Applied Energy 119 (2014) 497520

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Applied Energy
journal homepage: www.elsevier.com/locate/apenergy

Enzymatic biodiesel: Challenges and opportunities

Lew P. Christopher a,b,, Hemanathan Kumar c, Vasudeo P. Zambare d
a

Center for Bioprocessing Research and Development, South Dakota School of Mines and Technology, Rapid City, SD, USA Department of Civil and Environmental Engineering, South Dakota School of Mines and Technology, Rapid City, SD, USA c Department of Chemistry, Laboratory of Applied Chemistry, University of Jyvskyl, Finland d Biotechnology Division, Rossari Biotech Ltd., Naroli, Silvassa, India
b

h i g h l i g h t s
 A comprehensive overview of recent progress in enzymatic biodiesel production.  Critical assessment of merits and demerits of enzymatic biodiesel process.  Economic considerations and large scale developments in enzymatic biodiesel.  Current trends and future directions for enzymatic biodiesel R&D.

a r t i c l e

i n f o

a b s t r a c t
The chemical-catalyzed transesterication of vegetable oils to biodiesel has been industrially adopted due to its high conversion rates and low production time. However, this process suffers from several inherent drawbacks related to energy-intensive and environmentally unfriendly processing steps such as catalyst and product recovery, and waste water treatment. This has led to the development of the immobilized enzyme catalyzed process for biodiesel production which is characterized by certain environmental and economical advantages over the conventional chemical method. These include room-temperature reaction conditions, elimination of treatment costs associated with recovery of chemical catalysts, enzyme re-use, high substrate specicity, the ability to convert both free fatty acids and triglycerides to biodiesel in one step, lower alcohol to oil ratio, avoidance of side reactions and minimized impurities, easier product separation and recovery; biodegradability and environmental acceptance. This paper provides a comprehensive review of the current state of advancements in the enzymatic transesterication of oils. A thorough analysis of recent biotechnological progress is presented in the context of present technological challenges and future developmental opportunities aimed at bringing the enzyme costs down and improving the overall process economics towards large scale production of enzymatic biodiesel. As the major obstacles that impede industrial production of enzymatic biodiesel is the enzyme cost and conversion efciency, this topic is addressed in greater detail in the review. A better understanding and control of the underpinning mechanisms of the enzymatic biodiesel process would lead to improved process efciency and economics. The yield and conversion efciency of enzymatic catalysis is inuenced by a number of factors such as the nature and properties of the enzyme catalyst, enzyme and whole cell immobilization techniques, enzyme pretreatment, biodiesel substrates, acyl acceptors and their step-wise addition, use of solvents, operating conditions of enzymatic catalysis, bioreactor design. The ability of lipase to catalyze the synthesis of alkyl esters from low-cost feedstock with high free fatty acid content such as waste cooking oil, grease and tallow would lower the cost of enzymatic biodiesel. Discovery and engineering of new and robust lipases with high activity, thermostability and resistance to inhibition are needed for the establishment of a cost-effective enzymatic process. Opportunities to create a sustainable and eco-friendly pathway for production of enzymatic biodiesel from renewable resources are discussed. 2014 Elsevier Ltd. All rights reserved.

Article history: Received 31 August 2013 Received in revised form 21 November 2013 Accepted 6 January 2014 Available online 5 February 2014 Keywords: Biodiesel Lipase Enzyme-catalyzed transesterication Feedstock Glycerol

Corresponding author. Address: 501 E. St. Joseph Street, South Dakota School of Mines and Technology, Rapid City 57701, SD, USA. Tel.: +1 605 394 3385. E-mail address: lew.christopher@sdsmt.edu (L.P. Christopher).
0306-2619/$ - see front matter 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.apenergy.2014.01.017

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Contents 1. 2. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Biodiesel production methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Chemical production of biodiesel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Enzymatic production of biodiesel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Factors affecting enzymatic biodiesel production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Enzyme source. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Enzyme properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Enzyme and whole cell immobilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3.1. Enzyme immobilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3.2. Whole cell immobilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4. Enzyme pretreatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5. Biodiesel feedstock . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.6. Acyl acceptor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.7. Enzyme operating variables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.8. Bioreactor design. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.9. Glycerol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Current trends and future directions for enzymatic biodiesel R&D . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Economic considerations and large scale developments in enzymatic biodiesel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 498 499 499 501 501 501 502 503 503 503 504 506 508 509 510 510 511 513 513 514

3.

4. 5. 6.

1. Introduction Alternative fuels for internal combustion engines have recently attracted considerable attention due to the ever diminishing petroleum reserves and environmental consequences of increasing greenhouse gas emissions. The growing environmental concerns, tougher Clean Air Act Standards [1,2] and Renewable Fuel Standard (RFS) Mandates [3] are among the major drivers for development of alternative fuels from renewable resources that are sustainable and environmentally acceptable. The U.S. RFS sets a goal of 36 billion gallons of biofuels production by year 2022, of which 21 billion gallons should come from the so-called advanced biofuels and a minimum of 1 billion gallons of biodiesel. Over the last decade, biodiesel has attracted considerable attention as a renewable, biodegradable, non-toxic and clean-burning substitute for petroleum based diesel fuel which reduces emissions of carcinogenic compounds by as much as 85% compared to petrodiesel, essentially free of sulfur, polycyclic aromatic hydrocarbons and metals. The biodiesel properties such as cetane number, energy content, viscosity and phase changes are similar to those of petrodiesel fuel [46], however, the engines fueled with biodiesel emit signicantly fewer particulates, hydrocarbons, and less carbon monoxide than those engines operating on conventional diesel fuel. The greenhouse gas (GHG) emission of biodiesel (B100) are 4.5-times lower than gasoline, and 3-fold lower than petrodiesel and 85% ethanolgasoline fuel (E85). Although the NOx levels of biodiesel are slightly higher than those of conventional diesel fuel, biodiesel is believed to be eco-friendly alternatives to fossil fuels that can help reduce the risk of global warming by reducing the net carbon emissions to the atmosphere [79]. One of the great advantages of biodiesel is that it can be used in existing engines, vehicles and infrastructure with practically no changes [10]. B100 can be blended at any level with petroleum diesel, and its higher ash point makes it a safer fuel to use, handle, and store using existing diesel tanks and equipment. Biodiesel has excellent energy balance as compared to fossil fuels; biodiesel contains 3.2 times the amount of energy it takes to produce it [11]. The biodiesel production in the US increased dramatically in the past few years [12]. According to the National Diesel Board [13], there were 105 plants in operation as of early 2007 with a total production capacity of 864 million gallons and additional 1.7 billion gallons coming online from plants under construction. The

global biodiesel market is estimated to reach 37 billion gallons by 2016 with an average annual growth of 42%. In 2012, the US produced 1.143 billion gallons of biodiesel which was 14.3% more than what the RFS called for. In 2013, the biodiesel production in the US is projected to reach 1.28 billion gallons, a 10.7% increase over the 2012 blended gallons. Since 2005, the biomass-based diesel production increased more than 18-fold in the US only. The biodiesel industry in the US in on the rise, and federal agencies such as the U.S. Department of Transportation and Department of Defense invest in research and development to dramatically reduce dependence on foreign oil, and spur the creation of a domestic bio-industry for sustainable production of biofuels including biodiesel [14]. The cost of biodiesel, however, is currently approximately 30% higher than that of petroleum-based diesel [1517]. This is mainly due to the use of expensive, high quality and mostly non-rened virgin oils such as soybean, sunower, olive, palm, sh, jatropha, canola, cottonseed, peanut and linseed oil, known as rst generation biodiesel feedstock. In the enzyme-catalyzed biodiesel production, the high enzyme cost signicantly impacts the process protability. The cost of commercial enzyme products for industrial use is approximately $1,000/kg which is signicantly higher than that of the alkali catalyst ($0.62/kg) [18]. Biodiesel fuel is expensive in comparison with petroleum-based fuel as 6080% of the cost is associated with the feedstock oil [19]. Previous studies have estimated that biodiesel production costs range between $1.50 and $2.50 per gallon depending on the feedstock used in the production process. These costs exceeded the wholesale price of petroleum-based diesel by anywhere from $0.20 to $0.82 per gallon depending on the time period when these studies were conducted [20]. Currently, biodiesel production from soybean oil at $0.50/lb with a 7.35 lbs/gallon efciency is estimated to cost about $3.675/gallon [14]. Since the biodiesel production costs are proportional to the costs of the raw materials, use of alternate, low-cost feedstock such as waste frying oil, non-edible oil and oil extracted from other feedstocks such as waste restaurant oil, yellow grease, lard, animal fats and others, known as second generation biodiesel feedstock, instead of virgin oil is estimated to reduce the production cost and make it competitive in price with petroleum diesel [16,17]. Oils extracted from algae are particularly viewed as the most sustainable feedstock for the future due to the signicantly higher yields, 15,000 gal/acre/year, as compared to only 48 gal/acre/year

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for soybean oil, 113 gal/acre/year, for peanut oil, and 127 gal/acre/ year, for canola oil (also known as rapeseed oil). Rudolph Diesel, the inventor of the internal compression ignition engine now known as the Diesel engine, rst used vegetable oils (such as peanut oil) to fuel his engine [21,22]. The vegetable oils performed well in short term engine tests [23,24], but failed in long term operations [21,25,26]. Problems such as cooking of injectors, carbon deposits, oil ring sticking, thickening and gelling of the lubricating oils were encountered. High viscosity (almost 10 times that of No. 2 diesel fuel) and a tendency for polymerization within the cylinder appeared to be the root cause for many problems (such as high cloud point temperature), associated with the direct use of vegetable oils as fuel [23,2634]. In order to reduce these difculties, the idea of converting the vegetable oils into their ester derivatives was introduced. Reacting triglycerides (TGs) in oil with an alcohol in presence of a catalyst can produce esters of free fatty acids (FFA) such as fatty acid methyl esters (FAME). As a result, the viscosity of these alkyl esters is reduced, however, their cetane numbers and heating values remain unchanged. FAME are termed biodiesel and the process is known as trans-esterication [35,36]. The catalysts used for transesterication of biodiesel are alkali, acid (chemical) and enzyme (biobased). The alkali-catalyzed process gives higher conversion of TG at short reaction times. The foremost drawback of the alkali process is its sensitivity towards FFA in oils that leads to soap formation during the transesterication process. The acid-catalyzed processes are insensitive to FFA, however, with a drawback of slower reaction rates. Furthermore, the use of chemical catalysts can cause technical problems related to biodiesel purication and separation from catalysts and the glycerol by-product [37,38]. To minimize the problems associated with the use of acid and/ or alkali for biodiesel production, a lipase-catalyzed process has been proposed and extensively researched in the last decade [18]. Advantages of the enzyme-based biodiesel process include: (1) simplied production process; (2) lower energy consumption; (3) higher purity of glycerol by-product; (4) no soap formation in the system; (5) easy separation and reuse of immobilized enzymes. Although the enzyme catalyst has some drawbacks, mainly associated with lower reaction rates, higher costs, and loss of activity or enzyme inhibition, the enzymatic route for biodiesel production is nowadays considered as an environmentally-friendly alternative that is becoming more realistic as new, more efcient and less expensive enzymes are being developed [39]. To minimize enzyme costs, the enzymatic catalyst can be reused by immobilization, which leads to improved efciency of biodiesel recovery. In addition, the enzyme catalytic activities can be enhanced by screening and selection of high alcohol-tolerant microorganisms and by genetic engineering [40].

