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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 # Springer Science + Business Media, LLC 2009
Abstract This review paper provides an overview regarding the main aspects of microbial lipases production. The most important microbial lipase-producing strains for submerged and solid-state fermentations are reviewed as well as the main substrates, including the use of agroindustrial residues. Current process techniques (batch, repeated-batch, fed-batch, and continuous mode) are discussed and the main bioreactors configurations are also presented. Furthermore, the present review paper shows a general overview about the development of mathematical models applied to lipase production. Finally, some future perspectives on lipase production are discussed with special emphasis on lipase engineering and the use of mathematical models as a useful tool for process improvement and control. Keywords Review . Lipase production . Substrates . Process conduction . Bioprocess modeling
Introduction Lipases (triacylglycerol acylhydrolases EC 188.8.131.52) are a class of hydrolase which catalyze the hydrolysis of
H. Treichel (*) : D. de Oliveira : M. Di Luccio : J. V. Oliveira Department of Food Engineering, URI—Campus de Erechim, P.O. Box 743, CEP 99700-000 Erechim, Rio Grande do Sul, Brazil e-mail: firstname.lastname@example.org M. A. Mazutti Faculty of Food Engineering, Department of Food Engineering, UNICAMP, P.O. Box 6121, CEP 13083-862 Campinas, São Paulo, Brazil
triglycerides to glycerol and free fatty acids over an oil– water interface. In addition, lipases catalyze the hydrolysis and transesterification of other esters as well as the synthesis of esters and exhibit enantioselective properties. The ability of lipases to perform very specific chemical transformation (biotransformation) has make them increasingly popular in the food, detergent, cosmetic, organic synthesis, and pharmaceutical 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 biocatalysts with proven potential for contributing to the multibillion dollar underexploited lipid technology bio-industry and have been used in in situ lipid metabolism and ex situ multifaceted industrial applications (Joseph et al. 2008). The number of available lipases has increased since the 1980s. This is mainly a result of the huge achievements made in the cloning and expression of enzymes from microorganisms, as well as of an increasing demand for these biocatalysts with novel and specific properties such as specificity, stability, pH, and temperature (Bornscheuer et al. 2002; Menoncin et al. 2009). Lipases are produced by animals, plants, and microorganisms. Microbial lipases have gained special industrial attention due to their stability, selectivity, and broad substrate specificity (Dutra et al. 2008; Griebeler et al. 2009). Many microorganisms are known as potential producers of extracellular lipases, including bacteria, yeast, and fungi (Abada 2008). Fungal species are preferably cultivated in solid-state fermentation (SSF), while bacteria and yeast are cultivated in submerged fermentation (SmF; Dutra et al. 2008). The importance of lipases can be observed by the great number of published articles recently. In fact, over the last few years, there has been a progressive increase in the
Wolski et al. (2008) studied the lipase production by Penicillium simplicissimum and obtained an activity of 30 U gds−1. An agar plate medium containing bile salts and olive oil emulsion was employed for isolating and growing fungi in primary screening assay. respectively (Diaz et al. . (2008) reported the use of response surface methodology to optimize the lipase production by submerged fermentation using immobilized biomass of a newly isolated Penicillium sp. The industrial demand for new sources of lipases with different catalytic characteristics stimulates the isolation and selection of new strains. Cihangir and Sarikaya (2004) isolated a strain of Aspergillus sp. (2006) isolated 59 lipase-producing fungal strains from Brazilian savanna soil using enrichment culture techniques. Eleven strains were considered and. simplicissimum were isolated from the babassu oil industry. ionic liquids.. vegetable oil processing factories. and the optimum activity was 29 U mL−1. (2008) investigated the lipase production by Penicillium verrucosum and the optimum activity was about 40 U gram of dry substrate−1 (gds). Teng and Xu (2008) investigated the lipase production by Rhizopus chinensis and obtained. The main microorganisms used before 2004 have already been well described in the reports of Gupta et al.. by SSF and SmF. Kempka et al. cultivation conditions. Microbial Lipases Sources Lipases are ubiquitous in nature and are