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  • Enzymatic biodiesel: Chal

    Lew P. Christopher a,b,, HemanataCenter for Bioprocessing Research and Development, SobDepartment of Civil and Environmental Engineering, SocDepartment of Chemistry, Laboratory of Applied ChemidBiotechnology Division, Rossari Biotech Ltd., Naroli, Sil

    h i g h l i g h t s

    rogressrits ofle dever enzym

    ons, elimination of treatment costs associated with recovery of chemicaligh substrate specicity, the ability to convert both free fatty acids and triglyc-

    free fatty acid content such as waste cooking oil, grease and tallow would lower the cost of enhermostabic process. O

    nities to create a sustainable and eco-friendly pathway for production of enzymatic biodiesrenewable resources are discussed.

    2014 Elsevier Ltd. All rights re

    Corresponding author. Address: 501 E. St. Joseph Street, South Dakota School ofMines and Technology, Rapid City 57701, SD, USA. Tel.: +1 605 394 3385.

    Applied Energy 119 (2014) 497520

    Contents lists available at ScienceDirect

    Applied Energy

    journal homepage: www.elseE-mail address: [email protected] (L.P. Christopher).biodiesel. Discovery and engineering of new and robust lipases with high activity, tresistance to inhibition are needed for the establishment of a cost-effective enzymati0306-2619/$ - see front matter 2014 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.apenergy.2014.01.017zymaticlity andpportu-el from

    served.LipaseEnzyme-catalyzed transestericationFeedstockGlycerol

    erides to biodiesel in one step, lower alcohol to oil ratio, avoidance of side reactions and minimized impu-rities, easier product separation and recovery; biodegradability and environmental acceptance. Thispaper provides a comprehensive review of the current state of advancements in the enzymatic transeste-rication of oils. A thorough analysis of recent biotechnological progress is presented in the context ofpresent technological challenges and future developmental opportunities aimed at bringing the enzymecosts down and improving the overall process economics towards large scale production of enzymaticbiodiesel. As the major obstacles that impede industrial production of enzymatic biodiesel is the enzymecost and conversion efciency, this topic is addressed in greater detail in the review. A better understand-ing and control of the underpinning mechanisms of the enzymatic biodiesel process would lead toimproved process efciency and economics. The yield and conversion efciency of enzymatic catalysisis inuenced by a number of factors such as the nature and properties of the enzyme catalyst, enzymeand whole cell immobilization techniques, enzyme pretreatment, biodiesel substrates, acyl acceptorsand their step-wise addition, use of solvents, operating conditions of enzymatic catalysis, bioreactordesign. The ability of lipase to catalyze the synthesis of alkyl esters from low-cost feedstock with highKeywords:Biodiesel

    perature reaction conditicatalysts, enzyme re-use, h A comprehensive overview of recent p Critical assessment of merits and deme Economic considerations and large sca Current trends and future directions fo

    a r t i c l e i n f o

    Article history:Received 31 August 2013Received in revised form 21 November 2013Accepted 6 January 2014Available online 5 February 2014lenges and opportunities

    han Kumar c, Vasudeo P. Zambare d

    uth Dakota School of Mines and Technology, Rapid City, SD, USAuth Dakota School of Mines and Technology, Rapid City, SD, USAstry, University of Jyvskyl, Finlandvassa, India

    in enzymatic biodiesel production.enzymatic biodiesel process.lopments in enzymatic biodiesel.atic biodiesel R&D.

    a b s t r a c t

    The chemical-catalyzed transesterication of vegetable oils to biodiesel has been industrially adopteddue to its high conversion rates and low production time. However, this process suffers from severalinherent drawbacks related to energy-intensive and environmentally unfriendly processing steps suchas catalyst and product recovery, and waste water treatment. This has led to the development of theimmobilized enzyme catalyzed process for biodiesel production which is characterized by certain envi-ronmental and economical advantages over the conventional chemical method. These include room-tem-vier .com/locate /apenergy

  • . . .

    . . .. . .. . .. . .. . .. . .. . .. . .. . .. . .. . .. . .. . .. . .. . .. . .c bi. . .. . .

    substitute for petroleum based diesel fuel which reduces emissions

    epebfuel. T4.5-tiand 8biodiebiodie

    Onexisticsand shc

    virgin oils such as soybean, sunower, olive, palm, sh, jatropha,

    duction, the high enzyme cost signicantly impacts the process

    and make it competitive in price with petroleum diesel [16,17].

    ed Epast few years [12]. According to the National Diesel Board [13],

    thplias excellent energy balance as compared to fossil fuels; biodieselontains 3.2 times the amount of energy it takes to produce it [11].The biodiesel production in the US increased dramatically in the

    feedstocks such as waste restaurant oil, yellow grease, lard, animalfats and others, known as second generation biodiesel feedstock,instead of virgin oil is estimated to reduce the production costereroduon gd its higher ash point makes it a safer fuel to use, handle,tore using existing diesel tanks and equipment. Biodiesel

    costs of the rawmaterials, use of alternate, low-cost feedstock suchas waste frying oil, non-edible oil and oil extracted from otherhanges [10]. B100 can be blended at any level with petroleum die-el, an

    Since the biodiesel production costs are proportional to thee of the great advantages of biodiesel is that it can be used inng engines, vehicles and infrastructure with practically no

    $0.50/lb with a 7.35 lbs/gallon efciency is estimated to cost about$3.675/gallon [14].that can help reduce the risk of global warming by reducing thenet carbon emissions to the atmosphere [79].

    gallon depending on the time period when these studies were con-ducted [20]. Currently, biodiesel production from soybean oil atonoxide than those engines operating on conventional dieselhe greenhouse gas (GHG) emission of biodiesel (B100) aremes lower than gasoline, and 3-fold lower than petrodiesel5% ethanolgasoline fuel (E85). Although the NOx levels ofsel are slightly higher than those of conventional diesel fuel,sel is believed to be eco-friendly alternatives to fossil fuels

    expensive in comparison with petroleum-based fuel as 6080% ofthe cost is associated with the feedstock oil [19]. Previous studieshave estimated that biodiesel production costs range between$1.50 and $2.50 per gallon depending on the feedstock used inthe production process. These costs exceeded the wholesale priceof petroleum-based diesel by anywhere from $0.20 to $0.82 perrgy content, viscosity and phase changes are similar to those ofetrodiesel fuel [46], however, the engines fueled with biodieselmit signicantly fewer particulates, hydrocarbons, and less car-on m

    protability. The cost of commercial enzyme products for indus-trial use is approximately $1,000/kg which is signicantly higherthan that of the alkali catalyst ($0.62/kg) [18]. Biodiesel fuel isand metals. The biodiesel properties such as cetane number, en-of carcinogenic compounds by as much as 85% compared to petro-diesel, essentially free of sulfur, polycyclic aromatic hydrocarbons

    canola, cottonseed, peanut and linseed oil, known as rst genera-tion biodiesel feedstock. In the enzyme-catalyzed biodiesel pro-Contents

    1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2. Biodiesel production methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

    2.1. Chemical production of biodiesel . . . . . . . . . . . . . . . . . . . . . . .2.2. Enzymatic production of biodiesel . . . . . . . . . . . . . . . . . . . . . .

    3. 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 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

    4. Current trends and future directions for enzymatic biodiesel R&D .5. Economic considerations and large scale developments in enzymati6. Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

    References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

    1. Introduction

    Alternative fuels for internal combustion engines have recentlyattracted considerable attention due to the ever diminishing petro-leum reserves and environmental consequences of increasinggreenhouse 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 developmentof alternative fuels from renewable resources that are sustainableand environmentally acceptable. The U.S. RFS sets a goal of 36 bil-lion gallons of biofuels production by year 2022, of which 21 bil-lion gallons should come from the so-called advanced biofuelsand a minimum of 1 billion gallons of biodiesel.

    Over the last decade, biodiesel has attracted considerable atten-tion as a renewable, biodegradable, non-toxic and clean-burning

    498 L.P. Christopher et al. / Appliwere 105 plants in operation as of early 2007 with a totalction capacity of 864 million gallons and additional 1.7 bil-allons coming online from plants under construction. The. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 498

    . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 499. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 499. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 501. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 501. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 501. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 502. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 503. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 503. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 503. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 504. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 506. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 508. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 509. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 510. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 510. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 511odiesel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 513. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 513. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 514

    global biodiesel market is estimated to reach 37 billion gallonsby 2016 with an average annual growth of 42%. In 2012, the USproduced 1.143 billion gallons of biodiesel which was 14.3% morethan what the RFS called for. In 2013, the biodiesel production inthe US is projected to reach 1.28 billion gallons, a 10.7% increaseover the 2012 blended gallons. Since 2005, the biomass-based die-sel production increased more than 18-fold in the US only. The bio-diesel industry in the US in on the rise, and federal agencies such asthe U.S. Department of Transportation and Department of Defenseinvest in research and development to dramatically reduce depen-dence on foreign oil, and spur the creation of a domestic bio-indus-try 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 mainlydue to the use of expensive, high quality and mostly non-rened

    nergy 119 (2014) 497520Oils extracted from algae are particularly viewed as the most sus-tainable feedstock for the future due to the signicantly higheryields, 15,000 gal/acre/year, as compared to only 48 gal/acre/year

  • 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 igni-tion engine now known as the Diesel engine, rst used vegetableoils (such as peanut oil) to fuel his engine [21,22]. The vegetableoils performed well in short term engine tests [23,24], but failedin long term operations [21,25,26]. Problems such as cooking ofinjectors, carbon deposits, oil ring sticking, thickening and gellingof the lubricating oils were encountered. High viscosity (almost10 times that of No. 2 diesel fuel) and a tendency for polymeriza-tion within the cylinder appeared to be the root cause for manyproblems (such as high cloud point temperature), associated withthe direct use of vegetable oils as fuel [23,2634].

