REVIEW PAPER

A Review on Microbial Lipases Production

Helen Treichel & Débora de Oliveira &

Marcio A. Mazutti & Marco Di Luccio &

J. Vladimir Oliveira

Received: 28 November 2008 /Accepted: 11 March 2009 /Published online: 25 March 2009# Springer Science + Business Media, LLC 2009

Abstract This review paper provides an overview regard-ing the main aspects of microbial lipases production. Themost important microbial lipase-producing strains forsubmerged and solid-state fermentations are reviewed aswell as the main substrates, including the use of agro-industrial residues. Current process techniques (batch,repeated-batch, fed-batch, and continuous mode) are dis-cussed and the main bioreactors configurations are alsopresented. Furthermore, the present review paper shows ageneral overview about the development of mathematicalmodels applied to lipase production. Finally, some futureperspectives on lipase production are discussed with specialemphasis on lipase engineering and the use of mathematicalmodels as a useful tool for process improvement andcontrol.

Keywords Review . Lipase production . Substrates .

Process conduction . Bioprocess modeling

Introduction

Lipases (triacylglycerol acylhydrolases EC 3.1.1.3) are aclass of hydrolase which catalyze the hydrolysis of

triglycerides to glycerol and free fatty acids over an oil–water interface. In addition, lipases catalyze the hydrolysisand transesterification of other esters as well as the synthesisof esters and exhibit enantioselective properties. The ability oflipases to perform very specific chemical transformation(biotransformation) has make them increasingly popular inthe food, detergent, cosmetic, organic synthesis, and pharma-ceutical industries (Park et al. 2005; Gupta et al. 2007;Grbavcic et al. 2007; Franken et al. 2009).

Lipases have emerged as one of the leading biocatalystswith proven potential for contributing to the multibilliondollar underexploited lipid technology bio-industry andhave been used in in situ lipid metabolism and ex situmultifaceted industrial applications (Joseph et al. 2008).The number of available lipases has increased since the1980s. This is mainly a result of the huge achievementsmade in the cloning and expression of enzymes frommicroorganisms, as well as of an increasing demand forthese biocatalysts with novel and specific properties such asspecificity, stability, pH, and temperature (Bornscheuer etal. 2002; Menoncin et al. 2009).

Lipases are produced by animals, plants, and micro-organisms. Microbial lipases have gained special industrialattention due to their stability, selectivity, and broad sub-strate specificity (Dutra et al. 2008; Griebeler et al. 2009).Many microorganisms are known as potential producers ofextracellular lipases, including bacteria, yeast, and fungi(Abada 2008). Fungal species are preferably cultivated insolid-state fermentation (SSF), while bacteria and yeastare cultivated in submerged fermentation (SmF; Dutra etal. 2008).

The importance of lipases can be observed by the greatnumber of published articles recently. In fact, over the lastfew years, there has been a progressive increase in the

Food Bioprocess Technol (2010) 3:182–196DOI 10.1007/s11947-009-0202-2

H. Treichel (*) :D. de Oliveira :M. Di Luccio : J. V. OliveiraDepartment of Food Engineering, URI—Campus de Erechim,P.O. Box 743, CEP 99700-000 Erechim, Rio Grande do Sul,Brazile-mail: helen@uricer.edu.br

M. A. MazuttiFaculty of Food Engineering, Department of Food Engineering,UNICAMP,P.O. Box 6121, CEP 13083-862 Campinas, São Paulo, Brazil

number of publications related to industrial applications oflipase-catalyzed reactions, performed in common organicsolvents, ionic liquids, or even in non-conventional solvents.The present review is focused on lipase production discussingthe main microorganisms, substrates, and process operationsused in this specific field.

Microbial Lipases Sources

Lipases are ubiquitous in nature and are produced byseveral plants, animals, and microorganisms. Lipases ofmicrobial origin represent the most widely used class ofenzymes in biotechnological applications and organicchemistry. A review of the most recent (from 2004 to thepresent) potential microorganisms for lipase production inboth submerged and solid-state fermentations are reportedin Table 1. The main microorganisms used before 2004have already been well described in the reports of Gupta etal. (2004) and Sharma et al. (2001).

Filamentous Fungi

Some of the most commercially important lipase-producingfungi are recognized as belonging to the genera Rhizopussp., Aspergillus sp., Penicillium sp., Geotrichum sp., Mucorsp., and Rhizomucor sp. Lipase production by filamentousfungi varies according to the strain, the composition of thegrowth medium, cultivation conditions, pH, temperature,and the kind of carbon and nitrogen sources (Cihangir andSarikaya 2004).

The industrial demand for new sources of lipases withdifferent catalytic characteristics stimulates the isolation andselection of new strains. Lipase-producing microorganismshave been found in different habitats such as industrial wastes,vegetable oil processing factories, dairy plants, and soilcontaminated with oil and oilseeds among others (Sharma etal. 2001). Colen et al. (2006) isolated 59 lipase-producingfungal strains from Brazilian savanna soil using enrichmentculture techniques. An agar plate medium containing bilesalts and olive oil emulsion was employed for isolatingand growing fungi in primary screening assay. Twenty-one strains were selected by the ratio of the lipolytic haloradius and the colony radius. Eleven strains wereconsidered and, among them, the strain identified asColletotrichum gloesporioides was the most productive. Inanother work, Cihangir and Sarikaya (2004) isolated astrain of Aspergillus sp. from soil samples from thedifferent regions of Turkey and obtained an expressiveactivity of 17 U mL−1.

In SmF, Teng and Xu (2008) investigated the lipaseproduction by Rhizopus chinensis and obtained, at theoptimized experimental conditions, a maximum lipase

activity of 14 U mL−1. Bapiraju et al. (2005) optimizedthe lipase production by the mutant strain of Rhizopus sp.and the optimum activity was 29 U mL−1. Kaushik et al.(2006) studied the production of an extracellular lipasefrom Aspergillus carneus and obtained a maximum activityof 13 U mL−1.

In SSF, Kempka et al. (2008) investigated the lipaseproduction by Penicillium verrucosum and the optimumactivity was about 40 U gram of dry substrate−1 (gds).Vargas et al. (2008) studied the lipase production byPenicillium simplicissimum and obtained an activity of30 U gds−1. Both P. verrucosum and P. simplicissimumwere isolated from the babassu oil industry.

A quantitative comparison between SmF and SSF isdifficult due to the difference in the methods used fordetermining the lipase activity. However, some qualitativeinformation presented in the literature can be of interest.For example, extracellular lipases were obtained usingRhizopus homothallicus with lipase activities of 1,500 Ugds−1 and 50 U mL−1, by SSF and SmF, respectively (Diazet al. 2006). Azeredo et al. (2007) obtained lipase activitiesof 17 U gds−1 and 12 U mL−1 for SSF and SmF, respectively,by cultivation of Penicillium restrictum.

