Biodiesel enzymatic reactions

Enzymatic Reactions for Production of Biodiesel Fuel and Their Application to the Oil and Fat Industry... 

Waste oils, from restaurants and household disposals and being creating serious problems of environmental control and food safety, have been considered as good raw material for biodiesel production. Immobilized Candida antarctica lipase was found to be effective for the methanolysis of waste oil. A three-step methanolysis protocol could be used to protect lipase from inactivation by methanol. Compared with one-step reaction, it needs a longer time to reach the reaction equilibrium. So, efforts should be made to increase enzymatic reaction rate. Reports on the enhancement of the activity of certain enzymes by applying ultrasonic irradiation on the enzymes led us to investigate its effects on the enzymatic transesterification of waste oil to biodiesel in a solvent free system. 

Klibanov AM (2001) Improving enzymes by using them in organic solvents. Nature 409 241-246 Koeller KM, Wong CH (2001) Enzymes for chemical synthesis. Nature 409 232-240 Knothe G, van Gerpen J, Krahl J (2005) The biodiesel handbook. AOCS Press, Champaign, 304 pp Koskinen AMP, Klibanov AM (1996) Enzymatic reactions in oiganic media. Blackie Academic Professional, London, 314 pp... 

Ester hydrolysis, esterification or acylation, and transesterification reactions play an important role in nature and organic synthesis including the synthesis of natural products [1, 2], Applications of hydrolytic or esterification reactions range from laboratory syntheses (e.g. with the acyl moiety as key intermediate or as protecting group) to industrial scale production of bulk chemicals, biodiesel or food (e.g. ester units as building blocks for polymerisation and polycondensation reactions, transesterification of triglycerides) [3, 4]. Transesterification reactions in nature include for example enzymatic reactions of lipases, esterases and other hydrolases that rely on the catalytic triad of serine proteases, which will be discussed in the first part of this section [5-8]. 

Shimada, Y., Watanabe, Y., Sugihara, A., and Tominaga, Y. 2002. Enzymatic alcoholysis for biodiesel fuel production and application of the reaction to oil processing./. Mol. Catal. B Enzym.,17,133-142. 

The effect of varying reaction temperature and substrate molar ratio at constant reaction time (12h), enzyme amount (30%), and added water content (10%) is shown in Figure 9.1. In general, an increase in substrate molar ratio led to lower yields at any temperature. It was concluded that a great deal of methanol inactivated Novozym 435 to synthesize the biodiesel. Similar results, that an excess of methanol decreased the enzymatic biodiesel catalyzed by Lipozyme IM77, was reported by our previous study (Shieh et al., 2003). 

The reaction is catalyzed by a variety of both acids and bases but simple bases such as NaOH and KOH are generally used for the industrial production of biodiesel [200, 201]. The vegetable oil feedstock, usually soybean or rapeseed oil, needs to be free of water (<0.05%) and fatty acids (<0.5%) in order to avoid catalyst consumption. This presents a possible opportunity for the application of enzymatic transesterification. For example, lipases such as Candida antarctica B lipase have been shown to be effective catalysts for the methanolysis of triglycerides. When the immobilized form, Novozyme 435, was used it could be recycled 50 times without loss of activity [201, 202]. The presence of free fatty acids in the triglyceride did not affect the enzymes performance. The methanolysis of triglycerides catalyzed by Novozyme 435 has also been successfully performed in scC02 as solvent [203]. 

Biodiesel (long-chain monoalkyl fatty acid esters), a renewable and green fuel, is made from vegetable oil or animal fats. At present, the high cost of biodiesel, of which the raw material amounts to 75%, prohibits its wide application. Compared with chemical method, enzymatic process seems to be a promising alternative because of its mild reaction conditions, easy recovery of product, being free of chemical wastes and low demangding on raw materials, which makes it possible to use waste oil as substrate for enzymatic production of biodiesel. 

Presently industrial production of biodiesel from waste edible oil is performed by chemical alkaline catalysts, but it has been found that high content of free fatty acid (FFA) in waste edible oil (FFA>2%) would decrease the yield sharply due to soaps formed in the process (7). Several studies showed that enzymatic methanolysis with waste edible oil was a promising alternative owing to its mild reaction condition and free of chemical waste (2, 5). However, this conventional protocol was associated with many drawbacks such as deactivation of lipase caused by methanol and absorption of glycerol to lipase surface, thus resulting in serious negative effect on the reaction (4,5). 