    In order to reduce these difculties, the idea of converting thevegetable oils into their ester derivatives was introduced. Reacting

    The following sections provide a state of the art review of thecurrent biodiesel process technologies with emphasis on the enzy-matic transesterication method. A comprehensive analysis ofrecent biotechnological advancements in the enzyme processbiodiesel is presented in the context of current technical challengesand future developmental opportunities aimed at bringing theenzyme costs down and improving the overall process economicstowards large scale production of enzymatic biodiesel.

    2. Biodiesel production methods

    Biodiesel can be made via three production routes: microemul-sions, thermal cracking (pyrolysis), and trans/inter/esterication[26]. The most cost-efcient method for production of biodiesel

    all process of alcoholysis includes three reversible reactions in

    k

    k

    k

    k

    k

    k

    L.P. Christopher et al. / Applied Energy 119 (2014) 497520 499triglycerides (TGs) in oil with an alcohol in presence of a catalystcan produce esters of free fatty acids (FFA) such as fatty acid methylesters (FAME). As a result, the viscosity of these alkyl esters is re-duced, however, their cetane numbers and heating values remainunchanged. FAME are termed biodiesel and the process is knownas trans-esterication [35,36]. The catalysts used for transesteri-cation of biodiesel are alkali, acid (chemical) and enzyme (bio-based). The alkali-catalyzed process gives higher conversion of TGat short reaction times. The foremost drawback of the alkali processis its sensitivity towards FFA in oils that leads to soap formationduring the transesterication process. The acid-catalyzed processesare insensitive to FFA, however, with a drawback of slower reactionrates. Furthermore, the use of chemical catalysts can cause techni-cal problems related to biodiesel purication and separation fromcatalysts 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 hasbeen 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 inthe system; (5) easy separation and reuse of immobilized enzymes.Although the enzyme catalyst has some drawbacks, mainly associ-ated with lower reaction rates, higher costs, and loss of activity orenzyme inhibition, the enzymatic route for biodiesel production isnowadays considered as an environmentally-friendly alternativethat is becoming more realistic as new, more efcient and lessexpensive enzymes are being developed [39]. To minimize enzymecosts, the enzymatic catalyst can be reused by immobilization,which leads to improved efciency of biodiesel recovery. In addi-tion, the enzyme catalytic activities can be enhanced by screeningand selection of high alcohol-tolerant microorganisms and bygenetic engineering [40].

    R1OCOOCOR2

    OCOR3 ROH

    HOOCOR2

    OCOR3 ROH

    ROHHOOH

    OCOR3

    +

    +

    +

    Triglyceride Alcohol

    Diglyceride

    Monoglyceride

    Alcohol

    Alcohol

    1.

    2.

    3.Fig. 1. Step-wise reaction pathways for biodiesel production (R is a small alkyl group,catalysts).R2OOCR

    R3OOCR

    HOOH

    OCOR3

    HOOH

    OH

    +

    +

    Ester

    Ester

    Monoglyceride

    Glycerol

    2

    5

    3

    6which di- and mono-glycerides are formed as intermediates[26,41]. From each TG molecule, three molecules of biodiesel andone molecule of glycerol are produced (Fig. 1). The process equilib-rium can be shifted toward product formation in excess of alcoholor by continuous product removal.

    2.1. Chemical production of biodiesel

    The base-catalyzed transesterication of oils and fats to biodieselproceeds at faster rates and greater yields than the acid-catalyzedprocess [4143]. Hence, biodiesel at yields of 9499% is convention-ally manufactured via chemical catalysis that uses sodium or potas-sium hydroxide at concentrations in the range of 0.51 wt%, amethanol to oil ratio of 6:1, and temperatures of 4580 C to com-pletely transesterify the lipids in several hours. The 6:1 ratio of acylacceptor to vegetable oil is higher than the stoichiometrically re-quired 3:1; this, however ensures excess ofmethanol in the reactionmixture and increased methyl ester yields. However, molar ratioshigher than 6:1 increase glycerol solubility and complicate glycerol

    HOOCOR2

    OCOR3R1OOCR +

    DiglycerideEster

    1

    4with higher quality is transesterication of vegetable oils and ani-mal fats. In lipid chemistry, transesterication is the catalytic pro-cess of exchanging the alkoxy group of an ester by an alcohol (acylacceptor) that converts the TG in oils to fatty acid alkyl esters(FAAE) and glycerol (Fig. 1). This process is also known as alcohol-ysis and makes use of short chain alcohols such as methanol andethanol as acyl acceptors. Interesterication is the transformationof TG to biodiesel in presence of an ester (such as methyl acetate)as the acyl acceptor. In this process, instead of glycerol, another tri-acylglycerol is formed as the by-product. Lastly, biodiesel can bealso produced by a direct esterication of FFA with alcohols to pro-duce FAAE and water as the by-product. Chemical (acids and/orbases) and biological (enzymes) agents serve as catalysts. The over-R1, R2 and R3 are fatty acid chains; k1, k2, k3, k4, k5, k6 are chemical or enzymatic

  • separation. Sodium hydroxide is the preferred alkaline catalyst dueto its lower price and higher biodiesel yields [44].

    Although biodiesel is currently exclusively produced at com-mercial scale utilizing alkali, mainly sodium hydroxide, there areprocess limitations that are considered drawbacks of chemical bio-diesel. These are normally associated with the presence of FFA inquantities higher than 0.5% that can lead to soap formation, andwith the presence of water exceeding 0.3% that can results in con-sumption of the catalyst thereby reducing the reaction yield.Saponication not only consumes the alkali catalyst, but the result-ing soaps can cause the formation of emulsions that create difcul-ties in downstream recovery of biodiesel, and can lower the ester

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

    Alcohol

    Oil

    (a)

    (b)

    Transesterification

    Alcohol

    Acidification Glycerol

    Alcohol

    Separation

    Crude Glycerol

    Evaporation

    Washing

    Drying

    Waste Water

    Alcohol

    Acid

    Catalyst(Acid)

    Catalyst(Base)

    Fatty Acid Alkyl Esters

    F

    Water

    Water

    Saponified product

    PreesterificationTransesterification

    Biodiesel

    Evaporation

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

    EnzymeGlycerol

    SeparationFig. 2. Biodiesel production from feedstocks containing high free fatBiodiesel

    Alcoholatty Acid Alkyl Esters

    Evaporationty acids: (a) two-step chemical process; (b) enzymatic process.

  • ed Eto biodiesel in presence of a base [4754]. A two-step acidbaseprocess has been developed to utilize oils such as used cookingoil and restaurant grease with a high FFA content (2050%) in bio-diesel production (Fig. 2a). The water formed in the acid-catalyzedesterication is separated from the organic layer of FAME prior tothe alkali-catalyzed step. The presence of hydroxide ions in waterincreases the saponication which produces soap by reaction ofthe hydroxide ions with esters, FFA and with other glycerides, thusrendering the downstream separation expensive and affecting theester yields [41,5558]. Liu et al. [59] studied the affect of water onsulfuric acid-catalyzed esterication of carboxylic acid and re-ported a signicant decrease in catalytic activity as water was pro-duced from the condensation of carboxylic acids and alcohols. Theacid-catalyzed process is corrosive in nature causing damage toequipment, proceeds at low rates, and requires long reaction timesto achieve conversion in excess of 90% [55]. For a full completion ofesterication (up to 99%), more than 48 h is normally needed[41,46,55]. However, the major demerit of the acidbase processis 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 pro-duction of biodiesel [19,37]: (1) side reactions of saponicationand hydrolysis affect biodiesel yield and purity; (2) the process,especially the acid pretreatment step, is energy and capital inten-sive; (3) recovery and purication of catalysts and glycerol isexpensive; (4) neutralization and waste water treatment is re-quired. Problems with separation and soap formation haveprompted research with heterogeneous non-enzymatic catalystssuch as amorphous zirconia, titanium-, aluminum-, and potas-sium-doped zirconias, metal oxides, hetero-polyacids, sulfatedzeolites and others [6062]. A mesoporous silica catalyst function-alized with hydrophobic allyl and phenyl was shown to hydropho-bically exclude water from the catalyst active sites while effectivelyesterifying FFA [63]. Compared to homogenous catalysts, heteroge-neous catalysts have the advantages of easier recovery and usabilityon substrates containing higher concentrations of FFA. However,some heterogeneous catalysts could be cost and energy intensivedue to requirements for high reaction temperatures and highalcohol to substrate molar ratios [64].

    2.2. Enzymatic production of biodiesel

    As elaborated in the previous section, the main disadvantages ofthe traditional acid/base transesterication methods are the highreaction temperatures, high energy consumption, corrosive natureof acids, glycerol purication and recovery, separation of catalystsand unreacted alcohol accompanied with additional washing stepsto remove impurities from biodiesel. These problems can be mini-mized and even eliminated by using environmentally-friendly bio-catalysts (enzymes) for biodiesel production (Fig. 2b). Enzymessuch as lipases offer a biological route of biodiesel production witha number of environmental and economic advantages over thechemical route [6568]:

    Use of mild reaction temperatures. High selectivity and specicity of trans/esterication towardssubstrates.

    Broader substrate range due to ability to esterify both glyceride-linked and non-esteried fatty acids in one step; use of loweralcohol to oil ratios.

    Avoidance of side reactions, easier separation and productrecovery due to the production of a glycerol side stream withminimal impurities and water content [12].

    L.P. Christopher et al. / Appli Elimination of treatment costs associated with recovery ofchemical catalysts.

    Enzyme biodegradability and environmental acceptability. Opportunity for enzyme reuse and improved stability throughenzyme immobilization.