Recently, some works reporting the use of immobilizedwhole biomass of filamentous fungi have also beenpublished. The immobilization is advantageous since itcan avoid biomass washout at high dilution rates. Also,high cell concentration in the reactor could be achieved andthe separation of biomass from the medium is favored(Elitol and Ozer 2000). Wolski et al. (2008) reported theuse of response surface methodology to optimize the lipaseproduction by submerged fermentation using immobilizedbiomass of a newly isolated Penicillium sp. At theoptimized experimental conditions, the authors reached alipase activity around 21 U mL−1, higher than the activityobtained by the same microorganism before immobiliza-tion. Yang et al. (2005) studied the repeated-batch lipaseproduction by immobilized mycelium of Rhizopus arrhizusin submerged fermentation. The lipase productivity increasedfrom 3 to 18 UmL−1 h−1, changing the process from batch torepeated-batch mode. Ellaiah et al. (2004) used the wholeimmobilized biomass of Aspergillus niger to produce lipaseand obtained similar activities for both free and immobilizedbiomass cultivations (4 U mL−1). Elitol and Ozer (2000)immobilized the whole cell of R. arrhizus and the rate oflipase production was constant through several repeated-batch experiments.

Yeast

According to Vakhlu and Kour (2006), the main terrestrialspecies of yeasts that were found to produce lipases are:Candida rugosa, Candida tropicalis, Candida antarctica,

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Candida cylindracea, Candida parapsilopsis, Candidadeformans, Candida curvata, Candida valida, Yarrowialipolytica, Rhodotorula glutinis, Rhodotorula pilimornae,Pichia bispora, Pichia mexicana, Pichia sivicola, Pichia

xylosa, Pichia burtonii, Saccharomycopsis crataegenesis,Torulaspora globosa, and Trichosporon asteroids. Thegenes that encode lipase in Candida sp., Geotrichum sp.,Trichosporon sp., and Y. lipolytica have been cloned and

Table 1 Microorganisms cited in the recent literature as potential lipase producers

Microorganism Source Reference

Submerged fermentation Solid-state fermentation

Rhizopus arrhizus Fungal Tan and Yin (2005), Yang et al. (2005)

Rhizopus chinensis Fungal Teng et al. (2009), Wang et al. (2008),Teng and Xu (2008)

Sun and Xu (2008)

Aspergillus sp. Fungal Cihangir and Sarikaya (2004)

Rhizopus homothallicus Fungal Diaz et al. (2006) Diaz et al. (2006)

Penicillium citrinum Fungal D’Annibale et al. (2006b)

Penicillium restrictum Fungal Azeredo et al. (2007) Palma et al. (2000),Azeredo et al. (2007)

Penicillium simplicissimum Fungal Vargas et al. (2008),Cavalcanti et al. (2005)

Penicillium verrucosum Fungal Pinheiro et al. (2008) Kempka et al. (2008)

Geotrichum sp. Fungal Yan and Yan (2008), Burkert et al. (2004)

Geotrichum candidum Fungal Burkert et al. (2005)

Aspergillus carneus Fungal Kaushik et al. (2006)

Rhizopus sp. Fungal Bapiraju et al. (2005) Martinez-Ruiz et al. (2008)

Aspergillus niger Fungal Dutra et al. (2008), Mala et al.(2007), Falony et al. (2006)

Rhizopus oryzae Fungal Cos et al. (2005), Surribas et al. (2007)

Colletotrichum gloesporioides Fungal Colen et al. (2006)

Rhodotorula mucilaginosa Yeast Potumarthi et al. (2008)

Yarrowia lipolytica Yeast Lopes et al. (2009), Alonso et al. (2005), Kar et al. (2008),Fickers et al. (2006), Amaral et al. (2007)

Dominguez et al. (2003)

Candida utilis Yeast Grbavcic et al. (2007)

Trichosporon asahii Yeast Kumar and Gupta (2008)

Candida rugosa Yeast Rajendran et al. (2008), Boareto et al. (2007),Puthli et al. (2006), Zhao et al. (2008)

Benjamin and Pandey (2001)

Candida cylindracea Yeast Kim and Hou (2006), D’Annibale et al. (2006b)

Candida sp. Yeast He and Tan (2006)

Aureobasidium pullulans Yeast Liu et al. (2008)

Saccharomyces cerevisiae Yeast Ciafardini et al. (2006)

Williopsis californica Yeast Ciafardini et al. (2006)

Acinetobacter radioresistens Bacterial Li et al. (2005)

Pseudomonas sp. Bacterial Kiran et al. (2008)

Pseudomonas aeruginosa Bacterial Ruchi et al. (2008) Mahanta et al. (2008)

Staphylococcus caseolyticus Bacterial Volpato et al. (2008)

‘Biopetro-4’ Bacterial Carvalho et al. (2008)

Bacillus stearothermophilus Bacterial Abada (2008)

Burkholderia cepacia Bacterial Fernandes et al. (2007)

Burkholderia multivorans Bacterial Gupta et al. (2007)

Serratia rubidaea Bacterial Immanuel et al. (2008)

Bacillus sp. Bacterial Ertugrul et al. (2007), Shariff et al. (2007),Nawani and Kaur (2007)

Bacillus coagulans Bacterial Alkan et al. 2007

Bacillus subtilis Bacterial Takaç and Marul (2008)

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over-expressed (Wang et al. 2007). Although lipases fromC. rugosa and C. antarctica have been extensively used indifferent fields, there are several recent publications report-ing the production of lipases by other yeasts, as shown inTable 1.

Potumarthi et al. (2008) collected marine soil samplesnear an oil extraction platform in the Arabian Sea. After theisolation of the colonies, they were transferred to platescontaining 2% tributyrin and incubated at 35 °C for3–4 days. The colonies that showed the largest hydrolysishalos zone were selected. The most effective strain for lipaseproduction was identified as Rhodotorula mucilaginosa(MTCC 8737) by its phenotypic characteristics. Kumar andGupta (2008) isolated 15 yeasts from petroleum and oilsludge areas in Delphi (India). The isolates were purified andchecked for their lipolytic potential. Among these yeaststrains, one strain was selected for further studies, based onthe largest halo of lipolysis. On the basis of sequencehomology, this strain was found to belong to Trichosporonasahii genus and share 99% homology with the alreadyexisting database.

Ciafardini et al. (2006) have discovered that freshlyproduced olive oil is contaminated by a rich micro-flora,capable of conditioning the physicochemical and organo-leptic characteristics of the oil, through the production ofenzymes. Among the microorganisms that were isolatedfrom this oil, several strains of yeasts were identified asSaccharomyces cerevisiae, Candida wickerhamii, Williopsiscalifornica, and Candida boidinii, of which S. cerevisiaeand W. californica showed good potential to produce lipase.The lipase activity in S. cerevisiae was noted to beintracellular, and extracellular in W. californica.

The three-phase olive oil extraction process generates adark-colored effluent, usually termed olive oil mill waste-water (OMW). D’Annibale et al. (2006b) investigated thevalorization of OMW by its use as a possible growthmedium for the microbial production of extracellular lipase.Among the 12 strains tested, the most promising strain wasC. cylindracea.