produced by several plants. Elitol and Ozer (2000) immobilized the whole cell of R. high cell concentration in the reactor could be achieved and the separation of biomass from the medium is favored (Elitol and Ozer 2000). Lipases of microbial origin represent the most widely used class of enzymes in biotechnological applications and organic chemistry. among them. Ellaiah et al. and microorganisms. (2006) studied the production of an extracellular lipase from Aspergillus carneus and obtained a maximum activity of 13 U mL−1. The immobilization is advantageous since it can avoid biomass washout at high dilution rates. the composition of the growth medium. (2007) obtained lipase activities of 17 U gds−1 and 12 U mL−1 for SSF and SmF. Vargas et al. Also. (2004) used the whole immobilized biomass of Aspergillus niger to produce lipase and obtained similar activities for both free and immobilized biomass cultivations (4 U mL−1). Geotrichum sp. a maximum lipase activity of 14 U mL−1. changing the process from batch to repeated-batch mode. Bapiraju et al. Twentyone strains were selected by the ratio of the lipolytic halo radius and the colony radius. 2001). Penicillium sp. Azeredo et al. verrucosum and P. animals.Food Bioprocess Technol number of publications related to industrial applications of lipase-catalyzed reactions. However.. (2005) studied the repeated-batch lipase production by immobilized mycelium of Rhizopus arrhizus in submerged fermentation. respectively. Yang et al. dairy plants. arrhizus and the rate of lipase production was constant through several repeatedbatch experiments.. In SmF. The lipase productivity increased from 3 to 18 U mL−1 h−1. For example.. Both P. In another work.500 U gds−1 and 50 U mL−1. At the optimized experimental conditions. the main terrestrial species of yeasts that were found to produce lipases are: Candida rugosa. Filamentous Fungi Some of the most commercially important lipase-producing fungi are recognized as belonging to the genera Rhizopus sp. Colen et al. substrates. Mucor sp. from soil samples from the different regions of Turkey and obtained an expressive activity of 17 U mL−1. and process operations used in this specific field. In SSF. The present review is focused on lipase production discussing the main microorganisms. extracellular lipases were obtained using Rhizopus homothallicus with lipase activities of 1. (2005) optimized the lipase production by the mutant strain of Rhizopus sp. A review of the most recent (from 2004 to the present) potential microorganisms for lipase production in both submerged and solid-state fermentations are reported in Table 1. Candida antarctica. and Rhizomucor sp. or even in non-conventional solvents. pH. Yeast According to Vakhlu and Kour (2006). Candida tropicalis. by cultivation of Penicillium restrictum. some works reporting the use of immobilized whole biomass of filamentous fungi have also been published. and the kind of carbon and nitrogen sources (Cihangir and Sarikaya 2004). Lipase production by filamentous fungi varies according to the strain. the strain identified as Colletotrichum gloesporioides was the most productive. some qualitative information presented in the literature can be of interest. higher than the activity obtained by the same microorganism before immobilization. 2006). Lipase-producing microorganisms have been found in different habitats such as industrial wastes. Aspergillus sp. performed in common organic solvents. and soil contaminated with oil and oilseeds among others (Sharma et al. (2004) and Sharma et al. (2001). the authors reached a lipase activity around 21 U mL−1. Kaushik et al. temperature. Recently. A quantitative comparison between SmF and SSF is difficult due to the difference in the methods used for determining the lipase activity. at the optimized experimental conditions.
(2005) Kiran et al. Amaral et al. Trichosporon sp. (2000). (2005). (2008). (2007) Colen et al. Mala et al. Fickers et al.. (2005) Teng et al. Zhao et al. (2007) Immanuel et al.. Saccharomycopsis crataegenesis. Nawani and Kaur (2007) Dominguez et al. (2008) Lopes et al. Burkert et al. (2006) Bapiraju et al. Yarrowia lipolytica. Azeredo et al. Cavalcanti et al. (2009). (2007). (2007) Solid-state fermentation Sun and Xu (2008) Diaz et al. (2005) Kaushik et al. Falony et al. (2008) Kim and Hou (2006). (2008) Carvalho et al.Food Bioprocess Technol Table 1 Microorganisms cited in the recent literature as potential lipase producers Microorganism Source Reference Submerged fermentation Rhizopus arrhizus Rhizopus chinensis Aspergillus sp. Shariff et al. (2006) Li et al. Torulaspora globosa. 