    The presence of FFA in the starting material poses a majorproblem for transesterication via traditional methods, while thisproblem can be easily overcome by using enzymes that can catalyzeboth esterication of FFA and transesterication of TGs. The FFAcontained in waste oils and fats can be completely converted to bio-diesel. In fact, the enzyme-catalyzed transesterication is moresuitable for use on FFA-rich feedstocks such as waste oils, greases,beef tallow and lard since the FFA are directly esteried enzymati-cally into FAME [12]. Therefore, lipase can be used on oils with var-iable chemical composition which broadens the feedstock base andis of great advantagewhenwaste oils and fats are considered for theestablishment of a cost-effective biodiesel production process[65,69]. However, there are several technical challenges that needto be overcome to improve the economic feasibility of the lipase bio-process: high cost of enzymes, loss of activity during the process, en-zyme inhibition by reactants and products, and slow reaction rates.

    3. Factors affecting enzymatic biodiesel production

    The yield and conversion efciency of enzymatic biodiesel isinuenced by a number of factors: the nature and properties ofthe enzyme catalyst; enzyme and whole cell immobilization tech-niques; enzyme pretreatment; biodiesel substrates; acyl acceptors,their step-wise addition and use of solvents; operating conditionsof enzymatic catalysis; and bioreactor design.

    3.1. Enzyme source

    Lipases are enzymes (biocatalysts) which carry hydrolysis of TGto glycerol and fatty acid, hence, they are categorized in the class ofhydrolases (acylglycerolacylhydrolases, EC 3.1.1.3) [70,71]. Theyare best dened as carboxylesterases that catalyze both the hydro-lysis and synthesis of long-chain acylglycerols [72]. These enzymesare ubiquitously present and based on their origin are classied asplant, animal or microbial lipases. Plant lipases have been reportedfrom papaya latex, rapeseed, oat and castor seeds [73]. Plantlipases arenot commercially usedwhereas the animal andmicrobiallipases are used extensively. Sources of the animal lipases are pan-creas of cattle, sheep, hogs and pigs [73]. Microbial lipases havegained wide industrial importance and they now share about 5% ofthe world enzyme market after proteases and carbohydrases [7478]. Lipasesofmicrobialoriginaremorestable thanplant andanimallipases and are available in bulk at lower cost compared to lipases ofother origin. Yeasts lipases are easy to handle and grow compared tobacterial lipases [79]. Among the yeast lipases, Candida rugosa hasgained good commercial importance. Themost commonly used bio-catalyst for biodiesel production are the microbial lipases that areproduced by a number of fungal, bacterial and yeast species(Table 1).

    As it can be seen fromTable 1, a large number ofmicrobial strainshave been used for lipase production, however, the most frequentlyreported enzyme sources are Candida sp., Pseudomonas sp. and Rhi-zopus sp.[132]. Lipase producing microorganisms have beenscreened from various sources including soil, marine water, wastewater and industrial wastes [76]. Traditionally, screening is carriedout on batch cultures using agar substrates which is time-consum-ing, however, continuous enrichment cultures overcome this prob-lem by using fermentors for growth and selection of desiredmicrobial isolates [133]. Soil isolates of Aspergillus, Mucor, Candidaand Sclerotina species were reported to produce lipase [129].

    nergy 119 (2014) 497520 501Lipase-producing strains of P. uorescens, P. alcaligenes, Enterobacterintermedium, Geotrichum asteroids and Bacillus acidophiluswere iso-lated from vegetable oil processing plants [129,134,135].

  • Table 1Lipase-producing microorganisms.

    Microbial type Microbial source Refs.

    Bacteria Achromobacter lipolyticum [77]Acinetobacter radioresistens [80]Acinetobacter calcoaceticus [81]Acinetobacter pseudoalcaligenes [82]Aeromonas hydrophila [83]Archaeglobus fulgidus [84]Bacillus acidocaldarius [72]Bacillus megaterium [76]Bacillus pumilus [72]Bacillus sp. [77]Bacillus stearothermophilus [85]Bacillus subtilis [86]Bacillus thermocatenulatus [87]Bacillus thermoleovorans [88]Burkholderia glumae [77]Chromobacterium viscosum [77]Enterococcus faecalis [89]Micrococcus freudenreichii [90]Moraxella sp. [73]Mycobacterium chelonae [91]Pasteurella multocida [92]Propionibacterium acnes [73]Propionibacterium avidium [93]Propionibacterium granulosum [93]Proteus vulgaris [73]Pseudomonas aeruginosa [90,94]Pseudomonas cepacia [95]Pseudomonas fragi [77,96]Pseudomonas mendocina [77]Pseudomonas nitroreducens var. thermotolerans [96]Pseudomonas sp. [97]Psychrobacter immobilis [98]Serratia marcescens [99]Staphylococcus aureus [77]Staphylococcus canosus [100]Staphylococcus epidermidis [101]Staphylococcus haemolyticus [102]Staphylococcus hyicus [87,103]Staphylococcus warneri [103]Staphylococcus xylosus [91]Sulfolobus acidocaldarius [73]Vibrio chloreae [73]Pseudomonas alcaligens [104]Chromobacterium visosum [105]Pseudomonas putida [106]Statphylococcus stolonifer [107]

    Fungi Alternaria brassicicola [108]Aspergillus fumigates [109]Aspergillus japonicas [110]Aspergillus nidulans [111]Candida antarctica [87]Mucor miehei [112]Penicilliumcyclopium [113]Rhizomucor miehei [114]Rhizopus arrhizus [82]Rhizopus chinensis [76]Rhizopus microsporous [76]Rhizopus niveus [115]Rhizopus nodosus [116]Rhizopus oryzae [117,118]Streptomyces cinnamomeus [119]Streptomyces exfoliates [98]Streptomyces fradiae [82]Streptomyces sp. [82]Aspergillus niger [120]Thermomyces lanuginous [121]Fusarium heterosporum [122]Humicola lanuginose [123]Oospora lactis [124]Rhizopus oryzae [125]

    Yeasts Candida deformans [87]Candida parapsilosis [112]Candida rugosa [126,127]

    502 L.P. Christopher et al. / Applied Energy 119 (2014) 4975203.2. Enzyme properties

    Microbial lipases are mostly extracellular with a molecularweight of 3050 kDa and a pH optimum in the slightly alkaline pHrange of 7.59 [136]. Lipases originate mainly from mesophilicand thermophilic microorganisms and have an optimum activityat 3550 C and 6080 C, respectively [129,135]. Most mesophiliclipases are unstable at temperatures above 70 C, whereas thermo-stable lipases show activity at up to 100 C in presence of organicsolvents and detergents [137]. For instance, thermostable lipasesfrom the hyperthermophilic archaea Pyrobaculum calidifonti [138]and Pyrococcus furiosus [139] and from the extreme thermophilicbacteria Thermoanaerobacter thermohydrosulfuricus and Caldanae-robacter subterraneus [140] exhibited lipase activity at 90 C. Inaddition, the Thermoanaerobacter and Caldanaerobacter lipaseswereshown to be resistant to organic solvents, whichmakes them strongcandidates for biodiesel production in water-free environments.

    The optimum expression of lipases depends on many factorsincluding carbon and nitrogen sources, growth conditions likepH, temperature, dissolved oxygen. Numerous literature sourcesare available on lipase producing microorganisms, methods for li-pase production and application [74,78,91,141]. As lipases are lar-gely inducible enzymes, the main factor for the expression of lipaseactivity is the carbon source which can be a lipid source or a carbonsource [142]. The lipid sources include triacylglycerols, short orlong chain fatty acids, hydrolyzable esters, tween, bile salts andglycerol [141,143] and the carbon sources are sugars, sugar alco-hol, polysaccharides, whey, amino acids and other complex sources[141]. For maximum lipase production, incubation period usuallyrange from few hours to few/several days based on the organism[141] and production method. Fungal species are preferably culti-vated in solid-state fermentation, while bacteria and yeast are bestproduced in submerged fermentation [144]. The lipase reactionsystems for biodiesel production are complex, consisting of twoimmiscible phases an aqueous phase containing enzyme, andan organic phase containing oil/fat. Lipases have the unique featureof acting on the interface between aqueous and non-aqueousphases and so they can catalyze many reactions including hydroly-sis, inter-esterication and alcoholysis [78].

    The mode of action of lipases in substrate transesterications tobiodiesel depends on their origin and specic properties. Lipasescatalyze the trans/esterication reaction between TG and acylacceptors (alcohols) through the formation of acyl-enzyme inter-

    Table 1 (continued)

    Microbial type Microbial source Refs.

    Candida quercitrusa [109]Pichia burtonii [128]Pichia sivicola [128]Pichia xylosa [129]Saccharomyces lipolytica [100]Geotrichum candidum [130]Yarrowia lipolytica NRRL YB-423 [131]mediates that subsequently donate the acyl moiety to produceFAAE [145]. The overall structure of the lipases can be describedas a structure with a central L-sheet with the active serine placedin a loop called catalytic elbow [146]. The activation which is oftennecessary for the lipase enzyme is the movement of a lid. Some en-zymes such as Thermomyces lanuginosus lipase have an active siteand a lid on the surface of the enzyme. Others like C. rugosa lipasehave an active site at the end of a tunnel containing the lid in itsexternal parts [147]. The structural properties of lipase from differ-ent sources might be the reason for showing different activity ondifferent oil substrates, hence, the need to optimize the process

  • ed Ebased on the selected enzymes and substrates for biodiesel pro-duction. Furthermore, the selection of a particular lipase for lipidmodication is based on the type of the desired modication andmay be position-specic modication of triacylglycerol, fattyacids-specic modication, modication by hydrolysis, and modi-cation by direct synthesis and transesterication [148]. Basedon their substrate specicity, lipases can be divided into threegroups: 1,3-specic, fatty acid-specic, and non-specic lipases.The 1,3-specic lipases release and hydrolyze ester bonds in theposition 1 and 3 of TGs [149]. Du et al. [150] reported the use ofT. lanuginosus 1,3-specic lipases to produce a transestericationyield of 90%. The fatty acid-specic lipases are known to hydrolyzeesters of long chain fatty acids with double bonds in cis-position atC9, whereas the non-specic lipases randomly cleave the acylgly-cerols in FFA [149]. For optimal biodiesel production, lipasesshould be able to convert all three forms of glycerides (mono-,di-, and tri-glycerides) to biodiesel, hence, they need to be non-ste-reospecic and to efciently catalyze the esterication of FFA [18].