Candida sp. is the most potential lipase producer fromyeasts reported in the literature. He and Tan (2006) used theresponse surface methodology to optimize culture mediumfor lipase production by the strain Candida sp. 99-125.After optimization, the authors reported the optimum lipaseactivity as 6,230 and 9,600 U mL−1 in shaken flasks andin a 5-L bioreactor, respectively. In a 30-L bioreactor, Tanet al. (2003) reached a maximum lipase activity of 8,300 UmL−1, thus showing that lipase activity values are highlyinfluenced by the microorganism, substrates, and theoperational conditions. In contrast to the high activitiesreached in the above-mentioned works, Rajendran et al.(2008) reported the optimum lipase activity of 3.8 U mL−1

by C. rugosa.

Bacteria

Among bacterial lipases being exploited, those fromBacillus exhibit interesting properties that make thempotential candidates for biotechnological applications.Bacillus subtilis, Bacillus pumilus, Bacillus licheniformis,Bacillus coagulans, Bacillus stearothermophilus, andBacillus alcalophilus are the most common bacterial lipases.In addition, Pseudomonas sp., Pseudomonas aeruginosa,Burkholderia multivorans, Burkholderia cepacia, andStaphylococcus caseolyticus are also reported as bacteriallipase producers (Table 1).

Ertugrul et al. (2007) isolated 17 bacterial strains thatcould grow on media based on OMW and selected the mostpromising strain for lipase production. After screening intributyrin agar medium, a strain of Bacillus sp. was identifiedas the best lipase producer. After the medium optimization,the intracellular activity found was 168 U mL−1. Kiran et al.(2008) isolated 57 heterotrophic bacteria from the marinesponge Dendrodoris nigra, of which 37% produced a clearhalo around the colonies on tributyrin agar plates for lipaseproduction. Particularly, the strain Pseudomonas MSI057exhibited large clean zones around the colonies. Then, thisstrain was selected for further studies, and after optimization,a maximum lipase activity was found as 750 U mL−1.Carvalho et al. (2008) isolated a bacterium strain frompetroleum-contaminated soil and codified as Biopetro-4.After investigation of several inducers on lipase activity,the maximum value obtained was 1,675 U mL−1 after 120 hof fermentation. Abada (2008) produced lipase from a strainof B. stearothermophilus AB-1 isolated from air andobtained a maximum lipase activity of 1,585 U mL−1 in48 h of fermentation.

Takaç and Marul (2008) isolated microbial cultures fromsoil enriched by periodic sub-culturing of samples innutrient broth containing 1% (v/v) tributyrin. The isolationprocess was performed by serial dilution samples ontributyrin agar (TBA) plates. Bacillus sp. was selectedtowards producing the largest opaque halo. Active colonieswere re-streaked on TBA agar for purification. Shariff et al.(2007) isolated a thermophilic bacterium, Bacillus sp. strainL2 from a hot spring in Perak, Malaysia. An extracellularthermostable lipase activity was detected through plate andbroth assays at 70 °C after 28 h of fermentation.

In most cases, bacteria are preferably cultivated in SmF,due to the high water activity required by the micro-organisms (higher than 0.9). There are few exceptions forbacteria grown in SSF. However, when the bacteria are welladapted to this solid medium, the production is normallyhigh. Mahanta et al. (2008) obtained a maximum lipaseactivity of 1,084 U gds−1 using a solvent tolerant P.aeruginosa PseA strain. Alkan et al. (2007) produced anextracellular lipase by B. coagulans and obtained a

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maximum lipase activity of 149 U gds−1 after 24 h offermentation. Fernandes et al. (2007) obtained a maximumlipase activity of 108 U gds−1 after 72 h of fermentation byB. cepacia.

Substrates for Lipase Production

Microbial lipases are mostly extracellular and their produc-tion is greatly influenced by medium composition besidesphysicochemical factors such as temperature, pH, anddissolved oxygen. The major factor for the expression oflipase activity has always been reported as the carbonsource, since lipases are inducible enzymes. These enzymesare generally produced in the presence of a lipid such as oilor any other inducer, such as triacylglycerols, fatty acids,hydrolysable esters, Tweens, bile salts, and glycerol (Guptaet al. 2004; Sharma et al. 2001). Lipidic carbon sourcesseem to be essential for obtaining a high lipase yield.However, nitrogen sources and essential micronutrientsshould also be carefully considered for growth andproduction optimization. These nutritional requirementsfor microbial growth are fulfilled by several alternativemedia as those based on defined compounds (syntheticmedium) like sugars, oils, and complex components such aspeptone, yeast extract, malt extract media, and also agro-industrial residues containing all the components necessaryfor microorganism development. A mix of these two kindsof media can also be used for the purpose of lipaseproduction. The main studies available in the literaturesince 2005 covering these subjects are presented below,divided by the kind of medium used.

Synthetic Medium

Generally, high productivity has been achieved by culturemedium optimization. Optimization of the concentration ofeach compound that constitutes a cultivation medium isusually a time-consuming procedure. The classical practiceof changing one variable at a time, while keeping othersconstant was found to be inefficient, since it does notexplain the interaction effects among variables and theireffects on the fermentation process (Haaland 1989; Khuriand Cornell 1987; Rodrigues and Iemma 2005). Anefficient and widely used approach is the application ofPlackett–Burman (PB) designs that allow efficient screen-ing of key variables for further optimization in a rationalway (Rodrigues and Iemma 2005).

An alkaline lipase from B. multivorans was producedafter 15 h of cultivation in a 14-L bioreactor. The mediumoptimization was carried out to lead to an increase of12-fold in lipase production. Initially, the effect of ninefactors, namely, concentrations of glucose, dextran, olive

oil, NH4Cl, trace metals, K2HPO4, MgCl2, and CaCl2 andinoculum density were studied using the Plackett–Burmanexperimental design. These components were varied in thebasal medium containing olive oil as inducer and yeastextract as nitrogen source. After the screening of the mostsignificant factors by the PB design, the optimization wascarried out in terms of the olive oil, glucose, and yeastextract concentrations, inoculum density, and fermentationtime. The optimal medium composition for the lipaseproduction was determined to be (percent w/v): glucose0.1, olive oil 3.0, NH4Cl 0.5, yeast extract 0.36, K2HPO4

0.1, MgCl2 0.01, and CaCl2 0.4 mM (Gupta et al. 2007).Kumar and Gupta (2008) compared the medium optimi-

zation for the yeast T. asahii by both one variable at a timeand statistical approach. A Plackett–Burman design forseven independent variables (glucose, olive oil, yeastextract, malt extract, MgCl2, and CaCl2 concentrationsand time) was applied to select the most significant factor.All variables significantly influenced lipase production,apart from glucose and CaCl2 concentrations. Responsesurface methodology indicated that the requirement of maltextract and yeast extract varied with the type of lipaseinducer used. The use of high malt and yeast extractconcentrations favored lipase production when corn oil wasused as substrate. On the other hand, the use of Tween 80as inductor inhibited the lipase production. Conversely,during kerosene induction both malt and yeast extractsrequirements were minimal.