2007 Takaç and Marul (2008) Candida cylindracea. (2008). Pichia sivicola. Aspergillus niger Rhizopus oryzae Colletotrichum gloesporioides Rhodotorula mucilaginosa Yarrowia lipolytica Candida utilis Trichosporon asahii Candida rugosa Candida cylindracea Candida sp. (2006). Candida valida. Rhodotorula glutinis. (2006) Palma et al. (2006) D’Annibale et al. (2008) Dutra et al. (2007). Boareto et al. (2008) Ciafardini et al. Rhodotorula pilimornae. (2008) Yan and Yan (2008). lipolytica have been cloned and . (2007) Vargas et al. Candida deformans. (2007) Grbavcic et al. Yang et al. Bacillus coagulans Bacillus subtilis Fungal Fungal Fungal Fungal Fungal Fungal Fungal Fungal Fungal Fungal Fungal Fungal Fungal Fungal Fungal Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Yeast Bacterial Bacterial Bacterial Bacterial Bacterial Bacterial Bacterial Bacterial Bacterial Bacterial Bacterial Bacterial Pinheiro et al. and Y. and Trichosporon asteroids. (2005). (2006) Potumarthi et al. D’Annibale et al. (2004) Burkert et al. (2007) Kumar and Gupta (2008) Rajendran et al. Geotrichum candidum Aspergillus carneus Rhizopus sp. (2005) Tan and Yin (2005). (2007). (2009). (2006) Ciafardini et al.. Pichia bispora. (2008) Abada (2008) Gupta et al. Surribas et al. (2006b) He and Tan (2006) Liu et al. The genes that encode lipase in Candida sp. Geotrichum sp. (2006b) Azeredo et al. (2007) Alkan et al. Pichia xylosa. (2007). (2008). (2008). (2008) Ertugrul et al. Teng and Xu (2008) Cihangir and Sarikaya (2004) Diaz et al. Pichia mexicana. (2008) Martinez-Ruiz et al. Wang et al. Aureobasidium pullulans Saccharomyces cerevisiae Williopsis californica Acinetobacter radioresistens Pseudomonas sp. (2003) Benjamin and Pandey (2001) Mahanta et al. (2006). Alonso et al. (2005) Kempka et al. (2008) Volpato et al. Kar et al. (2008) Ruchi et al. (2006) Cos et al. Pseudomonas aeruginosa Staphylococcus caseolyticus ‘Biopetro-4’ Bacillus stearothermophilus Burkholderia cepacia Burkholderia multivorans Serratia rubidaea Bacillus sp. Puthli et al. Candida parapsilopsis. (2008) Fernandes et al. (2008). Rhizopus homothallicus Penicillium citrinum Penicillium restrictum Penicillium simplicissimum Penicillium verrucosum Geotrichum sp. Pichia burtonii. Candida curvata.
Alkan et al. Bacillus subtilis. After the isolation of the colonies. cylindracea. and Candida boidinii. was selected towards producing the largest opaque halo. aeruginosa PseA strain. Among the microorganisms that were isolated from this oil..9). Bacillus sp. Active colonies were re-streaked on TBA agar for purification. After investigation of several inducers on lipase activity. (2003) reached a maximum lipase activity of 8. capable of conditioning the physicochemical and organoleptic characteristics of the oil. The most effective strain for lipase production was identified as Rhodotorula mucilaginosa (MTCC 8737) by its phenotypic characteristics. Shariff et al. After the medium optimization. There are few exceptions for bacteria grown in SSF. In contrast to the high activities reached in the above-mentioned works. Kumar and Gupta (2008) isolated 15 yeasts from petroleum and oil sludge areas in Delphi (India). and the operational conditions. this strain was selected for further studies. Takaç and Marul (2008) isolated microbial cultures from soil enriched by periodic sub-culturing of samples in nutrient broth containing 1% (v/v) tributyrin. Ertugrul et al. Mahanta et al. the most promising strain was C. and Bacillus alcalophilus are the most common bacterial lipases. and extracellular in W. Then. Bacteria Among bacterial lipases being exploited. After screening in tributyrin agar medium. D’Annibale et al. However. Burkholderia multivorans. cerevisiae was noted to be intracellular. Bacillus sp. Candida sp. based on the largest halo of lipolysis. The isolation process was performed by serial dilution samples on tributyrin agar (TBA) plates. Bacillus licheniformis. The colonies that showed the largest hydrolysis halos zone were selected. those from Bacillus exhibit interesting properties that make them potential candidates for biotechnological applications. Abada (2008) produced lipase from a strain of B. coagulans and obtained a . After optimization. Bacillus coagulans. Ciafardini et al. (2006b) investigated the valorization of OMW by its use as a possible growth medium for the microbial production of extracellular lipase. Bacillus stearothermophilus. there are several recent publications reporting the production of lipases by other yeasts. a maximum lipase activity was found as 750 U mL−1. Williopsis californica. Bacillus pumilus. rugosa. The isolates were purified and checked for their lipolytic potential.300 U mL−1. An extracellular thermostable lipase activity was detected through plate and broth assays at 70 °C after 28 h of fermentation. Burkholderia cepacia. the strain Pseudomonas MSI057 exhibited