    3.3. Enzyme and whole cell immobilization

    Immobilization is the process of attaching enzymes physicallyto a solid support so that the substrate is passed over the enzymesupport and can be converted to the product. Immobilization hasbeen increasingly used in industrial applications to facilitate sepa-ration of biocatalysts from the process stream, and hence, therecovery and purication of products [151]. Immobilized enzymesare preferred over free enzymes due to their prolonged enzyme-substrate contact that curtails redundant downstream and puri-cation processes [152]. There are many advantages of immobilizedenzyme over free enzyme: the repetitive use of a single batch ofenzymes is economical in the development of continuous biopro-cesses; rapid termination of the enzyme-substrate reaction byremoving the enzyme from the reaction solution; enzyme stabil-ization due to binding to support; no contamination of the productand enzyme [153]. Immobilization can dramatically affect enzymeproperties such as pH-dependence, temperature prole, resistanceto proteolytic digestion and denaturants, thermostability andkinetics. The chief issues for enzyme immobilization are selectionof support matrices and immobilization techniques that permitboth rapid enzyme activity and enzyme stability under the con-straints imposed by the substrate medium [154,155].

    A number of techniques and supports are available for the immo-bilization of enzymes on a variety of natural and synthetic supports[156]. The choice of the support and the technique depends on theenzyme nature, nature of the substrate and the type of reaction itis used [152,157]. For industrial application, support materials areselected based on the ow properties, low cost, non-toxicity andmaximum biocatalysts loading while retaining the desirable owcharacteristics, operational durability, availability and ease ofimmobilization [158]. The activity of the immobilized enzymemaybe reducedduring the immobilization procedure and as a resultof mass-transfer limitations. Enzyme immobilization techniqueshave been classied into three categories: entrapment, carrier bind-ing and cross-linking [159]. The economics of the immobilizationprocess depends on both the activity and the operational stability(activity integrated over the operational time) of the biocatalysts.

    3.3.1. Enzyme immobilizationImmobilization of lipases was carried out using entrapment

    [159], physical adsorption [160], ion exchange [161], and cross-linking [162]. Carriers for lipase immobilization include polyure-thane foam [163], silica [164], sepabeads [165], cellulosic nano-

    L.P. Christopher et al. / Applibers [166]. Criteria for selecting the immobilization techniqueand carrier depends on the source of lipase, the type of reactionsystem (aqueous, organic solvent or two-phase system) and theprocess conditions (pH, temperature and pressure). Based on theimmobilization technique and carrier, the bioreactor type (batch,stirred tank, membrane reactor, column and plug-ow) can be de-signed. The literature is replete with various lipase producingmicroorganisms, enzyme immobilization methods and physicalcarriers. The challenge will be to select a carrier and immobiliza-tion technique that will allow maximum lipase activity, retentionand stability on the oil substrate [167]. Adsorption is the mostwidely employed method for lipase immobilization. The most fre-quently immobilized enzymes used for commercial applicationsare the C. antartica lipase, immobilized on acrylic resin (Nov-ozym435), Mucormiehei lipase, immobilized on a macroporousion-exchange resin (Lipozyme IM), T. lanuginosus acrylic resin-immobilized lipase (Lipozyme TLIM), Rhizomucor miehei lipaseimmobilized on macroporous anion exchange resin (LipozymeRM IM), and Candida sp. 99125 lipase, immobilized on textilemembranes [168]. As lipase is deactivated by methanol adsorptiononto the immobilized enzyme, enzyme regeneration with higheralcohols such as butanol is required. Due to immiscibility of thelarge TG molecules with lower alcohols, diffusion problems to ac-cess the immobilized biocatalyst may arise due to the small poresof the carrier. This may cause internal transport problems therebyreducing the enzyme efciency [18].

    Recently, interest has focused on carrier-free immobilized li-pases by cross-linking of enzyme aggregates for use in a solvent-free biodiesel production [169]. This technique presents severalinteresting advantages over carrier-bound immobilized enzymesthat includes highly concentrated enzymatic activity, high stabilityof the produced superstructure, costs savings from omitting thesupport material, and no enzyme purication requirement [170].Lipase from Rhizopus oryzae was immobilized as crosslinked en-zyme aggregate via precipitation with ammonium sulfate directlyfrom the fermentation broth and simultaneous crosslinking withglutaraldehyde [171]. The cross-linked enzyme retained 91%activity after 10 cycles in aqueous medium. Another recentdevelopment in enzyme immobilization with potential use in bio-diesel production is the protein-coated microcrystals that consistof water-soluble micron-sized crystalline particles coated with abiocatalyst such as lipase [172]. Microcrystals coated with a C. ant-arctica lipase B retained nearly 90% of the enzyme initial activityover a period of one year at room temperature. The enzymeactivity of P. aeruginosa lipase protein-coated microcrystals wasenhanced tenfold over that of the free lipase enzyme [173]. A com-bination of lipase cross-linking and lipase coating was reported toimprove the operational, pH and thermostability as well as theorganic solvent tolerance of a Geotrichum sp. lipase in biodieselproduction [174].

    3.3.2. Whole cell immobilizationIn an attempt to avoid the complex enzyme recovery and puri-

    cation requirements for immobilizationof free (extracellular) lipase,immobilization of intracellular lipase, known aswhole cell immobi-lization, has been extensively researched [175]. Immobilization ofthe intracellular, cell- ormembrane-bound lipase is believed to offeran alternative and less expensive source of immobilized biocatalystfor biodiesel production. Considerably fewer steps are required toproduce whole-cell lipase using inexpensive and readily availableindustrial cultures. Consequently, this can signicantly reduce thecost of the transesterication process [175] as shown in Fig. 3whichcompares the two immobilization methods.

    Lipase-producing bacteria, fungi and yeasts have been immobi-lized to serve as a lipase biocatalyst (Table 2). Matsumoto et al.[183] developed whole cell biocatalysts by immobilizing R. oryzae

    nergy 119 (2014) 497520 503cells, permeabilized by air drying. This biocatalyst was used in athree-step addition of methanol (known as methanolysis) to plantoil in a solvent- and water-free system at 37 C. The FAME yield

  • e

    y

    bio

    C

    4332443333

    ed E(a)

    (b)

    Cultivation

    Purification of Lipas Extraction Adsorption Chromatograph Crystallization

    Cultivation &

    Immobilization

    Fig. 3. Immobilization methods used for production of enzymatic

    Table 2Whole cell lipase biocatalysts used for biodiesel production.

    Microbial source Support material Substrate

    P. uorescens Na-alginatea Jatropha oilPseudomonas sp. Na-alginate Used cottonseed oilR. mucilagenosa None Palm oilA. niger BSPsb Palm oilA. niger BSPs Palm oilA. niger BSPs + GAc Palm oilR. oryzae BSPs Used cooking oilR. oryzae BSPs + GA Jatropha oilR. orzyae BSPs Soybean oilR. orzyae BSPs + GA Soybean oil

    a Na-alginate, sodium alginate.

    504 L.P. Christopher et al. / Appliwas 71 wt% after 165 h of reaction. Some of the researcher reportedthat olive oil and oleic acid enhanced the methanolysis activity ofimmobilized 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) stabi-lized and maintained lipase activity at the same level even after sixbatches of methanolysis, with ester yields of 7083% that repre-sented a 20 to 33% increase over the control without cross-linking.Also, Tamalampudi et al. [181] compared the performance of wholecell R. oryzae immobilized on polyurethane biomass support parti-cles (BSPs) to Novozym435 in methanolysis of jatropha oil. Thepresence of water in jatropha oil had a positive effect on the meth-anolysis with immobilized cells reaching a maximum yield of 89%(Table 3) at 5% (v/v) water, whereas the activity of Novozym435was inhibited.

    Methanolysis of plant oil using a R. oryzaewhole cell biocatalystwas also investigated in shake asks and in a packed-bed reactor[66,185]. The latter provided better results by protecting cells fromphysical damage and excess methanol. The cells were immobilizedwithin 6 6 3 mm BSPs during batch cultivation in a 20 L air-liftbioreactor. Emulsication of the reaction mixture at a ow rate of25 L/h resulted in maximum FAME yields of 90%. R. oryzaecellsimmobilized with polyurethane BSPs and treated with GA havebeen extensively researched widely used as a whole cell biocatalystfor production of biodiesel from various sources of non-edible andlow-cost feedstock [180,182,183]. Oda et al. [229] cultured R. oryzaecells immobilized in BSPs in a 20 L air-lift bioreactor, and performedrepeated batches of methanolysis of soybean oil. They found thatthe hydrolytic activity of the whole-cell biocatalyst was intact after20 batches of methanolysis that produced 6580% ester yields. Liet al. [230] studied biodiesel production from different feedstocks(rened, crude and acidied rapeseed oil) in tertiary-butanol usingwhole cell R. oryzae cells immobilized in BSPs. They found that the

    b BSPs, biomass support particles.c GA, glutaraldehyde.Immobilization of Lipase Cross linking Covalent bonding Entrapment

    Immobilized Lipase Biocatalyst

    Whole Cell Lipase Biocatalyst

    diesel: (a) lipase immobilization; (b) whole cell immobilization.

    onditions Yield (%) Refs.