The following sections provide a state of the art review of the current biodiesel process technologies with emphasis on the enzymatic transesterication method. A comprehensive analysis of recent biotechnological advancements in the enzyme process biodiesel is presented in the context of current technical challenges and future developmental opportunities aimed at bringing the enzyme costs down and improving the overall process economics towards large scale production of enzymatic biodiesel. 2. Biodiesel production methods Biodiesel can be made via three production routes: microemulsions, thermal cracking (pyrolysis), and trans/inter/esterication [26]. The most cost-efcient method for production of biodiesel with higher quality is transesterication of vegetable oils and animal fats. In lipid chemistry, transesterication is the catalytic process of exchanging the alkoxy group of an ester by an alcohol (acyl acceptor) that converts the TG in oils to fatty acid alkyl esters (FAAE) and glycerol (Fig. 1). This process is also known as alcoholysis and makes use of short chain alcohols such as methanol and ethanol as acyl acceptors. Interesterication is the transformation of TG to biodiesel in presence of an ester (such as methyl acetate) as the acyl acceptor. In this process, instead of glycerol, another triacylglycerol is formed as the by-product. Lastly, biodiesel can be also produced by a direct esterication of FFA with alcohols to produce FAAE and water as the by-product. Chemical (acids and/or bases) and biological (enzymes) agents serve as catalysts. The overall process of alcoholysis includes three reversible reactions in which di- and mono-glycerides are formed as intermediates [26,41]. From each TG molecule, three molecules of biodiesel and one molecule of glycerol are produced (Fig. 1). The process equilibrium can be shifted toward product formation in excess of alcohol or by continuous product removal. 2.1. Chemical production of biodiesel The base-catalyzed transesterication of oils and fats to biodiesel proceeds at faster rates and greater yields than the acid-catalyzed process [4143]. Hence, biodiesel at yields of 9499% is conventionally manufactured via chemical catalysis that uses sodium or potassium hydroxide at concentrations in the range of 0.51 wt%, a methanol to oil ratio of 6:1, and temperatures of 4580 C to completely transesterify the lipids in several hours. The 6:1 ratio of acyl acceptor to vegetable oil is higher than the stoichiometrically required 3:1; this, however ensures excess of methanol in the reaction mixture and increased methyl ester yields. However, molar ratios higher than 6:1 increase glycerol solubility and complicate glycerol
OCOR 2 OCOR 3 Diglyceride

OCOR 2 1. R1 OCO OCOR 3 + ROH Alcohol Triglyceride

k1 k4

R 1OOCR Ester

HO

2.

HO

OCOR 2 OCOR3 Diglyceride OH

ROH Alcohol

k2 k5

OH R 2OOCR Ester + HO OCOR 3

Monoglyceride OH

3.

HO

OCOR3

ROH Alcohol

k3 k6

R 3OOCR Ester

HO Glycerol

OH

Monoglyceride

Fig. 1. Step-wise reaction pathways for biodiesel production (R is a small alkyl group, R1, R2 and R3 are fatty acid chains; k1, k2, k3, k4, k5, k6 are chemical or enzymatic catalysts).

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separation. Sodium hydroxide is the preferred alkaline catalyst due to its lower price and higher biodiesel yields [44]. Although biodiesel is currently exclusively produced at commercial scale utilizing alkali, mainly sodium hydroxide, there are process limitations that are considered drawbacks of chemical biodiesel. These are normally associated with the presence of FFA in quantities higher than 0.5% that can lead to soap formation, and with the presence of water exceeding 0.3% that can results in consumption of the catalyst thereby reducing the reaction yield. Saponication not only consumes the alkali catalyst, but the resulting soaps can cause the formation of emulsions that create difculties in downstream recovery of biodiesel, and can lower the ester

yields [45]. Therefore, to prevent saponication, an acid pretreatment step is introduced that esteries the FFA to FAME in presence of methanol. The acid step is normally carried out with 0.51.0% sulfuric acid at a higher temperature (60100 C) and alcohol/substrate molar ratio of approximately 30 to 1. Esterication of oils containing high FFA using acid catalyst results in reduction of FFA to less than 1 wt%. Other mineral acids such as hydrochloric acid, p-toluenesulfonic acid, methanesulfonic acid and phosphoric acid are also commonly used. For instance, Guan et al. [46] used ptoluenesulfonic acid as catalyst for a complete transesterication of corn oil to biodiesel in dimethyl ether. Following the initial conversion of FFA to FAME, the remaining TG are then transesteried

(a)
Alcohol

Preesterification Transesterification

Evaporation Oil Separation

Fatty Acid Alkyl Esters Water Evaporation Water Catalyst (Acid) Catalyst (Base)

Alcohol

Washing

Waste Water Alcohol Crude Glycerol

Drying

Acid

Acidification

Glycerol

Biodiesel

Saponified product

(b)
Alcohol

Transesterification

Evaporation Oil Separation

Fatty Acid Alkyl Esters Alcohol

Enzyme Glycerol

Biodiesel

Fig. 2. Biodiesel production from feedstocks containing high free fatty acids: (a) two-step chemical process; (b) enzymatic process.

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to biodiesel in presence of a base [4754]. A two-step acidbase process has been developed to utilize oils such as used cooking oil and restaurant grease with a high FFA content (2050%) in biodiesel production (Fig. 2a). The water formed in the acid-catalyzed esterication is separated from the organic layer of FAME prior to the alkali-catalyzed step. The presence of hydroxide ions in water increases the saponication which produces soap by reaction of the hydroxide ions with esters, FFA and with other glycerides, thus rendering the downstream separation expensive and affecting the ester yields [41,5558]. Liu et al. [59] studied the affect of water on sulfuric acid-catalyzed esterication of carboxylic acid and reported a signicant decrease in catalytic activity as water was produced from the condensation of carboxylic acids and alcohols. The acid-catalyzed process is corrosive in nature causing damage to equipment, proceeds at low rates, and requires long reaction times to achieve conversion in excess of 90% [55]. For a full completion of esterication (up to 99%), more than 48 h is normally needed [41,46,55]. However, the major demerit of the acidbase process is the increased number of process steps and required equipment, which in turn results in increased production cost. There are several drawbacks associated with the chemical production of biodiesel [19,37]: (1) side reactions of saponication and hydrolysis affect biodiesel yield and purity; (2) the process, especially the acid pretreatment step, is energy and capital intensive; (3) recovery and purication of catalysts and glycerol is expensive; (4) neutralization and waste water treatment is required. Problems with separation and soap formation have prompted research with heterogeneous non-enzymatic catalysts such as amorphous zirconia, titanium-, aluminum-, and potassium-doped zirconias, metal oxides, hetero-polyacids, sulfated zeolites and others [6062]. A mesoporous silica catalyst functionalized with hydrophobic allyl and phenyl was shown to hydrophobically exclude water from the catalyst active sites while effectively esterifying FFA [63]. Compared to homogenous catalysts, heterogeneous catalysts have the advantages of easier recovery and usability on substrates containing higher concentrations of FFA. However, some heterogeneous catalysts could be cost and energy intensive due to requirements for high reaction temperatures and high alcohol to substrate molar ratios [64]. 2.2. Enzymatic production of biodiesel As elaborated in the previous section, the main disadvantages of the traditional acid/base transesterication methods are the high reaction temperatures, high energy consumption, corrosive nature of acids, glycerol purication and recovery, separation of catalysts and unreacted alcohol accompanied with additional washing steps to remove impurities from biodiesel. These problems can be minimized and even eliminated by using environmentally-friendly biocatalysts (enzymes) for biodiesel production (Fig. 2b). Enzymes such as lipases offer a biological route of biodiesel production with a number of environmental and economic advantages over the chemical route [6568]:  Use of mild reaction temperatures.  High selectivity and specicity of trans/esterication towards substrates.  Broader substrate range due to ability to esterify both glyceridelinked and non-esteried fatty acids in one step; use of lower alcohol to oil ratios.  Avoidance of side reactions, easier separation and product recovery due to the production of a glycerol side stream with minimal impurities and water content [12].  Elimination of treatment costs associated with recovery of chemical catalysts.  Enzyme biodegradability and environmental acceptability.

 Opportunity for enzyme reuse and improved stability through enzyme immobilization. The presence of FFA in the starting material poses a major problem for transesterication via traditional methods, while this problem can be easily overcome by using enzymes that can catalyze both esterication of FFA and transesterication of TGs. The FFA contained in waste oils and fats can be completely converted to biodiesel. In fact, the enzyme-catalyzed transesterication is more suitable for use on FFA-rich feedstocks such as waste oils, greases, beef tallow and lard since the FFA are directly esteried enzymatically into FAME [12]. Therefore, lipase can be used on oils with variable chemical composition which broadens the feedstock base and is of great advantage when waste oils and fats are considered for the establishment of a cost-effective biodiesel production process [65,69]. However, there are several technical challenges that need to be overcome to improve the economic feasibility of the lipase bioprocess: high cost of enzymes, loss of activity during the process, enzyme inhibition by reactants and products, and slow reaction rates. 3. Factors affecting enzymatic biodiesel production The yield and conversion efciency of enzymatic biodiesel is inuenced by a number of factors: the nature and properties of the enzyme catalyst; enzyme and whole cell immobilization techniques; enzyme pretreatment; biodiesel substrates; acyl acceptors, their step-wise addition and use of solvents; operating conditions of enzymatic catalysis; and bioreactor design. 3.1. Enzyme source Lipases are enzymes (biocatalysts) which carry hydrolysis of TG to glycerol and fatty acid, hence, they are categorized in the class of hydrolases (acylglycerolacylhydrolases, EC 3.1.1.3) [70,71]. They are best dened as carboxylesterases that catalyze both the hydrolysis and synthesis of long-chain acylglycerols [72]. These enzymes are ubiquitously present and based on their origin are classied as plant, animal or microbial lipases. Plant lipases have been reported from papaya latex, rapeseed, oat and castor seeds [73]. Plant lipases are not commercially used whereas the animal and microbial lipases are used extensively. Sources of the animal lipases are pancreas of cattle, sheep, hogs and pigs [73]. Microbial lipases have gained wide industrial importance and they now share about 5% of the world enzyme market after proteases and carbohydrases [74 78]. Lipases of microbial origin are more stable than plant and animal lipases and are available in bulk at lower cost compared to lipases of other origin. Yeasts lipases are easy to handle and grow compared to bacterial lipases [79]. Among the yeast lipases, Candida rugosa has gained good commercial importance. The most commonly used biocatalyst for biodiesel production are the microbial lipases that are produced by a number of fungal, bacterial and yeast species (Table 1). As it can be seen from Table 1, a large number of microbial strains have been used for lipase production, however, the most frequently reported enzyme sources are Candida sp., Pseudomonas sp. and Rhizopus sp.[132]. Lipase producing microorganisms have been screened from various sources including soil, marine water, waste water and industrial wastes [76]. Traditionally, screening is carried out on batch cultures using agar substrates which is time-consuming, however, continuous enrichment cultures overcome this problem by using fermentors for growth and selection of desired microbial isolates [133]. Soil isolates of Aspergillus, Mucor, Candida and Sclerotina species were reported to produce lipase [129]. Lipase-producing strains of P. uorescens, P. alcaligenes, Enterobacter intermedium, Geotrichum asteroids and Bacillus acidophilus were isolated from vegetable oil processing plants [129,134,135].