large clean zones around the colonies. of which 37% produced a clear halo around the colonies on tributyrin agar plates for lipase production. (2006) have discovered that freshly produced olive oil is contaminated by a rich micro-flora. through the production of enzymes. Malaysia. and after optimization.8 U mL−1 by C. Particularly. was identified as the best lipase producer. the authors reported the optimum lipase activity as 6. Although lipases from C. this strain was found to belong to Trichosporon asahii genus and share 99% homology with the already existing database. is the most potential lipase producer from yeasts reported in the literature. thus showing that lipase activity values are highly influenced by the microorganism. and Staphylococcus caseolyticus are also reported as bacterial lipase producers (Table 1). respectively. the maximum value obtained was 1. strain L2 from a hot spring in Perak.230 and 9. Potumarthi et al. several strains of yeasts were identified as Saccharomyces cerevisiae. (2008) isolated 57 heterotrophic bacteria from the marine sponge Dendrodoris nigra. antarctica have been extensively used in different fields. In most cases. due to the high water activity required by the microorganisms (higher than 0. (2008) collected marine soil samples near an oil extraction platform in the Arabian Sea. 99-125. stearothermophilus AB-1 isolated from air and obtained a maximum lipase activity of 1. The three-phase olive oil extraction process generates a dark-colored effluent. the intracellular activity found was 168 U mL−1. Kiran et al.585 U mL−1 in 48 h of fermentation. californica. (2007) isolated 17 bacterial strains that could grow on media based on OMW and selected the most promising strain for lipase production.675 U mL−1 after 120 h of fermentation. usually termed olive oil mill wastewater (OMW). Among these yeast strains. In addition.084 U gds−1 using a solvent tolerant P. Tan et al. a strain of Bacillus sp. (2007) isolated a thermophilic bacterium. substrates. (2007) produced an extracellular lipase by B. rugosa and C. californica showed good potential to produce lipase. of which S. 2007). the production is normally high. Carvalho et al. when the bacteria are well adapted to this solid medium. (2008) isolated a bacterium strain from petroleum-contaminated soil and codified as Biopetro-4. On the basis of sequence homology. cerevisiae and W. In a 30-L bioreactor. Candida wickerhamii. He and Tan (2006) used the response surface methodology to optimize culture medium for lipase production by the strain Candida sp. (2008) obtained a maximum lipase activity of 1. (2008) reported the optimum lipase activity of 3. Rajendran et al.Food Bioprocess Technol over-expressed (Wang et al. Among the 12 strains tested. The lipase activity in S.600 U mL−1 in shaken flasks and in a 5-L bioreactor. they were transferred to plates containing 2% tributyrin and incubated at 35 °C for 3–4 days. as shown in Table 1. bacteria are preferably cultivated in SmF. Pseudomonas aeruginosa. one strain was selected for further studies. Pseudomonas sp.
A mix of these two kinds of media can also be used for the purpose of lipase production. since it does not explain the interaction effects among variables and their effects on the fermentation process (Haaland 1989. Takaç and Marul (2008) improved the lipase production by B. These enzymes are generally produced in the presence of a lipid such as oil or any other inducer.0. 2004. and dissolved oxygen. the effect of nine factors. fatty acids. while keeping others constant was found to be inefficient. and yeast extract concentrations. yeast extract.1. oils. and FeCl3⋅6H2O were found to have more significant influence on lipase production by C. After the screening of the most significant factors by the PB design. One percent of sesame oil afforded the highest activity with 80% and 98% enhancements with respect to 1% concentrations of linoleic acid and triolein as the favored fatty acid and triglyceride. An alkaline lipase from B. Rodrigues and Iemma 2005). NH4Cl. The authors observed that the use of xylose. divided by the kind of medium used. The optimized medium for improved lipase activity consisted of peptone. subtilis using different concentrations of lipidic carbon sources such as vegetable oils. namely. Fernandes et al. These components were varied in the basal medium containing olive oil as inducer and yeast extract as nitrogen source. and CaCl2 0. olive oil. Tweens. and fermentation time. Khuri and Cornell 1987. The major factor for the expression of lipase activity has always been reported as the carbon source. fatty acids. and MgSO4⋅7H2O. (2008) optimized the fermentation medium for lipase production by R.Food Bioprocess Technol maximum lipase activity of 149 U gds−1 after 24 h of fermentation. (2007) obtained a maximum lipase activity of 108 U gds−1 after 72 h of fermentation by B. pH. alanine. Sharma et al. the use of Tween 80 as inductor inhibited the lipase production. and triglycerides. All variables significantly influenced lipase production. The effect of 12 medium components was studied in 16 experimental trials. chinensis. K2HPO4. trace metals. On the other hand. Synthetic Medium Generally. Initially. dextran. K2HPO4. malt extract media. 