    0 C; 3:1 methanol: oil; hexane solvent; 48 h 72 [69]7 C; 6:1 methanol: oil; step-wise; 48 h 70 [176]0 C; 3:1 methanol: oil; 72 h 51 [177]5 C; 3:1 methanol: oil; step-wise; 72 h 87 [178]0 C; 3:1 methanol: oil; 3 step-wise; 72 h 69 [179]0 C; 3:1 methanol: oil; 3 step-wise; 72 h 69 [179]5 C; 3:1 methanol: oil; 3 step-wise; 72 h 98 [180]0 C; 3:1 methanol: oil; 72 h 89 [181]5 C; 3:1 methanol: oil; 3 step-wise; 72 h 8085 [182]5 C; 3:1 methanol: oil; 3 step-wise; 72 h 8992 [182]

    nergy 119 (2014) 497520reaction rate and nal ester yield weremuch greater when acidiedrapeseed oil was used. Furthermore, the increase in the FFA in oil,the presence of phospholipids, and the use of adsorbents for waterremoval improved the rate of reaction and biodiesel yields. Fromthis perspective, based on numerous literature reports, the lipase-producing R. oryzaeseems to show great promise for furtherresearch and development that could aid in reducing the cost ofbiodiesel production [181,230,231].

    In addition to R. oryzae, other lipase-producing fungal and yeastwhole cell catalysts have been also investigated in biodiesel pro-duction: a newly isolated strain of A. niger JN [178]; a methanol-tolerant yeast R. mucilagenosa [177]; Pseudomonas sp. includingP. zuorescens MTCC 103 [69,232]. A 72% biodiesel yield was ob-tained in transesterication of Jatropha oil with P. uorescensMTCC103 immobilized in sodium alginate gel (Table 3). The optimumconditions were determined as follows: neutral pH, 40 C, oil/methanol mole ratio of 1:4, 48 h [69,67]. Pseudomonas sp.was usedas a whole cell biocatalyst for biodiesel production from used cot-tonseed oil: the FAME yield of 70% was attained by a step-wiseaddition of methanol in excess (oil/methanol mole ratio of 1:6)for 48 h [176]. Pseudomonas sp. immobilized in sodium alginategel was recommended for industrial use [69,232]. Lu et al. [233]studied the transesterication of lard using immobilized Candidasp. 99125 whole cells via a three-step methanolysis. They re-ported that for processing 1 g of lard the optimum, loading condi-tions 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 obtainedand 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 onenzyme performance by: (1) providing a protective enzyme shield

  • Table 3Lipase-catalyzed transesterication of oils to biodiesel.

    Substrate Enzyme Operating conditions Solvent/water

    Acyl acceptor Yield(%)

    Refs.

    Jatropha oil E. aerogenes lipase 55 C; 48 h NSa Methanol 94 [186]

    Palm oil PS 30 Lipase 40 C; 8 h NS Ethanol 72 [187]NS t-Butanol 62NS 1-Butanol 42NS n-Propanol 42NS 1-Propanol 24

    Coconut oil PS 30 Lipase 40 C, 8 h NS Ethanol 35NS iso-Butanol 40NS 1-Butanol 40NS 1-Propanol 16

    Soybean oil Novozym435 40 C;14 h NS Methyl acetate 92 [188]

    Vegetable oils Lipozyme TL IM 25 C; 7 h NS Ethanol 84 [189]Novozym435 Methanol >99

    Plant oils R. oryzae lipase 35 C; 5 h Water (430%)

    Methanol 8090 [190]

    Rapeseed oil Lipozyme TL IM + Novozym435 35 C; 12 h t-Butanol Methanol 95 [191]

    Microalgae Candida sp. lipase IM 38 C; 12 h Hexane Methanol 98 [192]

    Sunower oil Pseudomonas lipase 45 C; 5 h Petroleumether

    Methanol 79 [193]

    65 C; 5 h Petroleumether

    Methanol 49

    45 C; 5 h Petroleumether

    Ethanol 99

    Jatropha oil Novozym435 50 C; 8 h NS 2-Propanol 92.8 [194]Karanj oil 91.7Sunower oil 93.4

    Jatropha oil Novozym435 50 C; 12 h NS Ethyl acetate 91.3 [195]Karanj oil 90Sunower oil 92.7

    Plant oil Novozym435 Continuous reaction Petroleumether

    Methanol 90 [196]

    Tallow oil Lipozyme IM60 45 C; 5 h Hexane Primaryalcohols

    94.898.5

    [197]

    Novozym435 NS Secondaryalcohols

    61.283.8

    Lipozyme IM60 NS Methanol 19.4Lipozyme IM60 NS Ethanol 65.5

    Soybean oil IM P. cepacia lipase 35 C; 1 h Water Methanol 67 [154]Ethanol 65

    Cottonseed oil IM C. antarctica lipase 50 C; 24 h t-Butanol Methanol 97 [198]

    Triolein Novozym435 6 h NS Butanol 100 [199]Lipozyme RM IM 100

    Soybean oil Novozym435 30 C; 3.5 h NS Methanol 97 [200]

    Soybean oil deodorizer distillate Novozym435 Molecular sieve adsorbentreaction

    t-Butanol Methanol 95 [201]

    Acid oil Novozym435 24 h NS Methanol 90 [202]

    Waste edible oil Novozym435 Fixed bed reactor NS Methanol 90 [203]

    Degummed soybean oil Novozym435 30 C; 6 h NS Methanol 93.8 [204]

    Sunower oil Novozym435 NS NS Methanol 97 [205]

    Sunower oil Novozym435 45 C; 50 h NS Methanol >99 [206]Methyl acetate 95.65

    Plant oil R. oryzae lipase NS NS Methanol 90 [175]

    Soybean oil IM R. oryzae lipase RTb; PBRc NS Methanol 90 [185]

    Triolein IM P. ourescens lipase 50 C; 25 h NS Butanol 90 [207]

    Soybean oil Lipozyme TL IM 40 C; 12 h NS Methanol 98 [208]

    Waste oil adsorbed on activatedbleaching earth

    C. cylindracea lipase 25 C; 12 h Diesel oil Methanol 97 [209]

    Palm oil from waste bleaching earths R. oryzae lipase 35 C; 96 h NS Methanol 55 [210]

    Grease PS 30 38.4 C; 2.47 h NS Ethanol 85.4 [211]

    Soybean oil Recombinant LipB68 P. uorescenslipase

    20 C; 12 h NS Methanol 92 [212]

    (continued on next page)

    L.P. Christopher et al. / Applied Energy 119 (2014) 497520 505

  • Ope

    30

    37

    40

    37

    NS

    60

    50

    RT;

    35

    35

    35

    ed ETable 3 (continued)

    Substrate Enzyme

    Rice bran oil Cryptococcus spp. S-2

    Waste activated bleaching earth C. cylindracea lipase

    Rened soybean oil Novozym435

    Rapeseed oil C. rugosa lipase

    Soybean oil Lipozyme IM-77

    Mowrah oil Lipozyme IM-20

    Mango oilKernel oilSal oil

    Sunower oil Lipozyme

    Fish oils C. antarctica lipase

    Soybean oil P. uorescens lipase

    Soybean oil C. rugosa lipase

    Soybean oil P. cepacia lipase

    506 L.P. Christopher et al. / Applito minimize enzyme deactivation caused by lower alcohols(methanol and ethanol) and glycerol [4,200,234], and (2) enhanc-ing enzyme activity by maintaining the lipase molecule conforma-tion in its active form, or reversing the conformation of the activeenzyme sites from close to open [167,235]. The overall effect of li-pase pretreatment is regeneration of enzyme activity and im-proved mass transfer and transesterication rates, resulting inenhanced productivity [236,237], however, the exact mechanismof 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-butanolaffected a ten-fold increase in Novozym435 activity [234]. Incomparison, the enzyme was completely deactivated by methanolwithout washing. Pretreatment of the completely deactivated en-zyme with tert-butanol and 2-butanol restored the lipase activitiesof their original levels of 56% and 75%, respectively. Incubation ofimmobilized C. antarctica in methyl oleate for 30 min, followedby a step-wise addition of methanol to soybean oil increased theFAME yields to 97% [200]. Shah et al. [239] reported a 45% increasein ester conversion (from 34% to 79%) of jatropha oil when animmobilized lipase from P. uorescens was pretreated via irradia-tion in presence of an aqueous buffer and organic solvents.Pretreatment of immobilized Novozym435 using ultrasonic irra-

    Sunower oil R. miehei lipase 40

    Sunower oil T. lanuginosus lipase 40

    Sunower oil IM C. antarctica lipase 50

    Soybean oil IM M. miehei lipase 35

    Sunower oil IM T. lanuginosus lipase 30

    Crude palm oil Lipozyme RM IM 50 Lipozyme TL IMLipozyme RM IMLipozyme TL IM

    Soybean oil deodorizer distillate Novozym435 50

    Jatropha oil IM P. uorescens lipase 40

    Corn oil P. expansum lipase 40

    Palm oil IM B. cepacia lipase 30

    Fats and oils IM T. lanuginosus lipase 50

    Restaurant grease IM T. lanuginosus lipase 50

    a NS, not specied.b RT, room temperature.c PBR, packed bed reactor.rating conditions Solvent/water

    Acyl acceptor Yield(%)

    Refs.