502 Table 1 Lipase-producing microorganisms. Microbial type Bacteria Microbial source

L.P. Christopher et al. / Applied Energy 119 (2014) 497520 Table 1 (continued) Microbial type Refs. [77] [80] [81] [82] [83] [84] [72] [76] [72] [77] [85] [86] [87] [88] [77] [77] [89] [90] [73] [91] [92] [73] [93] [93] [73] [90,94] [95] [77,96] [77] [96] [97] [98] [99] [77] [100] [101] [102] [87,103] [103] [91] [73] [73] [104] [105] [106] [107] [108] [109] [110] [111] [87] [112] [113] [114] [82] [76] [76] [115] [116] [117,118] [119] [98] [82] [82] [120] [121] [122] [123] [124] [125] [87] [112] [126,127] Achromobacter lipolyticum Acinetobacter radioresistens Acinetobacter calcoaceticus Acinetobacter pseudoalcaligenes Aeromonas hydrophila Archaeglobus fulgidus Bacillus acidocaldarius Bacillus megaterium Bacillus pumilus Bacillus sp. Bacillus stearothermophilus Bacillus subtilis Bacillus thermocatenulatus Bacillus thermoleovorans Burkholderia glumae Chromobacterium viscosum Enterococcus faecalis Micrococcus freudenreichii Moraxella sp. Mycobacterium chelonae Pasteurella multocida Propionibacterium acnes Propionibacterium avidium Propionibacterium granulosum Proteus vulgaris Pseudomonas aeruginosa Pseudomonas cepacia Pseudomonas fragi Pseudomonas mendocina Pseudomonas nitroreducens var. thermotolerans Pseudomonas sp. Psychrobacter immobilis Serratia marcescens Staphylococcus aureus Staphylococcus canosus Staphylococcus epidermidis Staphylococcus haemolyticus Staphylococcus hyicus Staphylococcus warneri Staphylococcus xylosus Sulfolobus acidocaldarius Vibrio chloreae Pseudomonas alcaligens Chromobacterium visosum Pseudomonas putida Statphylococcus stolonifer Alternaria brassicicola Aspergillus fumigates Aspergillus japonicas Aspergillus nidulans Candida antarctica Mucor miehei Penicilliumcyclopium Rhizomucor miehei Rhizopus arrhizus Rhizopus chinensis Rhizopus microsporous Rhizopus niveus Rhizopus nodosus Rhizopus oryzae Streptomyces cinnamomeus Streptomyces exfoliates Streptomyces fradiae Streptomyces sp. Aspergillus niger Thermomyces lanuginous Fusarium heterosporum Humicola lanuginose Oospora lactis Rhizopus oryzae Candida deformans Candida parapsilosis Candida rugosa Microbial source Candida quercitrusa Pichia burtonii Pichia sivicola Pichia xylosa Saccharomyces lipolytica Geotrichum candidum Yarrowia lipolytica NRRL YB-423 Refs. [109] [128] [128] [129] [100] [130] [131]

3.2. Enzyme properties Microbial lipases are mostly extracellular with a molecular weight of 3050 kDa and a pH optimum in the slightly alkaline pH range of 7.59 [136]. Lipases originate mainly from mesophilic and thermophilic microorganisms and have an optimum activity at 3550 C and 6080 C, respectively [129,135]. Most mesophilic lipases are unstable at temperatures above 70 C, whereas thermostable lipases show activity at up to 100 C in presence of organic solvents and detergents [137]. For instance, thermostable lipases from the hyperthermophilic archaea Pyrobaculum calidifonti [138] and Pyrococcus furiosus [139] and from the extreme thermophilic bacteria Thermoanaerobacter thermohydrosulfuricus and Caldanaerobacter subterraneus [140] exhibited lipase activity at 90 C. In addition, the Thermoanaerobacter and Caldanaerobacter lipases were shown to be resistant to organic solvents, which makes them strong candidates for biodiesel production in water-free environments. The optimum expression of lipases depends on many factors including carbon and nitrogen sources, growth conditions like pH, temperature, dissolved oxygen. Numerous literature sources are available on lipase producing microorganisms, methods for lipase production and application [74,78,91,141]. As lipases are largely inducible enzymes, the main factor for the expression of lipase activity is the carbon source which can be a lipid source or a carbon source [142]. The lipid sources include triacylglycerols, short or long chain fatty acids, hydrolyzable esters, tween, bile salts and glycerol [141,143] and the carbon sources are sugars, sugar alcohol, polysaccharides, whey, amino acids and other complex sources [141]. For maximum lipase production, incubation period usually range from few hours to few/several days based on the organism [141] and production method. Fungal species are preferably cultivated in solid-state fermentation, while bacteria and yeast are best produced in submerged fermentation [144]. The lipase reaction systems for biodiesel production are complex, consisting of two immiscible phases an aqueous phase containing enzyme, and an organic phase containing oil/fat. Lipases have the unique feature of acting on the interface between aqueous and non-aqueous phases and so they can catalyze many reactions including hydrolysis, inter-esterication and alcoholysis [78]. The mode of action of lipases in substrate transesterications to biodiesel depends on their origin and specic properties. Lipases catalyze the trans/esterication reaction between TG and acyl acceptors (alcohols) through the formation of acyl-enzyme intermediates that subsequently donate the acyl moiety to produce FAAE [145]. The overall structure of the lipases can be described as a structure with a central L-sheet with the active serine placed in a loop called catalytic elbow [146]. The activation which is often necessary for the lipase enzyme is the movement of a lid. Some enzymes such as Thermomyces lanuginosus lipase have an active site and a lid on the surface of the enzyme. Others like C. rugosa lipase have an active site at the end of a tunnel containing the lid in its external parts [147]. The structural properties of lipase from different sources might be the reason for showing different activity on different oil substrates, hence, the need to optimize the process

Fungi

Yeasts

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based on the selected enzymes and substrates for biodiesel production. Furthermore, the selection of a particular lipase for lipid modication is based on the type of the desired modication and may be position-specic modication of triacylglycerol, fatty acids-specic modication, modication by hydrolysis, and modication by direct synthesis and transesterication [148]. Based on their substrate specicity, lipases can be divided into three groups: 1,3-specic, fatty acid-specic, and non-specic lipases. The 1,3-specic lipases release and hydrolyze ester bonds in the position 1 and 3 of TGs [149]. Du et al. [150] reported the use of T. lanuginosus 1,3-specic lipases to produce a transesterication yield of 90%. The fatty acid-specic lipases are known to hydrolyze esters of long chain fatty acids with double bonds in cis-position at C9, whereas the non-specic lipases randomly cleave the acylglycerols in FFA [149]. For optimal biodiesel production, lipases should be able to convert all three forms of glycerides (mono-, di-, and tri-glycerides) to biodiesel, hence, they need to be non-stereospecic and to efciently catalyze the esterication of FFA [18]. 3.3. Enzyme and whole cell immobilization Immobilization is the process of attaching enzymes physically to a solid support so that the substrate is passed over the enzyme support and can be converted to the product. Immobilization has been increasingly used in industrial applications to facilitate separation of biocatalysts from the process stream, and hence, the recovery and purication of products [151]. Immobilized enzymes are preferred over free enzymes due to their prolonged enzymesubstrate contact that curtails redundant downstream and purication processes [152]. There are many advantages of immobilized enzyme over free enzyme: the repetitive use of a single batch of enzymes is economical in the development of continuous bioprocesses; rapid termination of the enzyme-substrate reaction by removing the enzyme from the reaction solution; enzyme stabilization due to binding to support; no contamination of the product and enzyme [153]. Immobilization can dramatically affect enzyme properties such as pH-dependence, temperature prole, resistance to proteolytic digestion and denaturants, thermostability and kinetics. The chief issues for enzyme immobilization are selection of support matrices and immobilization techniques that permit both rapid enzyme activity and enzyme stability under the constraints imposed by the substrate medium [154,155]. A number of techniques and supports are available for the immobilization of enzymes on a variety of natural and synthetic supports [156]. The choice of the support and the technique depends on the enzyme nature, nature of the substrate and the type of reaction it is used [152,157]. For industrial application, support materials are selected based on the ow properties, low cost, non-toxicity and maximum biocatalysts loading while retaining the desirable ow characteristics, operational durability, availability and ease of immobilization [158]. The activity of the immobilized enzyme may be reduced during the immobilization procedure and as a result of mass-transfer limitations. Enzyme immobilization techniques have been classied into three categories: entrapment, carrier binding and cross-linking [159]. The economics of the immobilization process depends on both the activity and the operational stability (activity integrated over the operational time) of the biocatalysts. 3.3.1. Enzyme immobilization Immobilization of lipases was carried out using entrapment [159], physical adsorption [160], ion exchange [161], and crosslinking [162]. Carriers for lipase immobilization include polyurethane foam [163], silica [164], sepabeads [165], cellulosic nanobers [166]. Criteria for selecting the immobilization technique and carrier depends on the source of lipase, the type of reaction system (aqueous, organic solvent or two-phase system) and the

process conditions (pH, temperature and pressure). Based on the immobilization technique and carrier, the bioreactor type (batch, stirred tank, membrane reactor, column and plug-ow) can be designed. The literature is replete with various lipase producing microorganisms, enzyme immobilization methods and physical carriers. The challenge will be to select a carrier and immobilization technique that will allow maximum lipase activity, retention and stability on the oil substrate [167]. Adsorption is the most widely employed method for lipase immobilization. The most frequently immobilized enzymes used for commercial applications are the C. antartica lipase, immobilized on acrylic resin (Novozym435), Mucormiehei lipase, immobilized on a macroporous ion-exchange resin (Lipozyme IM), T. lanuginosus acrylic resinimmobilized lipase (Lipozyme TLIM), Rhizomucor miehei lipase immobilized on macroporous anion exchange resin (Lipozyme RM IM), and Candida sp. 99125 lipase, immobilized on textile membranes [168]. As lipase is deactivated by methanol adsorption onto the immobilized enzyme, enzyme regeneration with higher alcohols such as butanol is required. Due to immiscibility of the large TG molecules with lower alcohols, diffusion problems to access the immobilized biocatalyst may arise due to the small pores of the carrier. This may cause internal transport problems thereby reducing the enzyme efciency [18]. Recently, interest has focused on carrier-free immobilized lipases by cross-linking of enzyme aggregates for use in a solventfree biodiesel production [169]. This technique presents several interesting advantages over carrier-bound immobilized enzymes that includes highly concentrated enzymatic activity, high stability of the produced superstructure, costs savings from omitting the support material, and no enzyme purication requirement [170]. Lipase from Rhizopus oryzae was immobilized as crosslinked enzyme aggregate via precipitation with ammonium sulfate directly from the fermentation broth and simultaneous crosslinking with glutaraldehyde [171]. The cross-linked enzyme retained 91% activity after 10 cycles in aqueous medium. Another recent development in enzyme immobilization with potential use in biodiesel production is the protein-coated microcrystals that consist of water-soluble micron-sized crystalline particles coated with a biocatalyst such as lipase [172]. Microcrystals coated with a C. antarctica lipase B retained nearly 90% of the enzyme initial activity over a period of one year at room temperature. The enzyme activity of P. aeruginosa lipase protein-coated microcrystals was enhanced tenfold over that of the free lipase enzyme [173]. A combination of lipase cross-linking and lipase coating was reported to improve the operational, pH and thermostability as well as the organic solvent tolerance of a Geotrichum sp. lipase in biodiesel production [174]. 3.3.2. Whole cell immobilization In an attempt to avoid the complex enzyme recovery and purication requirements for immobilization of free (extracellular) lipase, immobilization of intracellular lipase, known as whole cell immobilization, has been extensively researched [175]. Immobilization of the intracellular, cell- or membrane-bound lipase is believed to offer an alternative and less expensive source of immobilized biocatalyst for biodiesel production. Considerably fewer steps are required to produce whole-cell lipase using inexpensive and readily available industrial cultures. Consequently, this can signicantly reduce the cost of the transesterication process [175] as shown in Fig. 3 which compares the two immobilization methods. Lipase-producing bacteria, fungi and yeasts have been immobilized to serve as a lipase biocatalyst (Table 2). Matsumoto et al. [183] developed whole cell biocatalysts by immobilizing R. oryzae cells, permeabilized by air drying. This biocatalyst was used in a three-step addition of methanol (known as methanolysis) to plant oil in a solvent- and water-free system at 37 C. The FAME yield

504

L.P. Christopher et al. / Applied Energy 119 (2014) 497520

(a)
Cultivation Purification of Lipase Extraction Adsorption Chromatography Crystallization Immobilization of Lipase Cross linking Covalent bonding Entrapment Immobilized Lipase Biocatalyst

(b)
Cultivation & Immobilization Whole Cell Lipase Biocatalyst

Fig. 3. Immobilization methods used for production of enzymatic biodiesel: (a) lipase immobilization; (b) whole cell immobilization.