2007). respectively.36. A Plackett–Burman design for seven independent variables (glucose. olive oil. and . Response surface methodology indicated that the requirement of malt extract and yeast extract varied with the type of lipase inducer used. MgCl2. olive oil. such as triacylglycerols. and glycerol (Gupta et al. and complex components such as peptone. Rajendran et al. inoculum density. Lipidic carbon sources seem to be essential for obtaining a high lipase yield. nitrogen sources and essential micronutrients should also be carefully considered for growth and production optimization. cepacia. and CaCl2 and inoculum density were studied using the Plackett–Burman experimental design. In order to improve the productivity of lipase.01. Optimization of the concentration of each compound that constitutes a cultivation medium is usually a time-consuming procedure. Glucose. The use of high malt and yeast extract concentrations favored lipase production when corn oil was used as substrate. apart from glucose and CaCl2 concentrations.4 mM (Gupta et al. the optimization was carried out in terms of the olive oil. K2HPO4 0.5. Wang et al. and CaCl2 concentrations and time) was applied to select the most significant factor. multivorans was produced after 15 h of cultivation in a 14-L bioreactor. 2001). high productivity has been achieved by culture medium optimization. (2008) used the Plackett–Burman statistical experimental design to evaluate the fermentation medium components. concentrations of glucose. maltose. tryptophan. asahii by both one variable at a time and statistical approach. stearothermophilus AB-1. and also agroindustrial residues containing all the components necessary for microorganism development. The optimal medium composition for the lipase production was determined to be (percent w/v): glucose 0. malt extract. Substrates for Lipase Production Microbial lipases are mostly extracellular and their production is greatly influenced by medium composition besides physicochemical factors such as temperature. olive oil. The classical practice of changing one variable at a time. Conversely. However. rugosa. glucose. yeast extract 0. hydrolysable esters. NH4Cl 0. An efficient and widely used approach is the application of Plackett–Burman (PB) designs that allow efficient screening of key variables for further optimization in a rational way (Rodrigues and Iemma 2005). bile salts. yeast extract. The same authors tested the use of glucose as carbon source and verified that it presented an impressive effect on lipase production. peptone. olive oil 3. during kerosene induction both malt and yeast extracts requirements were minimal. These nutritional requirements for microbial growth are fulfilled by several alternative media as those based on defined compounds (synthetic medium) like sugars. The medium optimization was carried out to lead to an increase of 12-fold in lipase production. the effects of oils and oil-related substrates were assessed by orthogonal test and response surface methodology. Kumar and Gupta (2008) compared the medium optimization for the yeast T. MgCl2 0. phenylalanine. Abada (2008) produced lipase from B. MgCl2.1. The main studies available in the literature since 2005 covering these subjects are presented below. since lipases are inducible enzymes.
58-fold. maltose. . The lipase fermentation in a 5-L vessel reached 9. NH4Cl. He and Tan (2006) used the response surface methodology to optimize the culture medium for lipase production with Candida sp. NaNO3. and NaCl) were assessed by a Plackett–Burman design. KH2PO4. Soybean meal without supplements appears to be the best medium of those tested for lipase production. glucose. MgSO4. 