Wang et al. (2008) optimized the fermentation mediumfor lipase production by R. chinensis. In order to improvethe productivity of lipase, the effects of oils and oil-relatedsubstrates were assessed by orthogonal test and responsesurface methodology. The optimized medium for improvedlipase activity consisted of peptone, olive oil, maltose,K2HPO4, and MgSO4⋅7H2O. Rajendran et al. (2008) usedthe Plackett–Burman statistical experimental design toevaluate the fermentation medium components. The effectof 12 medium components was studied in 16 experimentaltrials. Glucose, olive oil, peptone, and FeCl3⋅6H2O werefound to have more significant influence on lipase produc-tion by C. rugosa.

Takaç and Marul (2008) improved the lipase productionby B. subtilis using different concentrations of lipidiccarbon sources such as vegetable oils, fatty acids, andtriglycerides. One percent of sesame oil afforded thehighest activity with 80% and 98% enhancements withrespect to 1% concentrations of linoleic acid and triolein asthe favored fatty acid and triglyceride, respectively. Thesame authors tested the use of glucose as carbon sourceand verified that it presented an impressive effect onlipase production. Abada (2008) produced lipase from B.stearothermophilus AB-1. The authors observed that theuse of xylose, tryptophan, alanine, phenylalanine, and

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potassium nitrate as supplement lead to the highest lipaseproduction.

Ruchi et al. (2008) carried out media optimizationthrough response surface methodology for cost-effectiveproduction of lipase by P. aeruginosa. The effects of 11media components (peptone, tryptone, NH4Cl, NaNO3,yeast extract, glucose, glycerol, xylose, arabic gum,MgSO4, and NaCl) were assessed by a Plackett–Burmandesign, and the most significant factors (arabic gum, MgSO4,tryptone, and yeast extract) optimized by the responsesurface methodology. After optimization, the lipase pro-duction was increased 5.58-fold, yielding an activity of4,580 U mL−1. Kaushik et al. (2006) used the responsesurface approach to investigate the production of anextracellular lipase from A. carneus. Interactions wereevaluated for five different variables (sunflower oil, glucose,peptone, agitation rate, and incubation period) and a 1.8-foldincrease in production was reported at optimized conditions.

Lin et al. (2006) investigated the influence of differentculture conditions, temperature, pH, carbon, nitrogen, min-eral sources, and vitamins on the production of lipase byAntrodia cinnamomea in submerged cultures. Nine carbonsources, 14 nitrogen sources, six mineral sources, and fivevitamins were investigated. The authors found that 5%glycerol, 0.5% sodium nitrate, and 0.1% thiamine providedthe best results. The lipase production reached 54 U mL−1

after 17 days of incubation.He and Tan (2006) used the response surface method-

ology to optimize the culture medium for lipase productionwith Candida sp. 99-125. In the first step, a Plackett–Burman design was used to evaluate the effects of differentcomponents in the culture medium (soybean oil, soybeanmeal, K2HPO4, KH2PO4, (NH4)2SO4, MgSO4, and Spam60). Soybean oil, soybean meal, and K2HPO4 concentra-tions have a significant influence on lipase production.Results were optimized using central composite designsand response surface analysis. The optimized conditionallowed lipase production to be increased from 5,000 to6,230 U mL−1 in a shaken flask system. The lipasefermentation in a 5-L vessel reached 9,600 U mL−1.

The use of pure synthetic medium in solid-state fermenta-tion has been hardly presented in the literature. For example,Martinez-Ruiz et al. (2008) reported the production of lipasefrom Rhizopus sp. using perlite (as inert support) supple-mented with urea, lactose, olive oil, K2HPO4, KH2PO4,MgSO4⋅7H2O, polyvinyl alcohol, and an oligoelement solu-tion. The activity reached was 75 U gram of inert support−1.

Agroindustrial Residues

Over the recent years, research on the selection of suitablesubstrates for fermentative process has mainly been focusedon agroindustrial residues, due to their potential advantages.

Utilization of agroindustrial wastes provides alternativesubstrates and may help solving pollution problems, whichotherwise might be caused by their disposal. The nature ofthe substrate is the most important factor affectingfermentative processes. The choice of the substrate dependsupon several factors, mainly related to cost and availability.Thus, process optimization may involve the screening ofseveral agroindustrial residues.

Many reports of SSF have been recently published, inwhich emphasis is given to the application of agriculturalby-products for the production of fine chemicals andenzymes, including lipases. Vargas et al. (2008) investigatedthe lipase production by P. simplicissimum using soybeanmeal as substrate supplemented with low-cost substrates:soybean oil, wastewater from a slaughterhouse (rich on oiland fat), corn steep liquor, and yeast hydrolyzed. Soybeanmeal without supplements appears to be the best medium ofthose tested for lipase production. Alkan et al. (2007) studiedthe effect of several agroindustrial residues (wheat bran, ricehusk, lentil husk, banana waste, watermelon waste, andmelon waste) on lipase production by SSF using B.coagulans. The best results were obtained using solid wastefrom melon supplemented with NH4NO3 and 1% olive oil.

Kempka et al. (2008) produced lipase from P. verrucosumby SSF using soybean meal, sugar cane molasses, corn steepliquor, yeast hydrolyzed, yeast extract, sodium chloride,soybean oil, castor oil, corn oil, olive oil, and peptone.Soybean meal was the best substrate. Mahanta et al. (2008)reported the lipase production by SSF with P. aeruginosaPseA using Jatropha seed cake supplemented with differentcarbon (starch, maltose, glucose) and nitrogen (peptone,NH4Cl, NaNO3) sources. Jatropha seed cake withoutsupplementation showed a lipase activity of 625 U gds−1.When supplemented with maltose, the activity reachedvalues of 976 U gds−1, while with NaNO3 the activity was1,084 U gds−1.

Mala et al. (2007) developed a SSF for lipase productionby A. niger MTCC 2594 using wheat bran and gingelly oilcake as substrates and supplement, respectively, and theresults showed that addition of gingelly oil cake to wheatbran increased the lipase activity by 36% and the activitywas 384 U gds−1. Dutra et al. (2008) monitored the biomassgrowth of A. niger in SSF for lipase production using thedigital image processing technique. The strain of A. nigerwas cultivated in SSF using wheat bran as support, whichwas enriched with 0.91% of ammonium sulfate. Theaddition of several vegetable oils (castor, soybean, olive,corn, and palm oil) was investigated to enhance lipaseproduction. A maximum lipase activity was obtained using2% of castor oil. Sun and Xu (2008) reported that acombined substrate of wheat flour with wheat bransupported both good biomass and enzyme production byR. chinensis in SSF.