    C; 96 h Water (40%) Methanol 80 [213]

    C; 3 h Diesel oil Methanol 100 [214]

    C; 10 h NS Methyl acetate 92 [215]

    C; 24 h 2-Ethyl-1-hexanol

    97 [216]

    n-Hexane Methanol 92.2 [217]

    C; 6 h Water (10%) Alcohols (C4C18)

    86.899.2

    [218]

    C; 5 h NS Ethanol 83 [219]

    22 h NS Ethanol 100 [220]

    C; 90 h NS Methanol 80 [190]

    C; 90 h NS Methanol 80

    C; 90 h NS Methanol 100

    nergy 119 (2014) 497520diation with vibration resulted in 96% methyl ester yields after verepeated cycles [240]. Pretreatment of R. oryzae lipase withsoybean oil before immobilization increased the lipase activity20-times over that of the non-treated lipase thereby maintainingit at levels exceeding 90% of its original activity after 10 reuses[241]. Salts as pretreatment agents including calcium or magne-sium chloride are thought to stabilize the protein molecule and en-hance 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 andeconomy of the process. First-generation biodiesel has been pro-duced from vegetable oils extracted from oilgeneous plants likesunower, soya, canola and palm (Table 3) [242]. At present, cano-la, soybean and palm oil constitute about 75% of the world vegeta-ble oil supply. In the rst half of 2013, they traded for $1150, $1050and $755 on average per metric ton, respectively. Canola oil is theprimary source for biodiesel production globally, whereas soybeanoil is the largest biodiesel feedstock in the US which is also ex-change-traded. Soybeans are widely grown in the US for their highprotein and lipid content. Because of the value of the products and

    C; 48 h NS Methanol 95.5 [221]

    C; 48 h NS Methanol 92.3

    C; 12 h NS Ethyl acetate 63.3 [222]

    C; 8 h n-Hexane Ethanol 95.6 [223]

    C; 6 h n-Hexane Ethanol 1635 [223]

    C; 4 h NS Methanol 12 [224]NS Methanol 15NS Ethanol 16NS Ethanol 25

    C; 1.5 h NS Ethanol 83.5 [225]

    C; 48 h n-Hexane Methanol 71 [69]

    C; 24 h Ionic liquids Methanol 86 [226]

    C; 72 h NS Methanol 100 [227]

    C; 48 h NS Ethanol/methanol

    70100 [228]

    C; 48 h NS Ethanol/methanol

    8090

  • ed Ethe ability to be used in crop rotations with nitrogen-intensivecrops such as corn, soybean oil now accounts for more than 50%of all bio-based oils in the US. Although soybean oil yields only48 gallons/acre/year, 2 billion gallons were harvested from morethan 30 million ha in 2002 [35]. Nowadays soybean oil makes up25% of the total soybean oil demand in the US [14]. Biodiesel qual-ity is directly inuenced by the fatty acid composition of vegetableoils [243]. Low cetane numbers have been associated with thepresence of unsaturated fatty acids (C18:2 and C18:3) that are typ-ically found in soybean oils, whereas saturated fatty acids such aspalmitic (C16:0) and stearic (C18:0) acid increase the cetane num-ber of biodiesel produced from palm oil [244].

    The main constraint for biodiesel production is the cost of thefeedstock. It has been estimated that up to 80% of the total biodie-sel production cost arises from the cost of raw material [245247].The high value of edible vegetable oils as a food product makesproduction of a cost-effective biodiesel fuel very challenging. Inaddition, the recent increase in the use of vegetable (edible) oilsfor biodiesel production has caused some major controversy owingto their inuence on the global imbalance to food market and foodsecurity [248]. Furthermore, there are large amounts of low-costfeedstock such as waste cooking oils and animal fats that couldbe converted to biodiesel [167] thereby also helping solve theproblem of waste oil disposal [249]. This has historically led tothe production of the second-generation biodiesel that is derivedfrom non-edible oils. In comparison to edible oils, the major short-comings in the use of non-edible oils are the relatively low oilyields (with a few exceptions) and feedstock quality. For example,the yield of palm oil peaks at 5000 kg oil/hectare, whereas yieldsfrom non-edible oils such as jatropha and pongamia pinnata are250-fold lower, and range from 100 to 2300 kg oil/hectare. There-fore, in some instances, the use of non-edible oils for biodiesel pro-duction would require large plantation areas. However, incomparison, the oil yield of jatropha oil is nearly 5-times higherthan that that of soybean oil (475 kg/hectare), whereas macaubacan produce 10-times more oil than soybean. Furthermore, dueto the presence of high FFA in non-edible oils, biodiesel productionfrom this feedstock, the use of enzyme catalysts has been favored[248]. Nevertheless, inedible oils are a viable alternative to ediblevegetable oils due to their lower price and wide adaptability withminimal production requirements. For instance, jatropha can growon waste, sandy and saline soils under a wide variety of climaticconditions (severe heat, low rainfall, high rainfall and frost), andproduce up to 60 wt% oil in its seeds and kernels [250]. Becauseof that, Jatropha oil, despite its toxicity to humans, is regarded asa promising potential feedstock for biodiesel production in Asia,Europe and Africa [244]. The advantages of non-edible oils overtheir edible counterparts are their lower price, their availabilityand portability, higher caloric value and lower sulfur and aro-matic content. Drawbacks of inedible oils include higher reactivitydue to higher content of unsaturated fatty acids, higher viscosityand carbon residue content, and lower volatility [251,252].

    The waste cooking oils are directly accessible for biodiesel pro-duction (Table 3) and their quantity is mainly relying on theamount of edible oil consumed. Cooking oils contain many typesof vegetable-based oils as well as rendered animal oils. There areenough used cooking oils and fats generated in the US annually,including 18 billion pounds of soybean oil and 11 billion poundsof animal fat, to produce an estimated 5 billion gallons of biodiesel[253]. The cost of waste cooking oil is calculated from the collec-tion, transportation and pretreatment costs, which are minimalcompared to those for edible oils. The physical properties andchemical composition of waste cooking oil vary depending on the

    L.P. Christopher et al. / Applioil source and the content of water (0.75%) and FFA (56%) thatis comparatively higher than virgin oil (

  • challenges related to harvesting and oil extraction from algalbiomass [247,261]. Harvesting contributes 2040% of the total costof biomass production [262]. In addition, the microalgae containhigh levels of polyunsaturated fatty acids with four or more doublebands [263] as eicosapentanoic acid (C20) and docosahexaenoicacid (C22) which are unstable and prone to oxidation during stor-age that diminishes biodiesel performance [256].

    As biodiesel runs in normal compression engines or diesel en-gines, its physicochemical properties should meet the qualityrequirements that are applicable to the conventional diesel fuel(petrodiesel) [256]. The US and European standards for biodieselare stipulated in the American Society for Testing and Materials(ASTM) D6751 and EN 14214, respectively [258]. Feedstockproperties like FFA composition and content, moisture content,

    as the type and amount of alcohol, biocatalyst, temperature, mixing

    reported by several researchers for soybean oil [198], jatropha oil[261] and canola oil [273]. Immobilized C. antarctica lipase medi-ated transesterications of vegetable oil with a maximum conver-sion of 95% achieved at a methanol/oil molar ratio of 3:1 [274].Methanolysis of Simarouba oil with a fungal immobilized lipaseproduced a maximum yield of 91.5% FAME using methanol as acylacceptor [275]. A 95% yield of methyl esters of rape seed oil wasachieved with methanol/oil molar ratio of 4:1 [191]. Biodiesel pro-duction from transgenic corn oil using Lipozyme TL IM peaked at a

    508 L.P. Christopher et al. / Applied Energy 119 (2014) 497520intensity used, the moisture and FFA content of the substrate, andmode of bioreactor operation [266]. Overall, the lipase biocatalystsrequire lower alcohol to oil ratio than chemical catalysts [181,267].Generally, molar ratios of alcohol to oil of between 3:1 and 6:1 havebeen commonly used [268270]. A further increase of the alcoholcontent beyond the optimum concentration does not improve thebiodiesel yield, however, it has a negative impact on the costs ofalcohol recovery [269,271]. High alcohol to oil molar ratios may in-hibit lipase activity and interfere in the separation of glycerol due toits increased solubility in ethanol [268,272]. The effect of methanolto oil ratio on the FAAE yield catalyzed by immobilized lipase was

    Table 4Properties of enzymatic biodiesel.

    Parameters Biodieselfromstillingiaoil [263]

    Biodieselfromjatrophaoil [262]

    Biodieselfromwastecookingoil [260]

    Biodieselstandard(ASTMD6751-03) [264]

    Density (g/cm3) 0.900 0.8924(20 C)

    0.870.90(15 C)

    Pour point (C) 5 15 to10

    Flash point (C) 137 82 195 P130Heating value (MJ/kg) 36.5 3340Viscosity (mm2/s) 4.81 8.2 9.12 1.96unsaponied compounds, impurities, etc. inuence the engineperformance of biodiesel [244,249]. A considerable amount of liter-ature has been published on testing the physiochemical propertiesof biodiesel using chemical catalysts [259]. Recent studies com-pared the properties of biodiesel obtained from enzymatic transe-sterication with those of chemically-catalyzed biodiesel andconcluded that enzymatic biodiesel closely meets the stringentquality requirements of EU and US standards [260,262]. Propertiesof enzymatic biodiesel such as density, ash point, pour point,heating value, viscosity and ash content have met the ASTM bio-diesel standard 6751-03 (Table 4).

    3.6. Acyl acceptor

    The commercial production of biodiesel requires the use of inex-pensive and readily available acyl acceptors such as methanol andethanol. Stoichiometrically, the alcoholysis of any oil requires threemoles of alcohol for each mole of oil. Since transesterication is areversible reaction, an excess of alcohol is required to shift the equi-librium to FAAE formation [265]. The yield of biodiesel increaseswith the increase in the alcohol concentration up to a certain con-centration, depending on the particular operating conditions such(40 C) (20 C) (40 C)Ash content (wt%) 0.037 0.003 60.02methanol to oil ratio of 6 to 1 [268].For most of the transesterication reactions, methanol is com-

    monly used because of its reactivity, volatile nature, and lower costthan other alcohols. However, methanol is mostly produced fromnon-renewable sources such as natural gas or coal, and is toxic.Most lipases can be at least partially inactivated by methanol thataffects their catalytic performance [234]. The degree of lipase deac-tivation is believed to be inversely proportional to the number ofcarbon atoms in the linear lower alcohols [4]. On the other hand,the rate of transesterication was reported to increase proportion-ally to the number of carbon atoms in the alcohol [250]. Therefore,ethanol as a longer carbon chain alcohol, presents an eco-friendlyand renewable alternative that is less inhibitory than methanol.However, with ethanol as the acyl acceptor, the lipase activity ofNovozym435 was completely lost after only six cycles of enzymereuse [195]. The enzyme activity of Novozym435 with differentsubstrates and acyl acceptors is shown in Table 5.