99-125. KH2PO4. watermelon waste. K2HPO4. castor oil. which was enriched with 0. The addition of several vegetable oils (castor. olive. sugar cane molasses. (2008) carried out media optimization through response surface methodology for cost-effective production of lipase by P. (2006) investigated the influence of different culture conditions. The strain of A. The choice of the substrate depends upon several factors. glucose) and nitrogen (peptone. The activity reached was 75 U gram of inert support−1. polyvinyl alcohol. carneus. soybean. a Plackett– Burman design was used to evaluate the effects of different components in the culture medium (soybean oil. simplicissimum using soybean meal as substrate supplemented with low-cost substrates: soybean oil. niger was cultivated in SSF using wheat bran as support. The use of pure synthetic medium in solid-state fermentation has been hardly presented in the literature. nitrogen. The effects of 11 media components (peptone. MgSO4.1% thiamine provided the best results. Dutra et al. six mineral sources. and K2HPO4 concentrations have a significant influence on lipase production. A maximum lipase activity was obtained using 2% of castor oil. (2008) monitored the biomass growth of A. corn steep liquor. NH4Cl. yielding an activity of 4. peptone. Soybean oil. glucose. After optimization. Results were optimized using central composite designs and response surface analysis. respectively. wastewater from a slaughterhouse (rich on oil and fat). pH. xylose. Martinez-Ruiz et al. Mala et al. which otherwise might be caused by their disposal. MgSO4⋅7H2O. yeast extract. soybean meal. and palm oil) was investigated to enhance lipase production. For example.230 U mL−1 in a shaken flask system. soybean meal. Alkan et al. and an oligoelement solution. research on the selection of suitable substrates for fermentative process has mainly been focused on agroindustrial residues.600 U mL−1. Kempka et al. 14 nitrogen sources. in which emphasis is given to the application of agricultural by-products for the production of fine chemicals and enzymes. mainly related to cost and availability. The nature of the substrate is the most important factor affecting fermentative processes. chinensis in SSF.580 U mL−1. K2HPO4. olive oil.8-fold increase in production was reported at optimized conditions. and incubation period) and a 1.Food Bioprocess Technol potassium nitrate as supplement lead to the highest lipase production. and peptone. (2008) investigated the lipase production by P. the activity reached values of 976 U gds−1. The lipase production reached 54 U mL−1 after 17 days of incubation. aeruginosa. glycerol. mineral sources. and vitamins on the production of lipase by Antrodia cinnamomea in submerged cultures. olive oil. carbon. Jatropha seed cake without supplementation showed a lipase activity of 625 U gds−1. Kaushik et al. The best results were obtained using solid waste from melon supplemented with NH4NO3 and 1% olive oil. Vargas et al. lactose. lentil husk. and the results showed that addition of gingelly oil cake to wheat bran increased the lipase activity by 36% and the activity was 384 U gds−1. coagulans. niger in SSF for lipase production using the digital image processing technique. yeast extract.084 U gds−1. Many reports of SSF have been recently published.5% sodium nitrate. (2008) produced lipase from P. corn steep liquor. and yeast hydrolyzed. Lin et al. agitation rate. (2007) developed a SSF for lipase production by A. arabic gum.000 to 6. 0. Interactions were evaluated for five different variables (sunflower oil. corn. banana waste. rice husk. Sun and Xu (2008) reported that a combined substrate of wheat flour with wheat bran supported both good biomass and enzyme production by R. verrucosum by SSF using soybean meal. sodium chloride. and five vitamins were investigated. niger MTCC 2594 using wheat bran and gingelly oil cake as substrates and supplement. including lipases. NaNO3) sources. due to their potential advantages. Nine carbon sources. and melon waste) on lipase production by SSF using B. (2008) reported the lipase production by SSF with P. soybean oil. and yeast extract) optimized by the response surface methodology. while with NaNO3 the activity was 1. aeruginosa PseA using Jatropha seed cake supplemented with different carbon (starch. corn oil. tryptone. yeast hydrolyzed. Soybean meal was the best substrate. Thus. tryptone. The optimized condition allowed lipase production to be increased from 5. Agroindustrial Residues Over the recent years. When supplemented with maltose. MgSO4. and Spam 60). temperature. Mahanta et al.91% of ammonium sulfate. and 0. process optimization may involve the screening of several agroindustrial residues. (2006) used the response surface approach to investigate the production of an extracellular lipase from A. (NH4)2SO4. the lipase production was increased 5. (2007) studied the effect of several agroindustrial residues (wheat bran. Utilization of agroindustrial wastes provides alternative substrates and may help solving pollution problems. (2008) reported the production of lipase from Rhizopus sp. The authors found that 5% glycerol. and the most significant factors (arabic gum. Ruchi et al. In the first step. using perlite (as inert support) supplemented with urea.