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Azeredo et al. (2007) investigated the effects of differentcarbon sources, mainly carbohydrates and lipids, to supportgrowth and lipase production by P. restrictum in SSF. Smalltrays containing 10 g of ground babassu cake as the basalmedium were supplemented with different carbon sources(babassu oil, olive oil, oleic acid, tributyrin, starch, andglucose). In all tested media, the carbon source concentra-tion was calculated to give a C/N ratio of 13.3. The use ofolive oil led to higher lipase activities.

Diaz et al. (2006) obtained extracellular lipases fromSSF and SmF by R. homothallicus. The SmF culturemedium consisted of corn steep liquor, peptone, K2HPO4,KH2PO4, and MgSO4 while in SSF the medium wascomposed of sugarcane bagasse as support, supplementedwith olive oil, lactose, urea, K2HPO4, and MgSO4. Cellcultures in SmF yielded a maximum extracellular lipaseactivity of 50 U mL−1 after 22 h of fermentation and in SSFcell cultures yielded a maximum lipase activity of 1,500 Ugds−1 after 12 h of fermentation. Falony et al. (2006) testedthe lipase production by A. niger in SSF using wheat branas support and supplemented with synthetic mediumcomposed of glucose, Na2HPO4, KH2PO4, MgSO4⋅7H2O,CaCl2, (NH4)2SO4, NH2CONH2, and olive oil. Cavalcantiet al. (2005) investigated the lipase production by SSF infixed-bed bioreactor using babassu cake supplemented withsugar cane molasses.

Pinheiro et al. (2008) investigated the lipase productionby P. verrucosum in submerged fermentation using amedium based on peptone, yeast extract, NaCl, and oliveoil and an industrial medium composed of corn steepliquor, yeast hydrolysate, NaCl, and olive oil. Whencomparing both tested media, the best results were obtainedusing the one based on peptone, yeast extract, NaCl, andolive oil. Potumarthi et al. (2008) employed molasses assole carbon source for lipase production by R. mucilaginosaMTCC 8737. A maximum lipase activity was verified using1% of molasses. The increase in molasses concentrationresulted in lower lipase activity, probably due to theenhancement on the medium viscosity.

Volpato et al. (2008) used the Plackett–Burman statisticaldesign and the central composite design in order to optimizeculture conditions for lipase production by S. caseolyticusstrain EX17 growing on raw glycerol, which was obtained asa by-product from the enzymatic synthesis of biodiesel. Thelipase activity was lower when compared to the literature butresults were very interesting, since it was shown that theexcess of raw glycerol obtained from biodiesel process canbe used for lipase production, which has potential applica-tion in the enzymatic biodiesel synthesis and other fields.

Immanuel et al. (2008) investigated the production ofextracellular lipase in submerged fermentation of Serratiarubidaea. The tryptone was replaced by low-cost equiv-alents such as yeast extract, skim milk power, casein,

protease, peptone, beef extract, and urea. The carbonsources tested on lipase production were sucrose, fructose,lactose, galactose, and starch. The effect of surfactants asinducers of lipase production was also evaluated usingTween 20, Tween 80, polyethylene glycol 300, and Triton100. The triglycerides tested were olive oil, coconut oil,gingelly oil, tributyrin, and cod olive oil. Casein, starch,Tween 20, and gingelly oil were the most suitable nitrogensources, carbon sources, surfactant, and lipids, respectively.

Yan and Yan (2008) tested a combination of differentexperimental designs to optimize the production conditionsof cell-bound lipase from Geotrichum sp. A single factorialdesign showed that the most suitable carbon source was amixture of olive oil and citric acid and the most suitablenitrogen source was a mixture of corn steep liquor andNH4NO3. Burkert et al. (2004) studied the effects of carbonsource (soybean oil, olive oil, and glucose) and nitrogensource concentrations (corn steep liquor and NH4NO3) onlipase production by Geotrichum sp. using the methodologyof response surface reaching a lipase activity of 20 U mL−1.

D’Annibale et al. (2006b) evaluated the suitability ofOMW as a growth medium for lipase production in SmFusing Penicillium citrinum NRRL 1841, a versatile strainable to produce lipase on different kinds of OMW. Lipaseproduction by P. citrinum in OMW-based media wassignificantly stimulated by nitrogen addition, with ammo-nium chloride proving to be the most effective source. Incontrast, the addition of vegetable oils (olive, corn, andsoybean oil) did not significantly affect lipase production.Bapiraju et al. (2005) optimized the lipase production bythe mutant strain Rhizopus sp. BTNT-2 in SmF. Theoptimum substrates for lipase production were potato starchas a carbon source, corn steep liquor as a nitrogen source,and olive oil as lipid source.

Production Processes

Fermentative processes have been conducted in batch,repeated-batch, fed-batch, and continuous mode. The modeof operation is, to a large extent, dictated by the charac-teristics of the product of interest. This section will considerrecent applications of batch, repeated-batch, fed-batch,and continuous processes to lipase production in SmF andSSF. Table 2 summarizes the different forms of production,processes, and bioreactor configurations employed in thelast 10 years for lipase production both in SmF and SSF.

Batch Processes

Most papers reporting lipase production use batch mode inshaken flasks. However, there are a considerable number ofstudies focusing on the use of bubble, airlift, and stirring

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bioreactors. Main characteristics and application of thesebioreactors will be reviewed in this section, as the use offlasks in batch mode for lipase production has already beenpresented in previous sections of this work.

Kar et al. (2008) investigated the influence of extracel-lular factors (namely, methyl oleate dispersion in the broth,dissolved oxygen variations, and pH fluctuations) on lipaseproduction by Y. lipolytica in a 20-L batch bioreactor indifferent scale-down apparatus, which have been designedto mimic the environmental behavior of each factor inlaboratory conditions. These systems allow reproducing thehydrodynamic phenomena encountered in large-scaleequipment for the three specified factors on microbialgrowth, extracellular lipase production, and the induction ofthe gene LIP2 encoding for the main lipase of Y. lipolytica.Among the set of environmental factors investigated, thedissolved oxygen fluctuations generated in a controlledscale-down reactor has led to the more pronouncedphysiological effect, decreasing the LIP2 gene expressionlevel. The other environmental factors observed in apartitioned scale-down reactor, i.e., methyl oleate disper-sion and pH fluctuations, led to a less severe stressinterpreted only by a decrease in microbial yield and hencein the extracellular lipase-specific production rate.

Potumarthi et al. (2008) studied the influence of mediaand process parameters (aeration and agitation) on fer-mentation broth rheology and biomass formation in 1.5-Lstirred tank reactor for lipase production using R. mucila-ginosa MTCC 8737. A maximum lipase activity of 72 UmL−1 was obtained during 96 h of fermentation at 2 vvm,200 rpm, pH 7, and 25 °C. Lipase yield with respect tosubstrate, YP/S, was 25.71 U mg−1; with respect to biomass,YP/X, 10.9 U mg−1; and biomass yield on substrate, YX/S,was 2.35 mg mg−1.