    Enzyme inhibition by methanol can be overcome by overdosingthe lipase, which however is not a cost-effective solution to theproblem [18]. It was also reported that some lipases (from Pseudo-monas) were more tolerant to alcohol than others (e.g. from T.lanuginosus and R. miehei) [18,37]. Three strategies have beendeveloped to minimize alcohol inhibition: (1) stepwise or sequen-tial addition of alcohol or alcohol aliquots [224,277]; (2) use of sol-vents [195]; (3) use of alternative acyl acceptors such as longerchain alcohols and alkyl esters [215].

    The step-wise addition of alcohol maintains the alcohol concen-tration below the critical level to avoid loss of solubility in the oiland undesirable deactivation of the enzyme. Research has focusedon stepwise addition of methanol as the most commonly used acylacceptor with the greatest inhibitory effect on lipase. Shimadaet al. [274] rst used stepwise addition of methanol and attaineda 95% conversion after 50 cycles of operation. Watanabe et al.[278] reported a 90% yield using a two-step batch-wise additionof methanol. The yield was maintained even after 100 batches ofoperation. Similarly, a 95% conversion was observed following astep-wise addition of methanol after 50 batches [274]. A biodieselyield of as high as 97% was obtained from plant oil by a three-stepaddition of 0.33 M equivalents of methanol at 0.250.4 h intervals[200]. Kaieda et al. [190] reported that the stepwise addition ofmethanol prevented inhibition of non-regiospecic lipases suchas P. cepacia, C. rugosa and P. ourescens and regiospecic lipasessuch as R. oryzae. Yields of 90% were obtained by step-wise addi-tion of methanol to waste cooking oil [277]. Similarly, during batchand continuous transesterication with immobilized

    Table 5Acyl acceptors used with Novozym435 in biodiesel production.

    Plant oil Acylacceptor

    Conditions Enzyme activity Refs.

    Soybean Ethanol 25 C; 7 h 85% After 9batches

    [189]

    Olive Methanol Methanol step-wiseaddition

    70% After 8batches

    [276]Sunower Ethylacetate

    Immobilized enzyme;25 C

    85% After 12batches

    [195]

  • ed ENovozym435 using step-wise addition of methanol to wastecooking oil, 96% and 93% yields were achieved for the batch andcontinuous process, respectively, with lipase retaining full activityafter 20 days of continuous operation [196]. Yields of 98% were re-ported for transesterication of soybean oil with T. lanuginosus li-pase using a step-wise addition of methanol, while iso-propanolwas used to remove glycerol [208]. A 34% increase in the conver-sion was achieved through a stepwise addition of methanol com-pared to a batch methanolysis of olive oil [279].

    Solvents exert multiple effects on both reactants and products inbiodiesel production. These effects include: (1) increased solubilityof alcohol thatprotects lipase fromdenaturation [262]; (2) increasedsolubility of glycerol that prevents lipase inhibition [198]; (3) crea-tion of a single phase reaction mixture that improves mass transferand reaction rates, reduces viscosity, and stabilizes immobilized en-zymes [4,18]. The enzyme stabilization is due to increased wateractivity in the vicinity of the enzyme that is caused by the sol-ventwater immiscibility. The polar, less hydrophobic solvents arenot suitable for biocatalytic processes since they can distort thewater microlayer around the enzyme, inuencing its native struc-ture and leading to denaturation [280]. Organic solvents such ashexane, ionic liquids and t-butanol have been widely used[70,198]. Most of the literature concentrated on the use of solventswith commercial enzymes for production of biodiesel from vegeta-ble oils. Transesterication of sunower oil with methanol, ethanoland butanol was studied using Novozym435 with and withoutpetroleum ether as the solvent [193]. The petroleum ether did notimpact theproduct yields in presence of ethanol andbutanol (as acylacceptors), however, it was required for biodiesel production withmethanol. Similar ndings pointed out to the need of a solvent (hex-ane) in a study of methanolysis of soybean oil, rapeseed oil, tallow,and recycled restaurant grease catalyzed byM. miehei lipase (Lipo-zyme IM 60) and C. antarctica (SP 435) [197]. This was attributedto the inhibition of immobilized enzymes by methanol. The use ofa common solvent for bothmethanol and oil has proved to be effec-tive [191,198,207]. Iso et al. [207] investigated the effects of 1,4-dioxane, benzene, chloroform and tetrahydrofuran as commonsolvents for methanolysis with lipase enzymes fromP. uorescens (Lipase AK), P. cepacia (Lipase PS), M. javanicus(Lipase M), C. rugosa (Lipase AY) and R. niveus (Newlase F). Higherconversionsof rapeseedoil of up to 97%were reportedusingmixtureof 2-ehtyl-1-hexanol [216]. Lipase AK achieved high conversionrates without loss of activity when 1,4-dioxanewas used as solvent.Use of tert-butanol resulted in higher yields of methyl esters[191,198]. Studies showed that often excess alcohols are used toshift the equilibrium towards products [37,41]. Although similarbiodiesel yields have been obtained in presence and absence of sol-vent [18,262,280], the use of solvents signicantly increases thereaction rate in comparison to solvent-free systems. However, theuse of solvents is not economically and environmentally favoredas it requires the addition of a solvent recovery unit for separatingthe organic solvent from the reaction mixture, which leads to in-crease in cost of recovery, losses and gas emissions [247].

    Acyl acceptor alternatives to methanol include primary, second-ary, straight chained and branched alcohols such as isopropanol[281], t-butanol [191,198], and octanol [67]. The choice of alcoholcan inuence the cold ow properties [282] and lubricity [283] ofbiodiesel. The longer chain alcohols have shown higher yields thanmethanol [64] as lipases are known to have a higher afnity to-ward long-chain than short-chain alcohols [65].

    3.7. Enzyme operating variables

    L.P. Christopher et al. / AppliGenerally, transesterication is carried out below the boilingpoint 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 reactiontemperatures reduce oil viscosity, increase the reaction rates andshorten the reaction time [41]. As most lipases have their temper-ature optimum between 30 and 60 C, the enzymatic transesteri-cation is mostly conducted in that temperature range. The freelipase enzymes, especially bacterial lipases, are known for higherthermal stability [67]. Lipase immobilization to solid supports de-creases the effect of temperature deactivation which leads to im-proved thermostability [18] and increased temperature optimumof lipase [284]. Overall, the optimum temperature is inuencedby the enzyme stability, alcohol to oil molar ratio and the type oforganic solvent used [250]. The initial rate of reaction increaseswith reaction temperature, however, conversion rates decrease attemperatures above the optimal due to thermal deactivation of li-pase [285,286]. A broad range of operational temperatures hasbeen employed depending on the enzyme source: 2555 C forC. antarctica IM lipase (temperature optimum of 40 C) [287];2070 C for P. cepacia IM lipase (50 C) [199]; 2060 C for R. chin-ensis whole-cell IM lipase (30 C) [288]. The optimal temperaturefor lipid transesterications with a mixture of R. oryzae and C. rug-osa IM lipases was 45 C [289]. Most frequently, the optimum oper-ating temperature for lipases has been reported to be 40 C onaverage [290].

    Bacterial lipases have neutral or alkaline pH optima [141,291],whereas lipases from yeasts and fungi are more active at the nearneutral to slightly acidic pH [292294]. However, some lipases areactive and stable over a broad pH range (pH 312) [295]. Overall,bacterial lipases have been reported to exhibit a higher activityand thermostability than other microbial lipases [141,284,291].The protein conformation of lipase is affected by pH, and uponimmobilization, the optimum pH for reactions catalyzed by lipasesis slightly shifted toward more alkaline values due to the partialopening of the lid at the enzyme active site [284]. In recent years,research has focused on the use of lipase-producing fungi as asource 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 cellbiocatalyst for industrial applications [66].

    Overall, the reaction times needed for enzymatic transesteri-cation, although dependant on reaction temperature, reactantand catalyst concentrations, are generally longer (36 h on average)than those of the chemically-catalyzed process (9 h) [19]. A simpli-ed model of the reaction behavior can be presented to explain theaccumulation of FAME as a function of the reaction time: (1) a slowinitial rate to allow for mixing and diffusion of alcohol into the oil;(2) rate of fatty acid esters conversion increases exponentially withreaction time [41,45]; (3) reaction rate reaches a peak and pro-ceeds at a steady state [43,216]; (4) reaction rate declines [18].Immobilized B. cepacia lipase produced a 90% biodiesel from usedJatropha oil yield after a 12 h reaction time [297]. Methanolysisof Simarouba oil and vegetable oils with immobilized lipase wasoptimum at 36 h [275] and 48 h [274], respectively. Extendingthe reaction time beyond that required for optimum biodiesel pro-duction can reduce the biodiesel yields due to enzyme inhibitionby 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-boundwater which is best dened as water activity [250,300]. The opti-mal protein conformation is disturbed by removal of water thatsurrounds the enzyme macromolecule, whereas excess of waterforms water droplets within the enzyme active site. The hydrolysisreaction, that competes transesterication, is catalyzed by increas-

    nergy 119 (2014) 497520 509ing amounts of water in the reaction mixture [301]. Generally,minimum amount of water is necessary for biocatalytic activitythat is specic for a given lipase [167]. No transesterication is pos-

  • ed Esible in absence of water as the formation of enzyme substratecomplexes (lipase-oil) proceeds only in presence of oilwaterinterface. A optimum water content, minimum hydrolysis andmaximum transesterication occur [154,167]. The reaction ratewas reported to increase proportionally to the water content inmany 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 transeste-rication by Candida sp. 99125 lipase [285].