The use of olive oil led to higher lipase activities. (2004) studied the effects of carbon source (soybean oil. and stirring . NaCl. verrucosum in submerged fermentation using a medium based on peptone. A maximum lipase activity was verified using 1% of molasses. starch. Na2HPO4. NaCl. galactose. yeast extract. Tween 20. KH2PO4. to a large extent. Batch Processes Most papers reporting lipase production use batch mode in shaken flasks. caseolyticus strain EX17 growing on raw glycerol. The tryptone was replaced by low-cost equivalents such as yeast extract. (2005) investigated the lipase production by SSF in fixed-bed bioreactor using babassu cake supplemented with sugar cane molasses. Cavalcanti et al. Pinheiro et al. and cod olive oil. and continuous mode. and glucose) and nitrogen source concentrations (corn steep liquor and NH4NO3) on lipase production by Geotrichum sp. Small trays containing 10 g of ground babassu cake as the basal medium were supplemented with different carbon sources (babassu oil. BTNT-2 in SmF. and glucose). using the methodology of response surface reaching a lipase activity of 20 U mL−1. and continuous processes to lipase production in SmF and SSF. The lipase activity was lower when compared to the literature but results were very interesting. supplemented with olive oil. which was obtained as a by-product from the enzymatic synthesis of biodiesel. dictated by the characteristics of the product of interest. and olive oil and an industrial medium composed of corn steep liquor. Lipase production by P. coconut oil. and gingelly oil were the most suitable nitrogen sources. MgSO4⋅7H2O. Casein. (2008) investigated the lipase production by P. protease. (NH4)2SO4. and urea. (2008) used the Plackett–Burman statistical design and the central composite design in order to optimize culture conditions for lipase production by S. Table 2 summarizes the different forms of production. homothallicus.Food Bioprocess Technol Azeredo et al. The triglycerides tested were olive oil. repeated-batch. and bioreactor configurations employed in the last 10 years for lipase production both in SmF and SSF. olive oil. there are a considerable number of studies focusing on the use of bubble. (2008) investigated the production of extracellular lipase in submerged fermentation of Serratia rubidaea. niger in SSF using wheat bran as support and supplemented with synthetic medium composed of glucose. mucilaginosa MTCC 8737. which has potential application in the enzymatic biodiesel synthesis and other fields. tributyrin. and olive oil. to support growth and lipase production by P. fructose. The increase in molasses concentration resulted in lower lipase activity. surfactant. Bapiraju et al. peptone. NH2CONH2. airlift. However. (2006b) evaluated the suitability of OMW as a growth medium for lipase production in SmF using Penicillium citrinum NRRL 1841.3. yeast extract. NaCl. The optimum substrates for lipase production were potato starch as a carbon source. A single factorial design showed that the most suitable carbon source was a mixture of olive oil and citric acid and the most suitable nitrogen source was a mixture of corn steep liquor and NH4NO3. and MgSO4. carbon sources. K2HPO4. the addition of vegetable oils (olive. Potumarthi et al. respectively. Volpato et al. corn steep liquor as a nitrogen source. CaCl2. and MgSO4 while in SSF the medium was composed of sugarcane bagasse as support. the carbon source concentration was calculated to give a C/N ratio of 13. and olive oil as lipid source. mainly carbohydrates and lipids. and olive oil. Tween 80.500 U gds−1 after 12 h of fermentation. since it was shown that the excess of raw glycerol obtained from biodiesel process can be used for lipase production. In contrast. Production Processes Fermentative processes have been conducted in batch. polyethylene glycol 300. (2008) employed molasses as sole carbon source for lipase production by R. beef extract. starch. Burkert et al. fed-batch. and starch. and olive oil. (2007) investigated the effects of different carbon sources. oleic acid. olive oil. gingelly oil. processes. skim milk power. When comparing both tested media. yeast hydrolysate. (2005) optimized the lipase production by the mutant strain Rhizopus sp. restrictum in SSF. (2006) obtained extracellular lipases from SSF and SmF by R. and soybean oil) did not significantly affect lipase production. casein. Yan and Yan (2008) tested a combination of different experimental designs to optimize the production conditions of cell-bound lipase from Geotrichum sp. The SmF culture medium consisted of corn steep liquor. Immanuel et al. D’Annibale et al. Diaz et al. peptone. a versatile strain able to produce lipase on different kinds of OMW. the best results were obtained using the one based on peptone. lactose. and lipids. probably due to the enhancement on the medium viscosity. This section will consider recent applications of batch. fed-batch. repeated-batch. with ammonium chloride proving to be the most effective source. tributyrin. K2HPO4. citrinum in OMW-based media was significantly stimulated by nitrogen addition. lactose. In all tested media. KH2PO4. The mode of operation is. Falony et al. urea. (2006) tested the lipase production by A. The carbon sources tested on lipase production were sucrose. and Triton 100. corn. Cell cultures in SmF yielded a maximum extracellular lipase activity of 50 U mL−1 after 22 h of fermentation and in SSF cell cultures yielded a maximum lipase activity of 1. The effect of surfactants as inducers of lipase production was also evaluated using Tween 20.
