionyl chloride, acetyl chloride, p-toluenesulfonic acid, benzenesulfonic acid, methanesulfonic acid, dimethylsulfate and sulfuric acid were investigated as catalysts for the production of biodiesel because acids have higher tolerance to water and free fatty acids in oils and can simultaneously catalyze both the esterification and transesterification reactions. Sulfuryl chloride was found to be an effective catalyst for production of biodiesel from soybean oil, its waste oil and microalgal lipids.     

Lipase catalyzed methanolysis to produce biodiesel: Optimization of the biodiesel production

A lipase from Candida sp., suitable for transesterification of fats and oils to produce fatty acid methyl ester (FAME), was immobilized on a cheap cotton membrane, in this paper. The conversion ratio of salad oil to biodiesel could reach up to 96% with the optimal reaction conditions. Continuous reaction in a fixed bed reactor was also investigated. A three-step transesterification with methanol (methanolysis) of oil was conducted by using a series of nine columns packed with immobilized Candida sp. 99–125 lipase. As substrate of the first reaction step, plant or waste oil was used together with 1/3 molar equivalent of methanol against total fatty acids in the oil. Mixtures of the first- and second-step eluates and 1/3 molar equivalent of methanol were used for the second- and third-reaction steps. A hydrocyclone was used in order to on-line separate the by-product glycerol after every 1/3 molar equivalent of methanol was added. Petroleum ether was used as solvent (3/2, v/v of oil) and the pump was operated with a flow rate of 15 L/h giving an annual throughput of 100 t. The final conversion ratio of the FAME from plant oil and waste oil under the optimal condition was 90% and 92%, respectively. The life of the immobilized lipase was more than 10 days. This new technique has many strongpoints such as low pollution, environmentally friendly, and low energy costs.     

Effects of water on biodiesel fuel production by supercritical methanol treatment

In the conventional transesterification of fats/vegetable oils for biodiesel production, free fatty acids and water always produce negative effects, since the presence of free fatty acids and water causes soap formation, consumes catalyst and reduces catalyst effectiveness, all of which result in a low conversion. The objective of this study was, therefore, to investigate the effect of water on the yield of methyl esters in transesterification of triglycerides and methyl esterification of fatty acids as treated by catalyst-free supercritical methanol. The presence of water did not have a significant effect on the yield, as complete conversions were always achieved regardless of the content of water. In fact, the present of water at a certain amount could enhance the methyl esters formation. For the vegetable oil containing water, three types of reaction took place; transesterification and hydrolysis of triglycerides and methyl esterification of fatty acids proceeded simultaneously during the treatment to produce a high yield. These results were compared with those of methyl esters prepared by acid- and alkaline-catalyzed methods. The finding demonstrated that, by a supercritical methanol approach, crude vegetable oil as well as its wastes could be readily used for biodiesel fuel production in a simple preparation.     

Lipase-catalysed synthesis of ester oils from biodiesel by-products

Ester oils obtained from natural long-chain fatty acids and alcohols are versatile substitutes for many petroleum-based products. Their efficient synthesis with the solvent-free esterification of free fatty acids (FFA) from by-products of biodiesel fabrication and 2-ethyl-1-hexanol with immobilised lipase from Thermomyces lanuginosa was investigated. The immobilisation of the biocatalyst in static emulsion yielded a specific esterification activity that was higher by a factor of 4.9-9.4 than the activity of the native enzyme. Favourable properties of the silicone-based immobilisation matrix in terms of stability and immobilisation yield were observed. In biodiesel by-products, the immobilised lipase catalysed the esterification of FFA as well as the transesterification of residual fatty acid methyl esters (FAME) to the desired ester oils. A conversion of 90% FFA and 35% FAME gave a total yield of 60%. The inactivation coefficients during repeated use in a stirred-tank reactor with intermittent pressure reduction were exceptionally low.     

Influence of vegetable oils fatty acid composition on reaction temperature and glycerides conversion to biodiesel during transesterification

Presence of unreacted glycerides in biodiesel may reduce drastically its quality. This is why conversion of raw material in biodiesel through transesterification needs to readjust reaction parameter values to complete. In the present work, monitoring of glycerides transformation in biodiesel during the transesterification of vegetable oils was carried out. To check the influence of the chemical composition on glycerides conversion, selected vegetable oils covered a wide range of fatty acid composition. Reactions were carried out under alkali-transesterification in the presence of methanol. In addition, a multiple regression model was proposed. Results showed that kinetics depends on chemical and physical properties of the oils. It was found that the optimal reaction temperature depends on both length and unsaturation degree of vegetable oils fatty acid chains. Vegetable oils with higher degree of unsaturation exhibit faster monoglycerides conversion to biodiesel. It can be concluded that fatty acid composition influences reaction parameters and glycerides conversion, hence biodiesel yield and economic viability.     