Table 2 Whole cell lipase biocatalysts used for biodiesel production. Microbial source P. uorescens Pseudomonas sp. R. mucilagenosa A. niger A. niger A. niger R. oryzae R. oryzae R. orzyae R. orzyae
a b c

Support material Na-alginatea Na-alginate None BSPsb BSPs BSPs + GAc BSPs BSPs + GA BSPs BSPs + GA

Substrate Jatropha oil Used cottonseed oil Palm oil Palm oil Palm oil Palm oil Used cooking oil Jatropha oil Soybean oil Soybean oil

Conditions 40 C; 37 C; 30 C; 25 C; 40 C; 40 C; 35 C; 30 C; 35 C; 35 C; 3:1 6:1 3:1 3:1 3:1 3:1 3:1 3:1 3:1 3:1 methanol: methanol: methanol: methanol: methanol: methanol: methanol: methanol: methanol: methanol: oil; oil; oil; oil; oil; oil; oil; oil; oil; oil; hexane solvent; 48 h step-wise; 48 h 72 h step-wise; 72 h 3 step-wise; 72 h 3 step-wise; 72 h 3 step-wise; 72 h 72 h 3 step-wise; 72 h 3 step-wise; 72 h

Yield (%) 72 70 51 87 69 69 98 89 8085 8992

Refs. [69] [176] [177] [178] [179] [179] [180] [181] [182] [182]

Na-alginate, sodium alginate. BSPs, biomass support particles. GA, glutaraldehyde.

was 71 wt% after 165 h of reaction. Some of the researcher reported that olive oil and oleic acid enhanced the methanolysis activity of immobilized R. oryzae cells with a step-wise addition of methanol (in presence of 15% water) yielding 90% biodiesel [175,184,185]. Cross-linking of R. oryzae cells with 0.1% glutaraldehyde (GA) stabilized and maintained lipase activity at the same level even after six batches of methanolysis, with ester yields of 7083% that represented a 20 to 33% increase over the control without cross-linking. Also, Tamalampudi et al. [181] compared the performance of whole cell R. oryzae immobilized on polyurethane biomass support particles (BSPs) to Novozym435 in methanolysis of jatropha oil. The presence of water in jatropha oil had a positive effect on the methanolysis with immobilized cells reaching a maximum yield of 89% (Table 3) at 5% (v/v) water, whereas the activity of Novozym435 was inhibited. Methanolysis of plant oil using a R. oryzae whole cell biocatalyst was also investigated in shake asks and in a packed-bed reactor [66,185]. The latter provided better results by protecting cells from physical damage and excess methanol. The cells were immobilized within 6 6 3 mm BSPs during batch cultivation in a 20 L air-lift bioreactor. Emulsication of the reaction mixture at a ow rate of 25 L/h resulted in maximum FAME yields of 90%. R. oryzaecells immobilized with polyurethane BSPs and treated with GA have been extensively researched widely used as a whole cell biocatalyst for production of biodiesel from various sources of non-edible and low-cost feedstock [180,182,183]. Oda et al. [229] cultured R. oryzae cells immobilized in BSPs in a 20 L air-lift bioreactor, and performed repeated batches of methanolysis of soybean oil. They found that the hydrolytic activity of the whole-cell biocatalyst was intact after 20 batches of methanolysis that produced 6580% ester yields. Li et al. [230] studied biodiesel production from different feedstocks (rened, crude and acidied rapeseed oil) in tertiary-butanol using whole cell R. oryzae cells immobilized in BSPs. They found that the

reaction rate and nal ester yield were much greater when acidied rapeseed oil was used. Furthermore, the increase in the FFA in oil, the presence of phospholipids, and the use of adsorbents for water removal improved the rate of reaction and biodiesel yields. From this perspective, based on numerous literature reports, the lipaseproducing R. oryzaeseems to show great promise for further research and development that could aid in reducing the cost of biodiesel production [181,230,231]. In addition to R. oryzae, other lipase-producing fungal and yeast whole cell catalysts have been also investigated in biodiesel production: a newly isolated strain of A. niger JN [178]; a methanoltolerant yeast R. mucilagenosa [177]; Pseudomonas sp. including P. zuorescens MTCC 103 [69,232]. A 72% biodiesel yield was obtained in transesterication of Jatropha oil with P. uorescens MTCC 103 immobilized in sodium alginate gel (Table 3). The optimum conditions were determined as follows: neutral pH, 40 C, oil/ methanol mole ratio of 1:4, 48 h [69,67]. Pseudomonas sp. was used as a whole cell biocatalyst for biodiesel production from used cottonseed oil: the FAME yield of 70% was attained by a step-wise addition of methanol in excess (oil/methanol mole ratio of 1:6) for 48 h [176]. Pseudomonas sp. immobilized in sodium alginate gel was recommended for industrial use [69,232]. Lu et al. [233] studied the transesterication of lard using immobilized Candida sp. 99125 whole cells via a three-step methanolysis. They reported that for processing 1 g of lard the optimum, loading conditions are 0.2 g immobilized whole cell, 8 ml n-hexane as solvent, 20 wt% water, and 40 C. A biodiesel yield of 87.4% was obtained and the whole cell lipase was stable for 180 h of repeated use. 3.4. Enzyme pretreatment Pretreatment of lipase is believed to have a positive impact on enzyme performance by: (1) providing a protective enzyme shield

L.P. Christopher et al. / Applied Energy 119 (2014) 497520 Table 3 Lipase-catalyzed transesterication of oils to biodiesel. Substrate Jatropha oil Palm oil Enzyme E. aerogenes lipase PS 30 Lipase Operating conditions 55 C; 48 h 40 C; 8 h Solvent/ water NSa NS NS NS NS NS NS NS NS NS NS NS Water (4 30%) t-Butanol Hexane Petroleum ether Petroleum ether Petroleum ether NS Acyl acceptor Methanol Ethanol t-Butanol 1-Butanol n-Propanol 1-Propanol Ethanol iso-Butanol 1-Butanol 1-Propanol Methyl acetate Ethanol Methanol Methanol Methanol Methanol Methanol Methanol Ethanol 2-Propanol Yield (%) 94 72 62 42 42 24 35 40 40 16 92 84 >99 8090 95 98 79 49 99 92.8 91.7 93.4 91.3 90 92.7 90 94.8 98.5 61.2 83.8 19.4 65.5 67 65 97 100 100 97 95 90 90 93.8 97 >99 95.65 90 90 90 98 97 55 85.4 92

505

Refs. [186] [187]

Coconut oil

PS 30 Lipase

40 C, 8 h

Soybean oil Vegetable oils Plant oils Rapeseed oil Microalgae Sunower oil

Novozym435 Lipozyme TL IM Novozym435 R. oryzae lipase Lipozyme TL IM + Novozym435 Candida sp. lipase IM Pseudomonas lipase

40 C;14 h 25 C; 7 h 35 C; 5 h 35 C; 12 h 38 C; 12 h 45 C; 5 h 65 C; 5 h 45 C; 5 h

[188] [189] [190] [191] [192] [193]

Jatropha oil Karanj oil Sunower oil Jatropha oil Karanj oil Sunower oil Plant oil Tallow oil

Novozym435

50 C; 8 h

[194]

Novozym435

50 C; 12 h

NS

Ethyl acetate

[195]

Novozym435 Lipozyme IM60 Novozym435 Lipozyme IM60 Lipozyme IM60

Continuous reaction 45 C; 5 h

Petroleum ether Hexane NS NS NS

Methanol Primary alcohols Secondary alcohols Methanol Ethanol Methanol Ethanol Methanol Butanol Methanol Methanol Methanol Methanol Methanol Methanol Methanol Methyl acetate Methanol Methanol Butanol Methanol Methanol Methanol Ethanol Methanol

[196] [197]

Soybean oil Cottonseed oil Triolein Soybean oil Soybean oil deodorizer distillate Acid oil Waste edible oil Degummed soybean oil Sunower oil Sunower oil Plant oil Soybean oil Triolein Soybean oil Waste oil adsorbed on activated bleaching earth Palm oil from waste bleaching earths Grease Soybean oil

IM P. cepacia lipase IM C. antarctica lipase Novozym435 Lipozyme RM IM Novozym435 Novozym435 Novozym435 Novozym435 Novozym435 Novozym435 Novozym435 R. oryzae lipase IM R. oryzae lipase IM P. ourescens lipase Lipozyme TL IM C. cylindracea lipase R. oryzae lipase PS 30 Recombinant LipB68 P. uorescens lipase

35 C; 1 h 50 C; 24 h 6h 30 C; 3.5 h Molecular sieve adsorbent reaction 24 h Fixed bed reactor 30 C; 6 h NS 45 C; 50 h NS RTb; PBRc 50 C; 25 h 40 C; 12 h 25 C; 12 h 35 C; 96 h 38.4 C; 2.47 h 20 C; 12 h

Water t-Butanol NS NS t-Butanol NS NS NS NS NS NS NS NS NS Diesel oil NS NS NS

[154] [198] [199] [200] [201] [202] [203] [204] [205] [206] [175] [185] [207] [208] [209] [210] [211] [212]

(continued on next page)

506 Table 3 (continued) Substrate Enzyme

L.P. Christopher et al. / Applied Energy 119 (2014) 497520

Operating conditions

Solvent/ water Water (40%) Diesel oil NS

Acyl acceptor

Yield (%) 80 100 92 97 92.2 86.8 99.2

Refs.

Rice bran oil Waste activated bleaching earth Rened soybean oil Rapeseed oil Soybean oil Mowrah oil Mango oil Kernel oil Sal oil Sunower oil Fish oils Soybean oil Soybean oil Soybean oil Sunower oil Sunower oil Sunower oil Soybean oil Sunower oil Crude palm oil

Cryptococcus spp. S-2 C. cylindracea lipase Novozym435 C. rugosa lipase Lipozyme IM-77 Lipozyme IM-20

30 C; 96 h 37 C; 3 h 40 C; 10 h 37 C; 24 h NS 60 C; 6 h

Methanol Methanol Methyl acetate 2-Ethyl-1hexanol

[213] [214] [215] [216] [217] [218]

n-Hexane Water (10%)

Methanol Alcohols (C4 C18)

Lipozyme C. antarctica lipase P. uorescens lipase C. rugosa lipase P. cepacia lipase R. miehei lipase T. lanuginosus lipase IM C. antarctica lipase IM M. miehei lipase IM T. lanuginosus lipase Lipozyme Lipozyme Lipozyme Lipozyme RM IM TL IM RM IM TL IM

50 C; 5 h RT; 22 h 35 C; 90 h 35 C; 90 h 35 C; 90 h 40 C; 48 h 40 C; 48 h 50 C; 12 h 35 C; 8 h 30 C; 6 h 50 C; 4 h

NS NS NS NS NS NS NS NS n-Hexane n-Hexane NS NS NS NS NS n-Hexane Ionic liquids NS NS NS

Ethanol Ethanol Methanol Methanol Methanol Methanol Methanol Ethyl acetate Ethanol Ethanol Methanol Methanol Ethanol Ethanol Ethanol Methanol Methanol Methanol Ethanol/ methanol Ethanol/ methanol

83 100 80 80 100 95.5 92.3 63.3 95.6 1635 12 15 16 25 83.5 71 86 100 70100 8090

[219] [220] [190]

[221]

[222] [223] [223] [224]

Soybean oil deodorizer distillate Jatropha oil Corn oil Palm oil Fats and oils Restaurant grease
a b c

Novozym435 IM P. uorescens lipase P. expansum lipase IM B. cepacia lipase IM T. lanuginosus lipase IM T. lanuginosus lipase

50 C; 1.5 h 40 C; 48 h 40 C; 24 h 30 C; 72 h 50 C; 48 h 50 C; 48 h

[225] [69] [226] [227] [228]

NS, not specied. RT, room temperature. PBR, packed bed reactor.