Gupta et al. (2007) obtained an alkaline lipase from B.multivorans within 15 h of growth in a 14-L bioreactor. Anoverall 12-fold enhanced production (58 U mL−1) wasachieved after medium optimization. Fickers et al. (2006)reported the development of a process for the extracellularlipase secreted by Y. lipolytica. The enzyme production wascarried out in a 2,000-L bioreactor that led to a lipaseactivity of approximately 1,100 U mL−1 after 53 h offermentation. Puthli et al. (2006) investigated the fermen-tation kinetics for the synthesis of lipase by C. rugosa in abatch system in a 2-L triple impeller bioreactor. Thesestudies illustrated the influence of gas–liquid mass transfercoefficient on the cell growth and hence on the lipaseproduction. To maintain sufficient oxygen concentration forthe optimum cell growth and lipase activity, fermentationhas been carried out at 600 rpm and at different aerationrates. Gas flow rate of 50.34 cm3 s−1 yielded optimumproduction of lipase. He and Tan (2006) obtained amaximum lipase activity of 9,600 U mL−1 in a 5-Lbioreactor with Candida sp.

Burkert et al. (2005) compared the lipase production byGeotrichum candidum in both 3-L stirred tank and airliftbioreactors. In the stirred reactor, the optimum conditionsof agitation and aeration for lipase production were300 rpm and 1 vvm, leading to an activity of 20 U mL−1

in 54 h of fermentation. For the airlift bioreactor, the bestaeration condition was 2.5 vvm, which yielded similarlipase activity after 30 h of fermentation. In the absence ofmechanical agitation, lipase yields around 20 U mL−1 wereachieved in a shorter time, resulting in a productivity about60% higher compared to that obtained in the stirred reactor.D’Annibale et al. (2006b) investigated the lipase productionin shaken flasks, in a stirred tank (3 L), and in a bubblecolumn reactor (3 L). The lipase production was 1,230,

Table 2 Process modes and main bioreactor configuration employed for lipase production in the last 10 years

Process conduction Bioreactor References

Submerged fermentation

Batch STR Gupta et al. (2007), Burkert et al. (2005), Montesinos et al. (1997), Potumarthi et al. (2008), Coset al. (2005), Alonso et al. (2005), Gordillo et al. (1998a), Kar et al. (2008), Becker and Markl(2000), D’Annibale et al. (2006b), Puthli et al. (2006), Fickers et al. (2006), Zhao et al. (2008)

Airlift Burkert et al. (2005)

Bubble column D’Annibale et al. (2006a), Ghadge et al. (2005)

Repeated-batch STR Benjamin and Pandey (1997), Li et al. (2005), Yang et al. (2005)

Fed-batch STR Boareto et al. (2007), Montesinos et al. (1997), Gordillo et al. (1998a), Cos et al. (2005), Gordilloet al. (1998b), Ito et al. (2001), Surribas et al. (2007), Kim and Hou (2006), Ikeda et al. (2004)

Continuous STR Montesinos et al. (1997, 2003), Jensen et al. (2002), Gordillo et al. (1998a), Becker and Markl (2000)

Solid-state fermentation

Batch Packed-bed Cavalcanti et al. (2005), Benjamin and Pandey (2001), Diaz et al. (2006), Martinez-Ruiz et al. (2008)

Tray Mala et al. (2007)

STR stirred tank reactor

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735, and 430 U L−1 for shaken flasks, stirred reactor andbubble column, respectively.

Alonso et al. (2005) studied the lipase production in a2-L stirred tank reactor at different agitation speeds and airflow rates. The most pronounced effect of oxygen on lipaseproduction was determined by stirring rate. A maximumlipase activity was detected in the late stationary phase at200 rpm and air flow rate of 0.8 vvm, when the lipid sourcehad been fully consumed. Higher stirring rates resulted inmechanical and/or oxidative stress, while lower speedsseemed to limit oxygen levels. An increase in the availabilityof oxygen at higher air flow rates led to faster lipid uptake andanticipation of enzyme release in culture medium.

The same trend verified for lipase production in sub-merged fermentation is valid for solid-state fermentation:most of the works are reporting the lipase production in traybioreactors (conical flasks), using a few grams of substrate(Kempka et al. 2008; Vargas et al. 2008; Dutra et al. 2008;Alkan et al. 2007; Azeredo et al. 2007; Falony et al. 2006).Different from SmF, where there are variations in bioreactorconfiguration, SSF is mostly restricted to a packed-bedconfiguration. We have not found reports focusing on theuse of a rotating drum, intermittently agitated, or fluidizedbioreactor.

Cavalcanti et al. (2005) used a 30-g packed-bed bio-reactor to improve productivity and scaling-up of lipaseproduction using P. simplicissimum in solid-state fermenta-tion. The influence of temperature and air flow rate onenzyme production was assessed by statistical experimentaldesign, and an empirical model was fitted to experimentaldata. Higher lipase activities could be achieved at lowertemperature levels and higher air flow rate values. Amaximum lipase activity (26.4 U gds−1) was obtained at27 °C at an air flow rate of 0.8 L min−1. Diaz et al. (2006)reported the lipase production in a 50-g packed-bedbioreactor at 40 °C and 50 mL min−1 of air. A maximumlipase activity of 1,500 U gds−1 was achieved after 12 h offermentation. Mala et al. (2007) reported the lipaseproduction in a 1-kg tray bioreactor. The production waslower when compared to that obtained in a 100-g traybioreactor.

Repeated-Batch Processes

The repeated-batch processes combines the advantages offed-batch and batch processes, mainly making possible toconduct the process by long periods and improving theproductivity compared to the batch process.

The work of Yang et al. (2005) investigated the lipaseproduction by immobilized mycelium from R. arrhizus insubmerged fermentation using repeated-batch fermenta-tions. The time to replace the volume, the volume of thereplaced medium, and the optimal composition of the

medium were optimized. Immobilized cells showed highstability for repeated use. Nine repeated batches werecarried out in flasks for 140 h and six repeated batches ina 5-L bioreactor. The lipase productivity increased from3.1 U mL−1 h−1 in batch fermentation to 17.6 U mL−1 h−1

in repeated-batch fermentation.Li et al. (2005) used repeated fed-batch strategy to

produce lipase from Acinetobacter radioresistens. A con-stant cell concentration was shown to be a pre-requisite toextend the number of repeated cycles, and adequate cellgrowth rate was critical for obtaining high lipase yield. Inthe previous cited work, the authors also verified that thepH control presented a high influence on lipase production.Dissolved oxygen constant feeding, on the other hand,could be manipulated to allow adequate growth rate forefficient lipase production. The lipase productivity reached42,000 U h−1 in a 2.5-L bioreactor. Benjamin and Pandey(1997) carried out experiments in batch and repeated-batch(fed-batch type) for lipase production using immobilized C.rugosa cells in packed-bed bioreactor. A maximum enzymeactivity (17.9 U mL−1) was obtained when the fermentationwas carried out in repeated-batch mode using a feedmedium containing arabic gum and caprylic acid, keepingthe flow rate of the feed at 0.4 mL min−1 and allowing eachcycle to run for 12 h.