    3.8. Bioreactor design

    The batch stirred tank reactor (STR) [45,304] and packed-bedreactor (PBR) [196,277,278,305] are the most extensively studiedbioreactors for enzymatic biodiesel production. In STR, the enzyme(free or immobilized) is dispersed in the reaction mixture by agita-tion, whereas in the PBR, the immobilized enzyme is packed into acolumn. The batch STR is the simplest bioreactor type consisting ofa reactor and a propeller. It is suitable high-viscous solutions andimmobilized enzymes that are less sensitive to shear stress anddeactivation upon physical stirring. A solidliquid separation usingcentrifugation or ltration is normally applied at the end of theprocess to recover the immobilized enzyme. The STR operated ina batch mode has a low throughput due to the need to empty, cleanand reload the reactor before a new batch can start. The low pro-ductivity disadvantage of the batch STR can be eliminated by usinga continuous STR where the enzyme is retained in the reactor witha lter placed at the reactor outlet. The PBR can operate in batch orcontinuous mode by recirculating the reaction mixture. The recy-cling method is advantageous as it allows the substrate solutionto be passed through the column at a desired velocity. For indus-trial applications, the upward substrate ow is generally preferredover downward ow because it does not compress the enzyme bedthat results in blockages with poor oxygen transfer and pressuredrops [18]. In the continuous PBR, reaction mixture is continuouslypumped through the column and the enzyme can be effectively re-used without a prior separation. Advantages of using continuousPBR include high efciency, low cost and ease of construction,operation and maintenance. In addition, it allows for continuousremoval of glycerol and excess alcohol, and protects the enzymeparticles from mechanical shear stress [197,198]. The continuousPBR is superior to the batch PBR due to automated control andoperation, reduced labor costs, stable operating conditions, andeasy quality control of products [18,306]. Other bioreactor cong-urations include uid beds, expanding bed, recirculation mem-brane reactors, and static mixers [18]. In the membranebioreactors, the enzyme is immobilized onto at sheet or hollowber membranes; however, membrane fouling presents a problemthat is more challenging than the bed plugging in the PBR.

    Although most of the existing biodiesel plants are currentlyrunning in batch mode with stirred tank reactors, recent researchefforts have focused on optimization of PBR for use in differentenzymatic biodiesel production processes [30,179,203,307,308].Chen et al. [308] tested a PBR for continuous biodiesel productionusing methanolysis of soybean oil in a t-butanol solvent systemcatalyzed by Novozym435. A molar conversion of 83% was at-tained with no considerable decline in lipase activity in continuousoperation for 30 days at a ow rate of 0.1 ml/min, 52 C and a 4:1methanol to oil molar ratio. A 88% conversion was reached in acontinuous PBR system catalyzed by a lipase nanoparticles com-posite (lipase-Fe3O4) using a mixture of soybean oil, distilled water,methanol and n-hexane with volumetric ratio of 6:3:1:0.2, respec-tively, at a ow rate of 0.25 ml/min for 192 h [307]. PBR was used

    510 L.P. Christopher et al. / Applifor production of enzymatic biodiesel from waste cooking oil witha FAME yield of 90% using a solvent-free system [203]. Thestepwise addition of methanol prevented the deactivation of thelipase, which allowed a constant enzyme activity during 100 daysof operation. Transesterication of soybean oil in a PBR at a owrate of 25 l/h using a R. oryzae whole-cell immobilized catalystand a step-wise addition of methanol produced a maximum FAMEyield of 90% [184]. A whole-cell biocatalyst of A. niger immobilizedin a PBR produced 90% FAME from palm oil at a ow rate of 15 l/hby continuous recycling of the reaction mixture for 72 h [179]. Thebiodiesel yield dropped to 85% after 4 consecutive cycles. An opti-mal continuous production of biodiesel by methanolysis of soy-bean oil in a packed-bed reactor was developed using responsesurface methodology (RSM) and Box-Behnken design with immo-bilized lipase (Novozym435) as a catalyst in a t-butanol solventsystem [308]. The continuous process over 30 days showed noappreciable decrease in the molar conversion of 83.3%.

    Production of biodiesel from vegetable oils with a high FFA con-tent requires an additional step for separation of the reaction by-products from the biodiesel product. This can be achieved bywater-washing or water-free washing of biodiesel [309]. Thewater-free washing process is performed by a direct mixing ofthe transesterication reaction products and adsorbent particlesor of biodiesel washing pellets. The biodiesel uid containing theadsorbent particles subsequently requires ltration that is bothmonotonous and time consuming. Alternatively, a water-freewashing in PBR has been adopted as a method that effects auto-matic separation of the adsorbents. Several parameters are fac-tored into the design of a packed bed water-free washingbiodiesel separator such as the ease with which the biodieselstream ows through the bed; bed porosity; adsorption dynamicsof the adsorbents; ratio of convective to ow rate, etc. It should benoted that the washing requirement complicates the conventionalbiodiesel production, but is not a challenge in the production ofenzymatic biodiesel with minimal impurities [12].

    3.9. Glycerol

    Due to its hydrophilicity, glycerol, the co-product in biodieselproduction, is insoluble in the oils and gets easily adsorbed ontothe surface of the immobilized lipase. This creates mass transferlimitations that negatively impact lipase activity and stability.Adding silica gel into the reaction system to absorb the glycerolor periodically washing the lipase with organic solvents to regen-erate the lipase activity have been proposed. Studies by Bako-Belaet al. [205] employed dialysis for glycerol removal which resultedin a 97% conversion by methanolysis. However, these methods areimpractical for large-scale continuous production of biodiesel. Asdiscussed before, a novel interesterication process, that makesuse of methyl or ethyl acetate as the acyl acceptor instead of puremethanol, does not produce glycerol [215]. The triacetylglycerolco-product does not interfere with the enzyme catalyst or the bio-diesel main product, which also eliminates the need of complicateddownstream processing.

    The stoichiometric yield of glycerol is 10% (w/w) with respect tothe biodiesel produced. Following methanol recovery, glycerolwith up to 90% purity can be used as a marketable commodity.Most commercial biodiesel manufacturing companies are able tosend the glycerol to a glycerol recovery/rening facility. Puregrades of glycerol (99.7%) can be used as a raw material in otherindustrial sectors such as food, cosmetics, paints, pharmaceutics,paper, textiles, leather, toiletries, toothpaste, drugs, animal feed,plasticizers, tobacco, and emulsiers, and for the production ofvarious chemicals [310]. The increase in biodiesel production isexpected to result in excess of glycerol which has a limited marketthereby reducing the price of glycerol. This necessitates the

    nergy 119 (2014) 497520development of alternative and viable processes for glycerol as avalue-added co-product [311] that would impact positively onthe overall production cost of biodiesel [312]. For instance, there

  • is a growing interest in glycerol fermentation to methanol and eth-anol used in biodiesel production, which would improve the eco-nomics of the biodiesel process [189,313317].

    Fig. 4 shows some major economically-important biochemicalsthat can be produced from glycerol-based biorenery setup. Glyc-erol has been used as an alternative and inexpensive carbon sourcein the microbial production of various compounds such as citricacid, succinic acid, 1,3 propanediol, 2,3-butanediol, carotenoids,hydrogen, biosurfactants, etc. [318,319]. Using the yeast speciesYarrowia lipolytica, Rymowicz et al.[320] studied the productionof citric acid from raw glycerol derived from the biodiesel produc-tion of rapeseed oil. A maximum yield of 0.62 g citric acid/g of glyc-erol was reported. A nal concentration of 1,3-propanediol of51.3 g/l and 53.0 g/l was obtained with Klebsiella pneumoniae fromcrude glycerol (derived frommethanolysis of soybean oil) using al-kali- and lipase-catalyzed process, respectively [321]. Anotherstrain of K. pneumonia, ATCC 15380, was optimized for productionof 1,3 propanediol from glycerol derived from biodiesel productionof non-edible jatropha oil [322]. The bacterial strain E. coli AC-521produced lactic acid from crude glycerol with a high yield of0.9 mol/mol glycerol [323]. Anaerobic digestion of crude acidiedglycerol yielded 0.306 m3 methane/kg glycerol [324]. The natural

    4. Current trends and future directions for enzymatic biodieselR&D

    Recently, a novel method for production of biodiesel at reducedcosts has been reported [332]. This method employs the use of asolid whole cell biocatalyst obtained by solid state fermentation(SSF). The advantages associated with the use of a SSF-generatedbiocatalyst are twofold: (i) the microorganisms grow on low-costsubstrates (agro-industrial residues) maintaining low moisturecontent; (ii) the crude fermented solid material can be used di-rectly as a biocatalyst thereby omitting three processing steps: li-pase extraction, purication and immobilization. This leads to asignicant reduction of the lipase costs that translates into lowerbiodiesel production costs [333,334]. A SSF-derived lipase-contain-ing 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 Liuet al. [333] reported SSF production of a B. cepacia lipase on amixed substrate of sugarcane bagasse and sunower seed mealwith a catalytic activity of 72.3 units/g dry solids over a period of96 h. A novel mixed substrate for enhanced SSF production of li-pase from A. niger MTCC 2594 was developed for hydrolyzing tal-

    O

    Gly

    L.P. Christopher et al. / Applied Energy 119 (2014) 497520 511occurring polymer polyhydroxybutryate (PHB, also known as bac-terial polyester, can be synthesized from crude glycerol instead ofglucose as carbon source by Paracoccus denitricans and Cupria vid-usnecator JMP 134 bacterial strains [325].

    Using convention catalysts in addition to microbial conversion,chemical compounds with a diverse range of industrial applica-tions can be synthesized from biodiesel glycerol. These includeamong others acrolein[326], ethylene glycol [327], syngas [328],(2,2-dimethyl-1,3-dioxolan-4-yl) methyl acetate [329] and oligo-mers of glycerol [330]. A 74 mol% acrolein with 81% of selectivitywas synthesized from glycerol using an acid catalyst at supercriti-cal conditions (673 K and 34.5 MPa pressure) [331]. The fueladditive (2,2-dimethyl-1,3-dioxolan-4-yl) methyl acetate can beproduced from crude glycer


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