to minimize enzyme deactivation caused by lower alcohols (methanol and ethanol) and glycerol [4,200,234], and (2) enhancing enzyme activity by maintaining the lipase molecule conformation in its active form, or reversing the conformation of the active enzyme sites from close to open [167,235]. The overall effect of lipase pretreatment is regeneration of enzyme activity and improved mass transfer and transesterication rates, resulting in enhanced productivity [236,237], however, the exact mechanism of enzyme pretreatment is not clearly understood [238]. The most commonly used pretreatment agents include alcohols, solvents, ethers and salts. Washing with t-butanol and 2-butanol affected a ten-fold increase in Novozym435 activity [234]. In comparison, the enzyme was completely deactivated by methanol without washing. Pretreatment of the completely deactivated enzyme with tert-butanol and 2-butanol restored the lipase activities of their original levels of 56% and 75%, respectively. Incubation of immobilized C. antarctica in methyl oleate for 30 min, followed by a step-wise addition of methanol to soybean oil increased the FAME yields to 97% [200]. Shah et al. [239] reported a 45% increase in ester conversion (from 34% to 79%) of jatropha oil when an immobilized lipase from P. uorescens was pretreated via irradiation in presence of an aqueous buffer and organic solvents. Pretreatment of immobilized Novozym435 using ultrasonic irra-

diation with vibration resulted in 96% methyl ester yields after ve repeated cycles [240]. Pretreatment of R. oryzae lipase with soybean oil before immobilization increased the lipase activity 20-times over that of the non-treated lipase thereby maintaining it at levels exceeding 90% of its original activity after 10 reuses [241]. Salts as pretreatment agents including calcium or magnesium chloride are thought to stabilize the protein molecule and enhance its resistance toward methanol inhibition [235]. 3.5. Biodiesel feedstock The choice of feedstocks depends on the process chemistry, physical and chemical characteristics of virgin or used oils and economy of the process. First-generation biodiesel has been produced from vegetable oils extracted from oilgeneous plants like sunower, soya, canola and palm (Table 3) [242]. At present, canola, soybean and palm oil constitute about 75% of the world vegetable oil supply. In the rst half of 2013, they traded for $1150, $1050 and $755 on average per metric ton, respectively. Canola oil is the primary source for biodiesel production globally, whereas soybean oil is the largest biodiesel feedstock in the US which is also exchange-traded. Soybeans are widely grown in the US for their high protein and lipid content. Because of the value of the products and

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the ability to be used in crop rotations with nitrogen-intensive crops such as corn, soybean oil now accounts for more than 50% of all bio-based oils in the US. Although soybean oil yields only 48 gallons/acre/year, 2 billion gallons were harvested from more than 30 million ha in 2002 [35]. Nowadays soybean oil makes up 25% of the total soybean oil demand in the US [14]. Biodiesel quality is directly inuenced by the fatty acid composition of vegetable oils [243]. Low cetane numbers have been associated with the presence of unsaturated fatty acids (C18:2 and C18:3) that are typically found in soybean oils, whereas saturated fatty acids such as palmitic (C16:0) and stearic (C18:0) acid increase the cetane number of biodiesel produced from palm oil [244]. The main constraint for biodiesel production is the cost of the feedstock. It has been estimated that up to 80% of the total biodiesel production cost arises from the cost of raw material [245247]. The high value of edible vegetable oils as a food product makes production of a cost-effective biodiesel fuel very challenging. In addition, the recent increase in the use of vegetable (edible) oils for biodiesel production has caused some major controversy owing to their inuence on the global imbalance to food market and food security [248]. Furthermore, there are large amounts of low-cost feedstock such as waste cooking oils and animal fats that could be converted to biodiesel [167] thereby also helping solve the problem of waste oil disposal [249]. This has historically led to the production of the second-generation biodiesel that is derived from non-edible oils. In comparison to edible oils, the major shortcomings in the use of non-edible oils are the relatively low oil yields (with a few exceptions) and feedstock quality. For example, the yield of palm oil peaks at 5000 kg oil/hectare, whereas yields from non-edible oils such as jatropha and pongamia pinnata are 250-fold lower, and range from 100 to 2300 kg oil/hectare. Therefore, in some instances, the use of non-edible oils for biodiesel production would require large plantation areas. However, in comparison, the oil yield of jatropha oil is nearly 5-times higher than that that of soybean oil (475 kg/hectare), whereas macauba can produce 10-times more oil than soybean. Furthermore, due to the presence of high FFA in non-edible oils, biodiesel production from this feedstock, the use of enzyme catalysts has been favored [248]. Nevertheless, inedible oils are a viable alternative to edible vegetable oils due to their lower price and wide adaptability with minimal production requirements. For instance, jatropha can grow on waste, sandy and saline soils under a wide variety of climatic conditions (severe heat, low rainfall, high rainfall and frost), and produce up to 60 wt% oil in its seeds and kernels [250]. Because of that, Jatropha oil, despite its toxicity to humans, is regarded as a promising potential feedstock for biodiesel production in Asia, Europe and Africa [244]. The advantages of non-edible oils over their edible counterparts are their lower price, their availability and portability, higher caloric value and lower sulfur and aromatic content. Drawbacks of inedible oils include higher reactivity due to higher content of unsaturated fatty acids, higher viscosity and carbon residue content, and lower volatility [251,252]. The waste cooking oils are directly accessible for biodiesel production (Table 3) and their quantity is mainly relying on the amount of edible oil consumed. Cooking oils contain many types of vegetable-based oils as well as rendered animal oils. There are enough used cooking oils and fats generated in the US annually, including 18 billion pounds of soybean oil and 11 billion pounds of animal fat, to produce an estimated 5 billion gallons of biodiesel [253]. The cost of waste cooking oil is calculated from the collection, transportation and pretreatment costs, which are minimal compared to those for edible oils. The physical properties and chemical composition of waste cooking oil vary depending on the oil source and the content of water (0.75%) and FFA (56%) that is comparatively higher than virgin oil (<0.8% FFA) [248]. Waste cooking oils having less than 15% free fatty acids (FFA) as a by-

product of oxidation during cooking are considered yellow grease. If the FFA content is higher than 15%, as might occur particularly in the summer months during storage of waste grease, they are considered lower value brown grease. It has been reported that the FAME of oleic acid show greater stability and improved biodiesel properties [249], hence, waste cooking oils with high content of oleic acid would be a preferred biodiesel substrate. As discussed earlier, waste cooking oils with high FFA content can be easily converted to biodiesel in a single step as lipase is capable of catalyzing both transesterication of TG and interesterication of FFA [250]. Lipases exhibit greater stability and performance in FFA-rich substrates such as waste cooking oils than in TG-rich vegetable oils [251]. Moreover, crude vegetable oils contain appreciable amounts of phospholipids that were found to diminish the lipase performance during biodiesel production [204]. These phospholipids can be removed in a degumming pretreatment step which however adds to the cost of biodiesel production [252]. Animal feedstocks for biodiesel production include tallow, poultry fat, pork fat, yellow grease, lard and sh oil [253]. Animal feedstocks are priced favorably for a cost-efcient conversion to biodiesel, however, their availability is limited [252]. The U.S. Department of Agriculture predicts a continued growth of 1% per year with a guaranteed and steady supply of tallow and fats. Biodiesel produced from animal fats and vegetable oil has a similar chemical content with some variation in the component proportions [254] which however results in differences in properties of vegetable oil-derived and animal fat-derived biodiese. The cetane number of animal fat-derived biodiesel is up to 15% higher than that of soybean-based and conventional biodiesel. In addition to improving fuel combustion, increasing cetane levels often reduce emissions of NOx and particulate matter. Animal fat-based biodiesel also provides added lubricity, a measure of protective compounds in the fuel that reduce engine wear and tear. The higher percentage of saturated fats in animal fat-based biodiesel: (1) provides greater oxidative stability than its plant oil-based counterparts, reducing the risk of sedimentation [254]; (2) reduces the low-temperature uidity thereby leading to an increase in the cold lter plugging point of biodiesel [254] because the high levels of saturates crystallize more readily. Hence, the use of additives and blends with vegetable oil biodiesel could reduce temperatures below the cloud point. Currently, about 11 billion pounds of rendered fats are produced annually of which only a small fraction of 38% is used to produce about 116 million gallons of biodiesel. The tax incentive for biodiesel made from inedible animal fats and used vegetable oils is 50/gallon, and it takes about 7.5 lb of waste oils and fats for each gallon of biodiesel produced [255]. Microalgae are regarded as the biodiesel feedstock of the future and the only renewable source of renewable biodiesel that can meet the global demand for transportation fuels [256]. It has been estimated that only 2% of the existing US cropping area would be required to produce microalgae with 3050 wt% oil content that could potentially replace about a third of the US annual demand for transportation fuels. To completely replace all transportation fuels of about 180 billion gallons of gasoline and diesel, less than 5% of the cropping area would be needed for algae production [256]. Microalgae have lower water requirements compared to soybeans and canola [257]. They can accumulate more than 80% water-free oil of the algal dry biomass and produce over 250 times greater oil yields per acre than soybean water-free oil [256,258]. The exploitation of microalgae for biodiesel has some apparent advantages over other feedstocks: high oil yields; production from carbon dioxide and sunlight only; use of non-arable land. They can grow in salty and waste water, and have high growth rates with a generation time of 24 h [259,260] which allows multiple harvesting during the year. However, major obstacles to commercialization are the light intensity requirements, nitrogen deciency, and technological

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challenges related to harvesting and oil extraction from algal biomass [247,261]. Harvesting contributes 2040% of the total cost of biomass production [262]. In addition, the microalgae contain high levels of polyunsaturated fatty acids with four or more double bands [263] as eicosapentanoic acid (C20) and docosahexaenoic acid (C22) which are unstable and prone to oxidation during storage that diminishes biodiesel performance [256]. As biodiesel runs in normal compression engines or diesel engines, its physicochemical properties should meet the quality requirements that are applicable to the conventional diesel fuel (petrodiesel) [256]. The US and European standards for biodiesel are stipulated in the American Society for Testing and Materials (ASTM) D6751 and EN 14214, respectively [258]. Feedstock properties like FFA composition and content, moisture content, unsaponied compounds, impurities, etc. inuence the engine performance of biodiesel [244,249]. A considerable amount of literature has been published on testing the physiochemical properties of biodiesel using chemical catalysts [259]. Recent studies compared the properties of biodiesel obtained from enzymatic transesterication with those of chemically-catalyzed biodiesel and concluded that enzymatic biodiesel closely meets the stringent quality requirements of EU and US standards [260,262]. Properties of enzymatic biodiesel such as density, ash point, pour point, heating value, viscosity and ash content have met the ASTM biodiesel standard 6751-03 (Table 4). 3.6. Acyl acceptor The commercial production of biodiesel requires the use of inexpensive and readily available acyl acceptors such as methanol and ethanol. Stoichiometrically, the alcoholysis of any oil requires three moles of alcohol for each mole of oil. Since transesterication is a reversible reaction, an excess of alcohol is required to shift the equilibrium to FAAE formation [265]. The yield of biodiesel increases with the increase in the alcohol concentration up to a certain concentration, depending on the particular operating conditions such as the type and amount of alcohol, biocatalyst, temperature, mixing intensity used, the moisture and FFA content of the substrate, and mode of bioreactor operation [266]. Overall, the lipase biocatalysts require lower alcohol to oil ratio than chemical catalysts [181,267]. Generally, molar ratios of alcohol to oil of between 3:1 and 6:1 have been commonly used [268270]. A further increase of the alcohol content beyond the optimum concentration does not improve the biodiesel yield, however, it has a negative impact on the costs of alcohol recovery [269,271]. High alcohol to oil molar ratios may inhibit lipase activity and interfere in the separation of glycerol due to its increased solubility in ethanol [268,272]. The effect of methanol to oil ratio on the FAAE yield catalyzed by immobilized lipase was