Lipase-catalyzed biodiesel production with methyl acetate as acyl acceptor

Biodiesel is an alternative diesel fuel made from renewable biological resources. During the process of biodiesel production, lipase-catalyzed transesterification is a crucial step. However, current techniques using methanol as acyl acceptor have lower enzymatic activity; this limits the application of such techniques in large-scale biodiesel production. Furthermore, the lipid feedstock of currently available techniques is limited. In this paper, the technique of lipase-catalyzed transesterification of five different oils for biodiesel production with methyl acetate as acyl acceptor was investigated, and the transesterification reaction conditions were optimized. The operation stability of lipase under the obtained optimal conditions was further examined. The results showed that under optimal transesterification conditions, both plant oils and animal fats led to high yields of methyl ester: cotton-seed oil, 98%; rapeseed oil, 95%; soybean oil, 91%; tea-seed oil, 92%; and lard, 95%. Crude and refined cottonseed oil or lard made no significant difference in yields of methyl ester. No loss of enzymatic activity was detected for lipase after being repeatedly used for 40 cycles (ca. 800 h), which indicates that the operational stability of lipase was fairly good under these conditions. Our results suggest that cotton-seed oil, rape-seed oil and lard might substitute soybean oil as suitable lipid feedstock for biodiesel production. Our results also show that our technique is fit for various lipid feedstocks both from plants and animals, and presents a very promising way for the large-scale biodiesel production.     

Lipase-catalysed synthesis of ester oils from biodiesel by-products

Ester oils obtained from natural long-chain fatty acids and alcohols are versatile substitutes for many petroleum-based products. Their efficient synthesis with the solvent-free esterification of free fatty acids (FFA) from by-products of biodiesel fabrication and 2-ethyl-1-hexanol with immobilised lipase from Thermomyces lanuginosa was investigated. The immobilisation of the biocatalyst in static emulsion yielded a specific esterification activity that was higher by a factor of 4.9–9.4 than the activity of the native enzyme. Favourable properties of the silicone-based immobilisation matrix in terms of stability and immobilisation yield were observed. In biodiesel by-products, the immobilised lipase catalysed the esterification of FFA as well as the transesterification of residual fatty acid methyl esters (FAME) to the desired ester oils. A conversion of 90% FFA and 35% FAME gave a total yield of 60%. The inactivation coefficients during repeated use in a stirred-tank reactor with intermittent pressure reduction were exceptionally low.     

Selection of lipase producing yeasts for methanol-tolerant biocatalyst as whole cell application for palm-oil transesterification

Methanol-tolerant lipase producing yeast was successfully isolated and selected thorough ecological screening using palm oil-rhodamine B agar as one step-approach. All 49 lipase-producing yeasts exhibited the ability to catalyze esterification reaction of oleic acid and methanol at 3 molar equivalents. However, only 16 isolates catalyzed transesterification reaction of refined palm oil and methanol. Rhodotorula mucilagenosa P11I89 isolated from oil contaminated soil showed the strongest hydrolytic lipase activity of 1.2U/ml against palm oil. The production of oleic methyl ester and fatty acid methyl ester (FAME) of 64.123 and 51.260% was obtained from esterification and transesterification reaction catalyzed by whole cell of R. mucilagenosa P11I89 in the presence of methanol at 3 molar equivalents against the substrates, respectively. FAME content increased dramatically to 83.29% when 6 molar equivalents of methanol were added. Application of the methanol-tolerant-lipase producing yeast as a whole cell biocatalyst was effectively resolved major technical obstacles in term of enzyme stability and high cost of lipase, leading to the feasibility of green biodiesel industrialization.     

高含游离脂肪酸的香果树籽油制备生物柴油的方法

目前生物柴油因其环保和可再生利用资源的特性备受关注。多数生物柴油是通过甲醇和碱催化食用油得到的,而大量非食用油也可以制备生物柴油。本文报道用高含游离酸脂肪油快速高效低成本制备成其单酯的二步法工艺。先用1% H2SO4以少于1.5%量对甲醇和云南特产香果树（Lindera communis）籽的粗原料油以10∶1摩尔比组成的混合液酸催化酯化游离脂肪酸;之后再对醇和得到的油脂产品按摩尔比15∶1的混合液碱催化转化为单甲酯和甘油。本方法是一个直接甲脂化制备生物柴油的工艺简洁、降低成本的新技术。文中还讨论了该工艺影响转化效率的主要因素,如摩尔比,催化量,温度,反应时间和酸度。香果树生物柴油不重蒸,而其生物柴油的主要特性,如粘度、热值、比重、闪点、冷滤点等与生物柴油标准的匹配度,也做了报道,研究结果将为香果树生物柴油以非重蒸油料制备生物柴油产品,作为潜在的柴油燃料替代产品提供技术支撑。