A comparative study of biodiesel production with WDO-2 using three-step methanolysis and one-step transesterification with methyl acetate was also conducted here. As can be seen in Figure 5, the ME yield of the three-step methanolysis after reaction for 72 h was 69.1%, which was much lower than those of the one-step transesterification with or without addition of organic base depicted in Figure 4, suggesting that the enzymatic transesterification with methyl acetate was more effective than the enzymatic methanolysis in solvent free system for biodiesel production. 

Abstract Biodiesel is a fatly acid alkyl ester that can be derived fiom any v etable oil or animal fat via the process of transesterification. It is a renewable, biodegradable, and nontoxic fuel. In this paper, we have evaluated the efficacy of a transesterification process for rapeseed oil with methanol in the presence of an enzyme and tert-butanol, which is added to ameliorate the negative effects associated with excess methanol. The application of Novozym 435 was determined to catalyze the tiansesterification process, and a conversion of 76.1% was achieved under selected conditions (reaction temperature 40 °C, methanol/oil molar ratio 3 1, 5% (w/w) Novozym 435 based on the oil weight, water content 1% (w/w), and reaction time of 24h). It has also been determined that rapeseed oil can be converted to fatty acid methyl ester using this system, and the results of this study contribute to the body of basic data relevant to the development of continuous enzymatic processes. 

As compared to other catalyst types used in the production of biodiesel, enzymes have several advantages. They enable conversion under reaction conditions milder than those required for chemical catalysts. Moreover, in the enzymatic process, both the transesteiification of triglycerides and the esterification of fi e fatty acids occur in one process step. However, lipase-catalyzed transesterifications induce a series of drawbacks. As compared to conventional alkaline catalysis protocols, reaction efficiency tends to be rather poor, and thus enzymatic catalysis generally necessitates significantly longer reaction times and higher enzyme amounts. The primary obstacle to the application of enzymes in industrial processes is their relatively high cost [7]. 

In the enzymatic process utilized for the production of fatty acid methyl ester (biodiesel) from rapeseed oil, several factors can influence both the yield and rate. These factors include the reaction solvent, reaction temperature, reaction time, methanol/oil molar ratio, enzyme amount, and water content [7, 9, 12-14]. The initial step of this study involved the identification of factors likely to influence the conversion. 

Li, L., W. Du, D. Liu, L. Wang, and Z. Li. 2006. Lipase-Catalyzed Transesterification of Rapeseed Oils for Biodiesel Production with a Novel Organic Solvent as the Reaction Medium. Journal of Molecular Catalysis B Enzymatic 43 (l-4) 58-62. 

The most important challenge in enzymatic biodiesel production is the high cost of the lipase. Therefore, immobilization was considered in order to reduce overall biodiesel production costs. Lu et al. (2007) transesterified lard using immobilized Candida sp. 99-125 and found that the enzyme was reusable over seven repeated cycles (for 180 hours) with no significant decrease in activity. Also the production yield was higher than 80%. Modi et al. (2007) found a similar stability when ethyl acetate was used instead of alcohol they used Novozym 435. The immobilized enzyme was reused for 12 cycles without any loss in the activity. On the other hand, Shimada et al. (1999) reported more than 95% conversion even after 50 cycles (100 days) of the reaction. Table 6.7 shows a sununary of different lipases tested with different feedstocks, conditions and immobilization methods. 

The two common modes of reactor operations are batch and continuous modes. Batch reactors are commonly nsed for kinetics studies, which are important for process optimization and scale-np. System design in this mode is simple, controllable, and cheap. Typically, reaction snbstrates are first added to the reactor, followed by the immobilized lipase. CO2 is then introduced to the reactor and pressurized to the desired level. Figure 6.3 shows a schematic diagram of a setup used for enzymatic transesteriflcation of lamb fats and microalgae lipids to biodiesel using Novozym 435 under SC-CO2 (Taher, 2009, 2014). 

Or9aire, O., P. Buisson, and A. C. Pierre. 2006. Application of Silica Aerogel Encapsulated Lipases in the Synthesis of Biodiesel by Transesterification Reactions. Journal of Molecular Catalysis B Enzymatic 42 (3-4) 106-113. 

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