Table 4 Properties of enzymatic biodiesel. Parameters Biodiesel from stillingia oil [263] 0.900 Biodiesel from jatropha oil [262] Biodiesel from waste cooking oil [260] 0.8924 (20 C) 195 9.12 (20 C) 0.003 Biodiesel standard (ASTM D675103) [264] 0.87 0.90 (15 C) 15 to 10 P130 3340 1.96 (40 C) 60.02

reported by several researchers for soybean oil [198], jatropha oil [261] and canola oil [273]. Immobilized C. antarctica lipase mediated transesterications of vegetable oil with a maximum conversion of 95% achieved at a methanol/oil molar ratio of 3:1 [274]. Methanolysis of Simarouba oil with a fungal immobilized lipase produced a maximum yield of 91.5% FAME using methanol as acyl acceptor [275]. A 95% yield of methyl esters of rape seed oil was achieved with methanol/oil molar ratio of 4:1 [191]. Biodiesel production from transgenic corn oil using Lipozyme TL IM peaked at a methanol to oil ratio of 6 to 1 [268]. For most of the transesterication reactions, methanol is commonly used because of its reactivity, volatile nature, and lower cost than other alcohols. However, methanol is mostly produced from non-renewable sources such as natural gas or coal, and is toxic. Most lipases can be at least partially inactivated by methanol that affects their catalytic performance [234]. The degree of lipase deactivation is believed to be inversely proportional to the number of carbon atoms in the linear lower alcohols [4]. On the other hand, the rate of transesterication was reported to increase proportionally to the number of carbon atoms in the alcohol [250]. Therefore, ethanol as a longer carbon chain alcohol, presents an eco-friendly and renewable alternative that is less inhibitory than methanol. However, with ethanol as the acyl acceptor, the lipase activity of Novozym435 was completely lost after only six cycles of enzyme reuse [195]. The enzyme activity of Novozym435 with different substrates and acyl acceptors is shown in Table 5. Enzyme inhibition by methanol can be overcome by overdosing the lipase, which however is not a cost-effective solution to the problem [18]. It was also reported that some lipases (from Pseudomonas) were more tolerant to alcohol than others (e.g. from T. lanuginosus and R. miehei) [18,37]. Three strategies have been developed to minimize alcohol inhibition: (1) stepwise or sequential addition of alcohol or alcohol aliquots [224,277]; (2) use of solvents [195]; (3) use of alternative acyl acceptors such as longer chain alcohols and alkyl esters [215]. The step-wise addition of alcohol maintains the alcohol concentration below the critical level to avoid loss of solubility in the oil and undesirable deactivation of the enzyme. Research has focused on stepwise addition of methanol as the most commonly used acyl acceptor with the greatest inhibitory effect on lipase. Shimada et al. [274] rst used stepwise addition of methanol and attained a 95% conversion after 50 cycles of operation. Watanabe et al. [278] reported a 90% yield using a two-step batch-wise addition of methanol. The yield was maintained even after 100 batches of operation. Similarly, a 95% conversion was observed following a step-wise addition of methanol after 50 batches [274]. A biodiesel yield of as high as 97% was obtained from plant oil by a three-step addition of 0.33 M equivalents of methanol at 0.250.4 h intervals [200]. Kaieda et al. [190] reported that the stepwise addition of methanol prevented inhibition of non-regiospecic lipases such as P. cepacia, C. rugosa and P. ourescens and regiospecic lipases such as R. oryzae. Yields of 90% were obtained by step-wise addition of methanol to waste cooking oil [277]. Similarly, during batch and continuous transesterication with immobilized

Density (g/cm3)

Table 5 Acyl acceptors used with Novozym435 in biodiesel production. Plant oil Soybean Olive Sunower Acyl acceptor Ethanol Methanol Ethyl acetate Conditions 25 C; 7 h Methanol step-wise addition Immobilized enzyme; 25 C Enzyme activity 85% After 9 batches 70% After 8 batches 85% After 12 batches Refs. [189] [276] [195]

Pour point (C) Flash point (C) Heating value (MJ/kg) Viscosity (mm2/s) Ash content (wt%)

137 4.81 (40 C) 0.037

5 82 36.5 8.2

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Novozym435 using step-wise addition of methanol to waste cooking oil, 96% and 93% yields were achieved for the batch and continuous process, respectively, with lipase retaining full activity after 20 days of continuous operation [196]. Yields of 98% were reported for transesterication of soybean oil with T. lanuginosus lipase using a step-wise addition of methanol, while iso-propanol was used to remove glycerol [208]. A 34% increase in the conversion was achieved through a stepwise addition of methanol compared to a batch methanolysis of olive oil [279]. Solvents exert multiple effects on both reactants and products in biodiesel production. These effects include: (1) increased solubility of alcohol that protects lipase from denaturation [262]; (2) increased solubility of glycerol that prevents lipase inhibition [198]; (3) creation of a single phase reaction mixture that improves mass transfer and reaction rates, reduces viscosity, and stabilizes immobilized enzymes [4,18]. The enzyme stabilization is due to increased water activity in the vicinity of the enzyme that is caused by the solventwater immiscibility. The polar, less hydrophobic solvents are not suitable for biocatalytic processes since they can distort the water microlayer around the enzyme, inuencing its native structure and leading to denaturation [280]. Organic solvents such as hexane, ionic liquids and t-butanol have been widely used [70,198]. Most of the literature concentrated on the use of solvents with commercial enzymes for production of biodiesel from vegetable oils. Transesterication of sunower oil with methanol, ethanol and butanol was studied using Novozym435 with and without petroleum ether as the solvent [193]. The petroleum ether did not impact the product yields in presence of ethanol and butanol (as acyl acceptors), however, it was required for biodiesel production with methanol. Similar ndings pointed out to the need of a solvent (hexane) in a study of methanolysis of soybean oil, rapeseed oil, tallow, and recycled restaurant grease catalyzed by M. miehei lipase (Lipozyme IM 60) and C. antarctica (SP 435) [197]. This was attributed to the inhibition of immobilized enzymes by methanol. The use of a common solvent for both methanol and oil has proved to be effective [191,198,207]. Iso et al. [207] investigated the effects of 1,4dioxane, benzene, chloroform and tetrahydrofuran as common solvents for methanolysis with lipase enzymes from P. uorescens (Lipase AK), P. cepacia (Lipase PS), M. javanicus (Lipase M), C. rugosa (Lipase AY) and R. niveus (Newlase F). Higher conversions of rapeseed oil of up to 97% were reported using mixture of 2-ehtyl-1-hexanol [216]. Lipase AK achieved high conversion rates without loss of activity when 1,4-dioxane was used as solvent. Use of tert-butanol resulted in higher yields of methyl esters [191,198]. Studies showed that often excess alcohols are used to shift the equilibrium towards products [37,41]. Although similar biodiesel yields have been obtained in presence and absence of solvent [18,262,280], the use of solvents signicantly increases the reaction rate in comparison to solvent-free systems. However, the use of solvents is not economically and environmentally favored as it requires the addition of a solvent recovery unit for separating the organic solvent from the reaction mixture, which leads to increase in cost of recovery, losses and gas emissions [247]. Acyl acceptor alternatives to methanol include primary, secondary, straight chained and branched alcohols such as isopropanol [281], t-butanol [191,198], and octanol [67]. The choice of alcohol can inuence the cold ow properties [282] and lubricity [283] of biodiesel. The longer chain alcohols have shown higher yields than methanol [64] as lipases are known to have a higher afnity toward long-chain than short-chain alcohols [65]. 3.7. Enzyme operating variables Generally, transesterication is carried out below the boiling point of the alcohol used order to prevent the alcohol evaporation. In the case of methanol and ethanol, the boiling temperatures cor-

respond to 65 C and 78 C, respectively. Overall, higher reaction temperatures reduce oil viscosity, increase the reaction rates and shorten the reaction time [41]. As most lipases have their temperature optimum between 30 and 60 C, the enzymatic transesterication is mostly conducted in that temperature range. The free lipase enzymes, especially bacterial lipases, are known for higher thermal stability [67]. Lipase immobilization to solid supports decreases the effect of temperature deactivation which leads to improved thermostability [18] and increased temperature optimum of lipase [284]. Overall, the optimum temperature is inuenced by the enzyme stability, alcohol to oil molar ratio and the type of organic solvent used [250]. The initial rate of reaction increases with reaction temperature, however, conversion rates decrease at temperatures above the optimal due to thermal deactivation of lipase [285,286]. A broad range of operational temperatures has been employed depending on the enzyme source: 2555 C for C. antarctica IM lipase (temperature optimum of 40 C) [287]; 2070 C for P. cepacia IM lipase (50 C) [199]; 2060 C for R. chinensis whole-cell IM lipase (30 C) [288]. The optimal temperature for lipid transesterications with a mixture of R. oryzae and C. rugosa IM lipases was 45 C [289]. Most frequently, the optimum operating temperature for lipases has been reported to be 40 C on average [290]. Bacterial lipases have neutral or alkaline pH optima [141,291], whereas lipases from yeasts and fungi are more active at the near neutral to slightly acidic pH [292294]. However, some lipases are active and stable over a broad pH range (pH 312) [295]. Overall, bacterial lipases have been reported to exhibit a higher activity and thermostability than other microbial lipases [141,284,291]. The protein conformation of lipase is affected by pH, and upon immobilization, the optimum pH for reactions catalyzed by lipases is slightly shifted toward more alkaline values due to the partial opening of the lid at the enzyme active site [284]. In recent years, research has focused on the use of lipase-producing fungi as a source of extracellular lipase, or as a whole cell biocatalyst [19,277,296]. In terms of cost effectiveness of the biodiesel process, lamentous fungi have appeared as the most prominent whole cell biocatalyst for industrial applications [66]. Overall, the reaction times needed for enzymatic transesterication, although dependant on reaction temperature, reactant and catalyst concentrations, are generally longer (36 h on average) than those of the chemically-catalyzed process (9 h) [19]. A simplied model of the reaction behavior can be presented to explain the accumulation of FAME as a function of the reaction time: (1) a slow initial rate to allow for mixing and diffusion of alcohol into the oil; (2) rate of fatty acid esters conversion increases exponentially with reaction time [41,45]; (3) reaction rate reaches a peak and proceeds at a steady state [43,216]; (4) reaction rate declines [18]. Immobilized B. cepacia lipase produced a 90% biodiesel from used Jatropha oil yield after a 12 h reaction time [297]. Methanolysis of Simarouba oil and vegetable oils with immobilized lipase was optimum at 36 h [275] and 48 h [274], respectively. Extending the reaction time beyond that required for optimum biodiesel production can reduce the biodiesel yields due to enzyme inhibition by methanol and glycerol [275,298]. Water maintains the three dimensional structure of the enzyme [285] and can directly affect the enzyme structure and activity [299]. The latter is determined by the amount of enzyme-bound water which is best dened as water activity [250,300]. The optimal protein conformation is disturbed by removal of water that surrounds the enzyme macromolecule, whereas excess of water forms water droplets within the enzyme active site. The hydrolysis reaction, that competes transesterication, is catalyzed by increasing amounts of water in the reaction mixture [301]. Generally, minimum amount of water is necessary for biocatalytic activity that is specic for a given lipase [167]. No transesterication is pos-