Fed-Batch Processes

The fed-batch processes are characterized by the addition ofone or more nutrients to the bioreactor during the process,maintaining the products inside the bioreactor until the finalof fermentation. The fed-batch processes are amplyemployed to minimize the effects of the cell metabolismcontrol and, mainly, prevent the inhibition by substrate ormetabolic products.

Zhao et al. (2008) scaled up from 5- to 800-L fed-batchbioreactors for the high cell density fermentation of C.rugosa lipase in the constitutive Pichia pastoris expressionsystem. The fermentation conditions for both lab and pilotscale were optimized. The exponential feeding combinedwith pH control succeeded in small-scale studies, while atwo-stage fermentation strategy, which shifted at 48 h byfine tuning the culture temperature and pH, was consideredeffective in pilot-scale fermentation. A lipase activity ofapproximately 14,000 U mL−1 and a cell wet weight of ca.500 g L−1 at the 800-L scale were obtained.

Surribas et al. (2007) compared different fed-batchcultivation strategies for the production of Rhizopus oryzaelipase from P. pastoris in a 20-L bioreactor. Severaldrawbacks have been found using a methanol non-limitedfed-batch. Oxygen limitation appeared at early cell dryweight and high cell death was observed. A temperaturelimited fed-batch has been proposed to solve both prob-

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lems. However, a methanol non-limited fed-batch resultedin better productivities. Finally, a medium with low con-centration of salt was used to overcome cell deathproblems. A temperature-limited fed-batch was appliedthereafter to solve oxygen transfer limitations. This com-bined strategy resulted in lower productivities whencompared to a methanol non-limited fed-batch. However,the cultivation was extended for a longer time, and a 1.3-fold purer final product was obtained mainly due to celldeath reduction.

Kim and Hou (2006) cultivated C. cylindracea NRRLY-17506 to produce extracellular lipase from oleic acid as acarbon source. The highest lipase activity obtained in flaskculture was 3 U mL−1 after 48 h of fermentation. Fed-batchcultures (intermittent and stepwise feeding) were carried outin a 7.5-L bioreactor to improve cell concentration andlipase activity. For the intermittent feeding, the final cellconcentration was 52 g L−1 and the lipase activity was6.3 U mL−1 after 138.5 h of fermentation. Stepwise feedingwas carried out to simulate an exponential feeding and toinvestigate the effects of specific growth rate on cell growthand lipase production. The highest final cell concentrationobtained was 90 g L−1 when the set point of specific growthrate was 0.02 h−1 and the highest lipase activity was 23.7 UmL−1 at 179.5 h. High specific growth rate decreasedextracellular lipase production in the latter part of fed-batchcultures, due to build-up of over-supplied oleic acid.

Ikeda et al. (2004) developed a fed-batch fermentationprocess to enable the production of large amounts ofrecombinant human lysosomal acid lipase in Schizosac-charomyces. A feedback fed-batch system (5 L bioreactor)was used to determine the optimal feed rate of a 50%glucose solution used as carbon source. At the time of theinitial consumption of glucose in the batch-phase culture,the nutrient supply was automatically initiated by means ofmonitoring the respiratory rate change. The obtained profileof the feed rate was applied to the feed forward controlfermentation. Finally, the cells were grown up to 50 g drycell weight and the lipase expression was 16 U mL−1. Ito etal. (2001) investigated the lipase production by a two-stepfed-batch culture (2 L bioreactor) of an organic solvent-tolerant bacterium. The two-step lipase production com-prising a growth phase in fed-batch mode and a productionphase, in which lipase was induced by the addition of 5%stearic acid, was carried out. In the growth phase, themaximum cell concentration at 16 h was 30.2 g L−1 drycell, and lipase activity was 96 U mL−1 after 35 h, which isapproximately 40 times higher than the production levelobtained in flask culture.

Gordillo et al. (1998a) studied the lipase production byC. rugosa in a 6-L bioreactor using two different fed-batchoperational strategies to maximize lipase activity: constantsubstrate feeding rate and specific growth rate control. A

constant substrate feeding rate strategy showed that amaximum lipase activity (55 U mL−1) was reached at lowsubstrate feeding rates, whereas lipase tends to accumulateinside the cell at higher rates of substrate addition. In thesecond fed-batch strategy studied, a feedback controlstrategy has been developed based on the estimation ofstate variables (cells and specific growth rate) from themeasurement of indirect variables, such as carbon dioxideevolution rate by mass spectrometry. An on–off controllerwas then used to maintain the specific growth rate at thedesired value, adjusting the substrate feeding rate. A constantspecific growth rate strategy afforded higher final lipaseactivity (117 U mL−1) at low specific growth rates. With aconstant specific growth rate strategy, lipase production byC. rugosa was enhanced 10-fold compared to a batchoperation.

Continuous Processes

Montesinos et al. (2003) investigated the production ofextra- and intracellular lipases in continuous cultures of C.rugosa using pure or carbon source mixtures. Lipaseproductivity in continuous cultures increased by 50%compared to data obtained from batch fermentation andwas dependent on the dilution rate applied. Maximumyields relative to consumed substrate were obtained witholeic acid at low dilution rates. The authors found thatduring nitrogen limitation, lipase activity was suppressed.Results obtained were compared to previous data frombatch and fed-batch cultures for the purpose of selecting thebest process strategies for the lipase production with C.rugosa. The best lipase yields were obtained in fed-batchfermentation using oleic acid.

Jensen et al. (2002) studied the production of extracel-lular enzymes by the fungus Thermomyces lanuginosus inchemostat cultures at a dilution rate of 0.08 h−1 in relationto different ammonium concentrations in the feed medium.Under steady-state conditions, three growth regimes wererecognized and the production of several enzymes from T.lanuginosus was recorded under different nutrient limita-tions ranging from nitrogen to carbon/energy limitations.The range and the production of carbohydrate hydrolyzingenzymes and lipase increased from regime I (NH4Cl≤600 mg L−1) to regime III (NH4Cl≥1,200 mg L−1), whereasproduction of protease was the highest in regime II (600 mgL−1<NH4Cl<1,200 mg L−1).

Mathematical Modeling of Lipase Production

Some basic steps are required to scale-up lipase productionprocesses. The first one has been widely discussed in theliterature and includes the choice of a suitable microorgan-

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ism and substrates for lipase production. The second step isrelated to the choice of the bioreactor configuration forthe process development and the study, at laboratoryscale, of how the manipulated variables affect theperformance. The third step deals with the developmentand validation of mathematical models as a tool forscale-up, process control, and optimization. Finally, theanalysis of technical and economical viability of the processis required. In this section, it is focused on the main aspectsregarding the mathematical models for lipase productionreported in the last 10 years, and how these models havebeen used as a tool for process scale-up or processimprovement.