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sible in absence of water as the formation of enzyme substrate complexes (lipase-oil) proceeds only in presence of oilwater interface. A optimum water content, minimum hydrolysis and maximum transesterication occur [154,167]. The reaction rate was reported to increase proportionally to the water content in many studies [18,65,302,303]. The optimal water content for R. chinensis lipase [288], C. antarcticaand T. lanuginosalipase [191] was 2% whereas 20% water was required for maximum transesterication by Candida sp. 99125 lipase [285]. 3.8. Bioreactor design The batch stirred tank reactor (STR) [45,304] and packed-bed reactor (PBR) [196,277,278,305] are the most extensively studied bioreactors for enzymatic biodiesel production. In STR, the enzyme (free or immobilized) is dispersed in the reaction mixture by agitation, whereas in the PBR, the immobilized enzyme is packed into a column. The batch STR is the simplest bioreactor type consisting of a reactor and a propeller. It is suitable high-viscous solutions and immobilized enzymes that are less sensitive to shear stress and deactivation upon physical stirring. A solidliquid separation using centrifugation or ltration is normally applied at the end of the process to recover the immobilized enzyme. The STR operated in a batch mode has a low throughput due to the need to empty, clean and reload the reactor before a new batch can start. The low productivity disadvantage of the batch STR can be eliminated by using a continuous STR where the enzyme is retained in the reactor with a lter placed at the reactor outlet. The PBR can operate in batch or continuous mode by recirculating the reaction mixture. The recycling method is advantageous as it allows the substrate solution to be passed through the column at a desired velocity. For industrial applications, the upward substrate ow is generally preferred over downward ow because it does not compress the enzyme bed that results in blockages with poor oxygen transfer and pressure drops [18]. In the continuous PBR, reaction mixture is continuously pumped through the column and the enzyme can be effectively reused without a prior separation. Advantages of using continuous PBR include high efciency, low cost and ease of construction, operation and maintenance. In addition, it allows for continuous removal of glycerol and excess alcohol, and protects the enzyme particles from mechanical shear stress [197,198]. The continuous PBR is superior to the batch PBR due to automated control and operation, reduced labor costs, stable operating conditions, and easy quality control of products [18,306]. Other bioreactor congurations include uid beds, expanding bed, recirculation membrane reactors, and static mixers [18]. In the membrane bioreactors, the enzyme is immobilized onto at sheet or hollow ber membranes; however, membrane fouling presents a problem that is more challenging than the bed plugging in the PBR. Although most of the existing biodiesel plants are currently running in batch mode with stirred tank reactors, recent research efforts have focused on optimization of PBR for use in different enzymatic biodiesel production processes [30,179,203,307,308]. Chen et al. [308] tested a PBR for continuous biodiesel production using methanolysis of soybean oil in a t-butanol solvent system catalyzed by Novozym435. A molar conversion of 83% was attained with no considerable decline in lipase activity in continuous operation for 30 days at a ow rate of 0.1 ml/min, 52 C and a 4:1 methanol to oil molar ratio. A 88% conversion was reached in a continuous PBR system catalyzed by a lipase nanoparticles composite (lipase-Fe3O4) using a mixture of soybean oil, distilled water, methanol and n-hexane with volumetric ratio of 6:3:1:0.2, respectively, at a ow rate of 0.25 ml/min for 192 h [307]. PBR was used for production of enzymatic biodiesel from waste cooking oil with a FAME yield of 90% using a solvent-free system [203]. The stepwise addition of methanol prevented the deactivation of the

lipase, which allowed a constant enzyme activity during 100 days of operation. Transesterication of soybean oil in a PBR at a ow rate of 25 l/h using a R. oryzae whole-cell immobilized catalyst and a step-wise addition of methanol produced a maximum FAME yield of 90% [184]. A whole-cell biocatalyst of A. niger immobilized in a PBR produced 90% FAME from palm oil at a ow rate of 15 l/h by continuous recycling of the reaction mixture for 72 h [179]. The biodiesel yield dropped to 85% after 4 consecutive cycles. An optimal continuous production of biodiesel by methanolysis of soybean oil in a packed-bed reactor was developed using response surface methodology (RSM) and Box-Behnken design with immobilized lipase (Novozym435) as a catalyst in a t-butanol solvent system [308]. The continuous process over 30 days showed no appreciable decrease in the molar conversion of 83.3%. Production of biodiesel from vegetable oils with a high FFA content requires an additional step for separation of the reaction byproducts from the biodiesel product. This can be achieved by water-washing or water-free washing of biodiesel [309]. The water-free washing process is performed by a direct mixing of the transesterication reaction products and adsorbent particles or of biodiesel washing pellets. The biodiesel uid containing the adsorbent particles subsequently requires ltration that is both monotonous and time consuming. Alternatively, a water-free washing in PBR has been adopted as a method that effects automatic separation of the adsorbents. Several parameters are factored into the design of a packed bed water-free washing biodiesel separator such as the ease with which the biodiesel stream ows through the bed; bed porosity; adsorption dynamics of the adsorbents; ratio of convective to ow rate, etc. It should be noted that the washing requirement complicates the conventional biodiesel production, but is not a challenge in the production of enzymatic biodiesel with minimal impurities [12]. 3.9. Glycerol Due to its hydrophilicity, glycerol, the co-product in biodiesel production, is insoluble in the oils and gets easily adsorbed onto the surface of the immobilized lipase. This creates mass transfer limitations that negatively impact lipase activity and stability. Adding silica gel into the reaction system to absorb the glycerol or periodically washing the lipase with organic solvents to regenerate the lipase activity have been proposed. Studies by Bako-Bela et al. [205] employed dialysis for glycerol removal which resulted in a 97% conversion by methanolysis. However, these methods are impractical for large-scale continuous production of biodiesel. As discussed before, a novel interesterication process, that makes use of methyl or ethyl acetate as the acyl acceptor instead of pure methanol, does not produce glycerol [215]. The triacetylglycerol co-product does not interfere with the enzyme catalyst or the biodiesel main product, which also eliminates the need of complicated downstream processing. The stoichiometric yield of glycerol is 10% (w/w) with respect to the biodiesel produced. Following methanol recovery, glycerol with up to 90% purity can be used as a marketable commodity. Most commercial biodiesel manufacturing companies are able to send the glycerol to a glycerol recovery/rening facility. Pure grades of glycerol (99.7%) can be used as a raw material in other industrial sectors such as food, cosmetics, paints, pharmaceutics, paper, textiles, leather, toiletries, toothpaste, drugs, animal feed, plasticizers, tobacco, and emulsiers, and for the production of various chemicals [310]. The increase in biodiesel production is expected to result in excess of glycerol which has a limited market thereby reducing the price of glycerol. This necessitates the development of alternative and viable processes for glycerol as a value-added co-product [311] that would impact positively on the overall production cost of biodiesel [312]. For instance, there

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is a growing interest in glycerol fermentation to methanol and ethanol used in biodiesel production, which would improve the economics of the biodiesel process [189,313317]. Fig. 4 shows some major economically-important biochemicals that can be produced from glycerol-based biorenery setup. Glycerol has been used as an alternative and inexpensive carbon source in the microbial production of various compounds such as citric acid, succinic acid, 1,3 propanediol, 2,3-butanediol, carotenoids, hydrogen, biosurfactants, etc. [318,319]. Using the yeast species Yarrowia lipolytica, Rymowicz et al.[320] studied the production of citric acid from raw glycerol derived from the biodiesel production of rapeseed oil. A maximum yield of 0.62 g citric acid/g of glycerol was reported. A nal concentration of 1,3-propanediol of 51.3 g/l and 53.0 g/l was obtained with Klebsiella pneumoniae from crude glycerol (derived from methanolysis of soybean oil) using alkali- and lipase-catalyzed process, respectively [321]. Another strain of K. pneumonia, ATCC 15380, was optimized for production of 1,3 propanediol from glycerol derived from biodiesel production of non-edible jatropha oil [322]. The bacterial strain E. coli AC-521 produced lactic acid from crude glycerol with a high yield of 0.9 mol/mol glycerol [323]. Anaerobic digestion of crude acidied glycerol yielded 0.306 m3 methane/kg glycerol [324]. The natural occurring polymer polyhydroxybutryate (PHB, also known as bacterial polyester, can be synthesized from crude glycerol instead of glucose as carbon source by Paracoccus denitricans and Cupria vidusnecator JMP 134 bacterial strains [325]. Using convention catalysts in addition to microbial conversion, chemical compounds with a diverse range of industrial applications can be synthesized from biodiesel glycerol. These include among others acrolein[326], ethylene glycol [327], syngas [328], (2,2-dimethyl-1,3-dioxolan-4-yl) methyl acetate [329] and oligomers of glycerol [330]. A 74 mol% acrolein with 81% of selectivity was synthesized from glycerol using an acid catalyst at supercritical conditions (673 K and 34.5 MPa pressure) [331]. The fuel additive (2,2-dimethyl-1,3-dioxolan-4-yl) methyl acetate can be produced from crude glycerol with acetone using p-toluene sulfonic monohydrate catalyst. The addition of (2,2-dimethyl-1,3dioxolan-4-yl) methyl acetate to biodiesel was reported to recover the biodiesel viscosity and meet the U.S. and European fuel standards [329]. Dichlor-2-propanol was prepared directly from glycerol by the aid of heteropolyacid and acetic acid as catalysts [279].

4. Current trends and future directions for enzymatic biodiesel R&D Recently, a novel method for production of biodiesel at reduced costs has been reported [332]. This method employs the use of a solid whole cell biocatalyst obtained by solid state fermentation (SSF). The advantages associated with the use of a SSF-generated biocatalyst are twofold: (i) the microorganisms grow on low-cost substrates (agro-industrial residues) maintaining low moisture content; (ii) the crude fermented solid material can be used directly as a biocatalyst thereby omitting three processing steps: lipase extraction, purication and immobilization. This leads to a signicant reduction of the lipase costs that translates into lower biodiesel production costs [333,334]. A SSF-derived lipase-containing whole cell material, produced by Burkholderia cepacia LTEB11, was used for a direct catalytic synthesis of biodiesel in n-heptane [335] and co-solvent-free [336] systems A recent study by Liu et al. [333] reported SSF production of a B. cepacia lipase on a mixed substrate of sugarcane bagasse and sunower seed meal with a catalytic activity of 72.3 units/g dry solids over a period of 96 h. A novel mixed substrate for enhanced SSF production of lipase from A. niger MTCC 2594 was developed for hydrolyzing tallow at 85.2% for 24 h [337]. Another innovative approach, hydroesterication, was recently developed as a means to overcome problems related to biodiesel production from feedstocks containing high percentage of FFA and water [338]. The process of biodiesel production through hydroesterication occurs in two stages. In the rst stage, monoand tri-acylglycerols are hydrolyzed to FFA and glycerol. In the second, FFA are separated and esteried to biodiesel [339]. The hydroesterication process can be carried out by: (1) a supercritical hydrolysis and esterication (catalyst-free); (2) enzymaticchemical hydroestrication; and (3) enzymatic hydroesterication. The catalytic-free supercritical hydrolysis and supercritical esterication process (both conducted at a temperature of 270 C and pressure of 20 MPa) in presence of methanol resulted in biodiesel conversion rates in excess of 90% [340]. In a new hybrid (enzyme/chemical) hydroesterifcation process, biodiesel was produced through hydrolysis of physic nut oil by a plant enzyme (vegetable enzyme extracted from germinated physic nut seeds), followed by esterication of the generated FFA with methanol cat-

OH HO O Acrolein OH HO O O OH O OH Citricacid O O O O O OH

Glycerol

Catalytic conversions

(2,2-dimethyl-1,3-dioxolan-4-yl) methylacetate

Microbial conversions

OH OH Lacticacid

OH Cl Cl Dichloro-2-Propanol CO/H2 Syngas CH4 Methane R O HO OH 1,3 Propanediol

O n Poly(hydroxyalkanoates) (PHAs)

Fig. 4. Potential products derived from catalytic and microbial conversion of glycerol.

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alyzed by a heterogeneous acid (niobic acid in pellets) [339]. A nal product containing 91% FAME, 1% FFA and 7% of lipophilic compounds was obtained by enzymatic hydroesterication of acid oil, a byproduct from vegetable oil rening [341]. The initial hydrolysis step acid oil was carried with C. rugosa lipase and the subsequent esterication of FFA to FAME - by C. antarctica IM lipase. However, the hydroesterifcation was carried as a two-stage and energy-intensive process which, although ecofriendly, was considered a major economic drawback [338,341]. An alternative to hydroesterication comprised of a two-stage esterication and transesterication process for biodiesel production from brown grease was studied by Fan et al. [342]. The lipase deactivation by methanol was prevented by addition of biodiesel to the reaction mixture prior to initiation of the conversion process. A FAME yield greater than 95 wt% was attained after 15 cycles of operation. Methanol inhibition is addressed through the use of solvents, continuous removal of glycerol by dialysis or solvent extraction, and stepwise addition of methanol. However, the use of solvents adds to biodiesel costs, whereas the glycerol removal and the stepwise feeding of methanol are impractical and challenging on commercial scale. A permanent solution to the lipase inhibition by methanol is offered by recombinant DNA technology [343]. Protein engineering and directed evolution can improve the lipase specicity, thermostability, activity and methanol tolerance [344346]. A recent example for this effort is the high-level expression of a thermostable and methanol-tolerant lipase from Proteus sp. in E. coli. The recombinant strain was able to produce biodiesel in the presence of high water concentrations. Alternatively, the recombinant E. coli strain was used as a whole-cell biocatalyst. Directed evolution was subsequently employed to increase its thermostability and tolerance to methanol while retaining the wild-type lipase activity at ambient temperature [347]. One of the latest developments of thermostable and methanoltolerant lipases reports on a newly-constructed mutant of P. mirabillis lipase, known as Dieselzyme 4, that in comparison to the wild type enzyme, has a 30-fold improved thermostability and a 50-fold increased methanol tolerance of the half-inactivation time at 50 C in a 50% aqueous methanol [348]. The mutagenized lipase outperformed the commercially available lipase from B. cepacia. In another study, a robust recombinant whole cell thermo- and solvent-tolerant biocatalyst from A. oryzae (r-BTL) was developed for biodiesel production [349]. The r-BTL had an increased tolerance (up to 30%) toward organic solvents such as dimethyl carbonate, ethanol, methanol, 2-propanol or acetone at 50 C. Another recent breakthrough in enzymatic biodiesel production was the development of an enzyme-based bio-hybrid catalyst called Lipase@Organo-Si(HIPE) [39,350]. The bio-hybrid catalyst was used in continuous-ow, monolithic micro-reactors resulting in extremely high biodiesel yields at a superior recycling performance over the batch reactors. The turnover numbers and frequencies attained demonstrated the real potential of the catalyst for industrial applications The insolubility of methanol in oil was overcome by development of an innovative, enzyme-immobilized reactor that integrated a continuous STR with two PBR connected in series for a continuous biodiesel production omitting the use of organic solvents and intermittent addition of methanol to the system [351]. Recent research efforts for reducing reaction time and minimizing catalyst inhibition by methanol also focused on the construction of a semi-pilot, continuous system for enzymatic biodiesel synthesis based on the use of near-critical carbon dioxide as reaction medium [352]. The highest conversion of 99.9% was attained in only 4.5 h with Lipozyme TL IM at 30 C and 100 bar in a carbon dioxide environment through a continuous mixing of a 1:1.3 oil/ methanol mixture and a three-step addition of methanol. A foremost technical development was the use of a PBR coupled to a

glycerol separation system that produced a FAME yield of 94.3% with an efcient glycerol removal of 99.7% [353]. The production of syngas (synthesis gas) from glycerol is the furthermost possible application of glycerol. In literature, there are numerous catalytic processes to produce syngas from glycerol, which include autothermal reforming, aqueous phase reforming and supercritical water reforming [328,354,355]. Each process has different working conditions and various types of catalyst involved in the production of syngas [356]. As methanol used in the production of biodiesel is mostly derived from nonrenewable resources, syngas is exploited for methanol production as a sustainable alternative to natural gas [357,358] (Fig. 5a). The product quality depends on the glycerol purity, which is signicantly higher in the enzymatic than chemical process of biodiesel production. The use of a biocatalyst simplies glycerol separation even if lowquality feedstocks such as waste cooking oils are utilized. Despite their current high prices, acyl acceptors such as methyl or ethyl acetate are currently viewed as promising substitutes for alcohols [195]. The major advantage of the interesterication of TGs with methyl or ethyl acetate over the alcoholysis reaction is that no glycerol is formed which eliminates the need for downstream separation and glycerol recovery. Instead, a new, higher-value by-product of the interesterication with ethyl acetate, triacetylglycerol (triacetin), is formed, although separation and recovery of triacetin are still required. Triacetin has a higher value than glycerol and could help reduce the cost of biodiesel production [188]. Triacetin is an antifungal agent and can also be used as a xative in perfumery or as a solvent in basic dyes [302]. Conferring to the reaction stoichiometry, a single-phase interesterication product typically contains 80 wt% of biodiesel and 20 wt% triacetin [359]. Triacetin is miscible with biodiesel in all proportions and can be included in biodiesel formulations [360]. Casas et al. [359] studied the effects of triacetin on biodiesel quality and reported that the addition of triactin should not to be limited considering its positive effects on the fuel quality in accordance with ASTM D6751-09. However, the European norms (EN 14214:2008) limit the triacetin content in biodiesel mixtures to 3.510 wt% due its effect on increasing density and viscosity of the fuel. Interesterication also leads to the production of other byproducts like diactin, monoactin and glycerol in small fractions [361]. Casas et al. [360] reported that liquidliquid extraction with a three-stage washing can complete recover these byproducts from biodiesel-triacetin mixtures with a slight reduction in the triacetin content. In presence of ethyl acetate, interesterication of soybean oil with Novozym435 as biocatalyst and methyl acetate as acyl acceptor produced a biodiesel yield of 92% [188]. After 12 batches of enzyme reuse, Novozym435 retained its full activity on Jatropha, karanj and sunower oils [194]. The introduction of alternative acyl acceptors offers an innovative and eco-friendly approach to eliminating glycerol-associated problems (Fig 5b). Methanol is currently the less expensive alternative to biodiesel production. However, methanol is considered to be more toxic than ethanol, and to inhibit the enzyme biocatalysts. It is also not suitable for efcient biodiesel conversion of high-FFA feedstocks. On the other hand, ethyl acetate is currently more expensive than ethanol and methanol, however, as we consider the current and future trends in enzymatic biodiesel, the overall process and environmental sustainability of producing biodiesel from inexpensive raw materials and waste, and recovery/ conversion of triacetin to high-value products, this solvent appears as the natural, renewable and sustainable source of acyl acceptors for biodiesel production. Ethyl acetate is the derivative of two biodegradable and microbially-produced fermentation products, ethanol and acetic acid. In the present scenario, where environmental concerns and production costs are given high priorities, a solvent-free enzymatic process for industrial biodiesel production

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(a)

(b)

Fig. 5. Novel and eco-friendly biodiesel production processes using renewable acyl acceptors: (a) use of glycerol-derived biomethanol; (b) use of bioethanol-derived ethyl acetate.

utilizing renewable materials (enzymes, non-edible and waste oils, ethyl acetate as a green biochemical) would be encouraged as a sustainable, cost-effective and environmentally-sound alternative. With the increase in the production capacity of ethanol and focus on cellulosic, fermentable sugars from energy crops and agr-waste in the US and globally, the price of ethanol and acetic acid will be gradually decreasing which makes ethyl acetate the best candidate for a green biodiesel production. The use of ethyl acetate avoids the formation of glycerol which is an enzyme inhibitor and a by-product with a costly recovery. Biodiesel produced with ethyl acetate as acyl acceptor has a higher cetane number, lower pour and cloud points, higher ash and combustion points that improve cold starts, and lower smoke opacity and exhaust temperatures [195]. The use of ethyl acetate may also stimulate the development of new biodieseltriacetin blends to suppress knocking, improve performance and reduce tail pipe emissions of diesel engines [362].

which translates into about $0.03/kg biodiesel. In 2006, another Chinese company, Hainabaichuan Co. Ltd., launched production of enzymatic biodiesel at a 20,000 tons/year scale that was subsequently doubled to 40,000 tons per year in 2008. Biodiesel production is based on waste palm oil utilizing Novozym435 [168]. A new enzyme manufacturing company in Israel, TransBiodiesel, was started to provide the biodiesel market with enzyme-based catalysts at commercial quantities. In 2012, Piedmont Biofuels, North Carolina, developed a new technology (FAeSTER) for a continuous biodiesel production using immobilized or liquid enzyme. Challenges in the commercialization of enzymatic biodiesel relate to the retrot of existing conventional processes into enzymatic and the scale-up of technologies based on whole cell enzyme catalysts. Further progress in the development of innovative, inexpensive enzyme-immobilization techniques with low reaction time, higher enzyme stability and activity should be developed towards the global economic acceptance of large-scale enzymatic biodiesel production.

5. Economic considerations and large scale developments in enzymatic biodiesel 6. Conclusions The number of pilot and industrial scale plants for commercialization of enzymatic biodiesel signicantly increased in recent years (see also Introduction, paragraphs 46). Fjerbaek et al. [18] roughly estimated the cost of catalyst per kilogram of biodiesel produced as 0.14 US$ for Lipozyme IM, and 0.006 US$, for alkali catalyst. Based on a of 100 kg of oil to biodiesel, the cost of the alkali and enzyme (Novozym435) catalyst was estimated to be $0.62/kg and also approximately $1000/kg, respectively. In 2007, Lvming Co. Ltd., in Shanghai, China, established an enzymatic biodiesel production plant with a capacity of 10,000 tons using waste cooking oil as feedstock, and immobilized lipase of Candida sp. 99 125 as catalyst. The enzyme cost was 200 CNY per ton biodiesel, Due to the high conversion rates and short reaction times, the alkali-catalyzed process is currently the dominant large-scale method for biodiesel production from virgin oils. However, this process is characterized by several inherent process drawbacks including the difculties in glycerol separation and purication, catalyst recovery and recycling, saponication problems, the need to wash out biodiesel impurities, treat waste water and eliminate salts, and their energy-intensive nature. In addition, the production costs are high as virgin oil constitutes almost two-third of the total biodiesel price. Taking into account the biodiesel yield and quality, the alkali process does not offer the best economic and environ-

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L.P. Christopher et al. / Applied Energy 119 (2014) 497520 [16] Supple B, Howard-Hildige R, Gonzalez-Gomez E, Leahy J. The effect of steam treating waste cooking oil on the yield of methyl ester. J Am Oil Chem Soc 2002;79:1758. [17] Zhang Y, Dube M, McLean D, Kates M. Biodiesel production from waste cooking oil: 1. Process design and technological assessment. Bioresour Technol 2003;89:116. [18] Fjerbaek L, Christensen KV, Norddahl B. A review of the current state of biodiesel production using enzymatic transesterication. Biotechnol Bioeng 2009;102:1298315. [19] Parawira W. Biotechnological production of biodiesel fuel using biocatalysed transesterication: a review. Crit Rev Biotechnol 2009;29:8293. [20] Paulson N, Ginder R. The growth and direction of the biodiesel industry in the United States. Working Paper-Center for Agricultural and Rural Development, Iowa State University; 2007. p. 129. [21] Peterson CL, Wagner GL, Auld DL. Vegetable oil substitutes for diesel fuel. 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mental scenario for biodiesel production from low-cost feedstocks such as waste oils and animal fats that have high FFA and water; hence, for optimal results, a selective and specic enzymatic biocatalyst should be used. The acid catalysis converts both FFA and TGs to biodiesel, however, it is corrosive and the transesterication rates are signicantly lower (several thousand times) than those of the base catalysis with a requirement for higher reaction temperatures and a greater number of process equipment for separation and purication of biodiesel from glycerol. The drawbacks associated with the conventional chemical catalysis can be overcome with the introduction of an alternative, enzymatic route for biodiesel production. The enzymatic catalysis offers a number of environmental and economical advantages over the chemical method: room-temperature reaction conditions, elimination of treatment costs associated with recovery of chemical catalysts, enzyme re-use, high substrate specicity, single-step conversion of both FFA and TG to biodiesel, lower alcohol to oil ratio, avoidance of side reactions and minimized impurities, easier product separation and recovery; biodegradability and environmental acceptance. The ability of lipases to catalyze FAME synthesis from low-cost feedstock with high FFA (waste cooking oil, grease, tallow, etc.) would lower the cost of enzymatic biodiesel. The major demerits associated with the biocatalyst are the higher enzyme costs and enzyme inhibition by methanol. Enzyme costs can be reduced by discovery of new, more efcient lipase producers; strain improvement and use of highly productive recombinant strains for production of lipase with improved activity and methanol tolerance; optimization of fermentation media and parameters for lipase production; biocatalyst reuse through lipase and whole cell immobilization; lipase pretreatment for activity regeneration and extended lipase life, etc. Robust enzymes with high activity, thermostability and resistance to the harsh conditions of biodiesel production are needed. Such thermostable lipases produced from extremophiles are already employed as detergents in the textile industry.

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