Rajendran et al. (2008) proposed an unstructured kineticmodel to simulate the experimental data. The logisticmodel, the Luedeking–Piret model, and the modifiedLuedeking–Piret model were found to be suitable toefficiently predict cell mass, lipase production, and glucoseconsumption, respectively, with high correlation coefficient(R2). From the estimated values of the Luedeking–Piretmodel parameters, α and β, it was found that the lipaseproduction by C. rugosa is growth-associated.

Haider et al. (2008) modeled and optimized the lipaseproduction by a soil microorganism using artificial neuralnetwork (ANN) and genetic algorithm (GA) techniques,respectively. The ANN model, based on back propagationalgorithm, showed to be highly accurate in predicting thesystem with a correlation coefficient value close to 0.99.Optimization using GA, based on the ANN model, resultedin the following values of the media constituents: 9.991 mLL−1 oil, 0.100 g L−1 MgSO4, and 0.009 g L−1 FeSO4. Amaximum (7.69 U mL−1) of lipolytic activity at 72 h ofculture was obtained, using the ANN–GA method, whichwas found to be 8.8% higher than the maximum valuespredicted by a statistical regression-based optimizationtechnique response surface methodology.

Boareto et al. (2007) proposed an efficient hybrid neuralphenomenological model (HNM) for the lipase productionprocess by C. rugosa. The experimental data used corre-sponded to fed-batch operation with constant substrate feedrate. ANNs were trained to represent the aqueous andintracellular lipase activity and were further associated witha reduced version of the mechanistic model of the proposedHNM. When compared to experimental data, the HNMexhibited higher accuracy. The HNM can be used inprocess monitoring using on-line measurements of CO2

and substrate feed rate to infer enzyme activities and alsosubstrate and biomass concentrations.

Ghadge et al. (2005) investigated the effect of hydrody-namic flow parameters and interfacial flow parameters onthe activity of lipase in a bubble column reactor. Lipasesolution was subjected to hydrodynamic flow parameters in0.15 and 0.385 m bubble column reactors over a wide range

of superficial gas velocity. The flow parameters wereestimated using an in-house computational fluid dynamiccode based on the k-ε approach. The extent of lipasedeactivation in both columns was found to increase with anincrease in hydrodynamic and interfacial flow parameters.However, the use of the same values of the parameters, theextent of deactivation was different in the two columns.The rate of deactivation was found to follow a first-orderkinetics. An attempt was made to develop rationalcorrelations for the extent of deactivation as well as forthe deactivation constant. The rate of deactivation wasfound to be depending on the average turbulent normalstress and interfacial flow parameters, such as bubblediameter and bubble rise velocity.

Becker and Markl (2000) modeled olive oil degradationby the thermophilic lipolytic strain Bacillus thermoteovoransIHI-91 in chemostat and batch culture, to obtain a generalunderstanding of the underlying principles and limitationsof the process and to quantify its stoichiometry. Chemostatdata were successfully described using the Monod chemostatmodel extended by terms for maintenance requirementsand wall growth. Oleic acid accumulation observed duringbatch fermentation can be predicted using a modelinvolving growth-associated lipase production and oliveoil hydrolysis. Simulations confirmed that this accumu-lation was the cause for sudden growth cessationoccurring in batch fermentations with higher olive oilstarting concentrations. Further, an oscillatory behavior,as observed in some chemostat experiments, was pre-dicted using the latter model.

Gordillo et al. (1998a) developed a simple structuredmathematical model coupled with a methodology to estimatebiomass amounts, specific growth rate, and substrate amountsand applied to the production of C. rugosa lipase in batch,fed-batch, and continuous operations with a 10-fold increasein productivity relative to batch operation. Montesinos et al.(1997) proposed a simple structured mathematical modelcoupled with a methodology to estimate state variables andparameters and applied to C. rugosa batch and continuouslipase production. Process modeling was carried out usingAdvanced Continuous Simulation Language. Once modelparameters were determined, the whole model was validated,showing satisfactory results. For estimation of the beststrategy to improve lipase productivity, different simulationsof batch, fed-batch, and continuous cultures were performed.The maximum enzyme productivity was predicted incontinuous culture at moderate dilution rates and substratefeeding of 8 g L−1. However, the highest predicted lipaseactivity was reached in fed-batch cultures with a prefixedsubstrate feeding in order to maintain constant—at theiroptimal values—the relation substrate/biomass or the specificgrowth rate. The fed-batch process mode matched thesimulation results.

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Conclusions

Critical analysis of current literature shows that microbiallipases are one of the most produced enzymes. This reviewshowed that many researchers worldwide direct theiractivities to the screening of new lipase-producing micro-organisms and, subsequently, on the optimization of themedium composition and operational variables. All theseefforts are justified by the great versatility of lipase applica-tions. There has been much development on lipase productionby bioprocesses, mainly using submerged fermentation suchas the screening of high lipase producers, successful sub-stitution of synthetic medium by agroindustrial residues,scale-up of process, different process operation modes,strategies of bioreactor operation, and the use of mathematicalmodels as a tool for process optimization.

Though the screening of microorganism producers oflipases has afforded satisfactory results to the present, basedon our experience, we believe that the use of engineeringlipases will predominate in the near future since the productionof engineering lipases will allow attainment of enzymes withnew remarkable characteristics for a specific application.

The use of agroindustrial residues as substrates for lipaseproduction favors, undoubtedly, the reduction of productioncosts associated to substrates. However, systematic studiesshould be carried out to check whether the global productioncost is lowered, including the downstream step. Of course, insome cases, the difficulties imposed by the use of residues inthe purification of enzyme produced make its use unfeasible.

The fed-batch operation mode has provided the bestresults in terms of lipase activity and productivity. In allcases cited in this review, the feeding rate was notoptimized. An interesting alternative could be the use ofan optimization tool such as the dynamic optimization oroptimum control. This would allow the time domain to bepartitioned in N-subintervals for which the feeding ratecould be optimized. The application of a dynamic optimi-zation tool makes it possible to find the optimal profile offeeding rate that maximizes the production or productivity.It may be important to emphasize that the use of anoptimization tool would require a mathematical model ableto represent the process in a satisfactory and reliable way.

The results discussed in this review clearly demonstratethat SSF, as long as the cultivation volume is kept to a verysmall scale, yields good results in terms of processproductivity. However, SSF is difficult to scale-up due toexisting gradients (mainly in packed-bed bioreactor) intemperature, pH, moisture, oxygen, substrate, and inoculum.Considering the difficulties in handing the large volumebioreactors related to the lipase production by SSF, perhaps,it could be more practical to use SSF for the production atsmall scale; meanwhile, other bioreactor configurations areinvestigated, such as rotating drum, intermittently agitated,

or fluidized bed bioreactors. These configurations allow abetter uniformity of medium, decreasing considerably thegradients, though agitation can cause microorganisms death.

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