(2007) obtained an alkaline lipase from B. (2008). (2005) Benjamin and Pandey (1997). (2008).230. Becker and Markl (2000) Cavalcanti et al. (2005) Boareto et al. (1997. Surribas et al. (2006) investigated the fermentation kinetics for the synthesis of lipase by C. A maximum lipase activity of 72 U mL−1 was obtained during 96 h of fermentation at 2 vvm. i. methyl oleate dispersion and pH fluctuations. the dissolved oxygen fluctuations generated in a controlled scale-down reactor has led to the more pronounced physiological effect. and in a bubble column reactor (3 L). Ikeda et al.35 mg mg−1. (2006b) investigated the lipase production in shaken flasks.600 U mL−1 in a 5-L bioreactor with Candida sp. The other environmental factors observed in a partitioned scale-down reactor. (1998b). (1998a). (2007). (2005) D’Annibale et al. (2005). (2007) bioreactors. (2008) Mala et al. which yielded similar lipase activity after 30 h of fermentation. He and Tan (2006) obtained a maximum lipase activity of 9.. Fickers et al. lipolytica. Potumarthi et al. lipase yields around 20 U mL−1 were achieved in a shorter time. and pH fluctuations) on lipase production by Y. (2001). the optimum conditions of agitation and aeration for lipase production were 300 rpm and 1 vvm. (2006) reported the development of a process for the extracellular lipase secreted by Y. 10. Gordillo et al. Fickers et al. To maintain sufficient oxygen concentration for the optimum cell growth and lipase activity. Kim and Hou (2006). YX/S. Puthli et al. (2008) Burkert et al. Cos et al. Montesinos et al. and the induction of the gene LIP2 encoding for the main lipase of Y. In the absence of mechanical agitation. In the stirred reactor. (1998a). These systems allow reproducing the hydrodynamic phenomena encountered in large-scale equipment for the three specified factors on microbial growth. (1998a). YP/S.71 U mg−1. The lipase production was 1. lipolytica. Burkert et al. and 25 °C. The enzyme production was carried out in a 2. rugosa in a batch system in a 2-L triple impeller bioreactor. Becker and Markl (2000). was 2. (2005). (2008) studied the influence of media and process parameters (aeration and agitation) on fermentation broth rheology and biomass formation in 1. 200 rpm. (2004) Montesinos et al. For the airlift bioreactor. resulting in a productivity about 60% higher compared to that obtained in the stirred reactor. Gupta et al.5 vvm. An overall 12-fold enhanced production (58 U mL−1) was achieved after medium optimization. (2002). 2003). Cos et al. Ito et al.5-L stirred tank reactor for lipase production using R.100 U mL−1 after 53 h of fermentation. pH 7. (2008) investigated the influence of extracellular factors (namely.Food Bioprocess Technol 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 Repeated-batch Fed-batch Airlift Bubble column STR STR Continuous STR Solid-state fermentation Batch Packed-bed Tray STR stirred tank reactor Gupta et al. (1997). the best aeration condition was 2. Kar et al.000-L bioreactor that led to a lipase activity of approximately 1. (2006). Alonso et al. D’Annibale et al. fermentation has been carried out at 600 rpm and at different aeration rates. (2005). as the use of flasks in batch mode for lipase production has already been presented in previous sections of this work. led to a less severe stress interpreted only by a decrease in microbial yield and hence in the extracellular lipase-specific production rate. Diaz et al. in a stirred tank (3 L). Yang et al. dissolved oxygen variations. lipolytica in a 20-L batch bioreactor in different scale-down apparatus. These studies illustrated the influence of gas–liquid mass transfer coefficient on the cell growth and hence on the lipase production.9 U mg−1. Ghadge et al. Martinez-Ruiz et al. Gordillo et al.e. with respect to biomass. Lipase yield with respect to substrate. . (2006). methyl oleate dispersion in the broth. YP/X. (2007). (2007). Benjamin and Pandey (2001).34 cm3 s−1 yielded optimum production of lipase. Gordillo et al. (2006). decreasing the LIP2 gene expression level. leading to an activity of 20 U mL−1 in 54 h of fermentation. Jensen et al. Montesinos et al. (2005). Main characteristics and application of these bioreactors will be reviewed in this section. mucilaginosa MTCC 8737. Potumarthi et al. Puthli et al. (2005). Among the set of environmental factors investigated. and biomass yield on substrate. (1997). Zhao et al. which have been designed to mimic the environmental behavior of each factor in laboratory conditions. multivorans within 15 h of growth in a 14-L bioreactor. Gas flow rate of 50. extracellular lipase production. D’Annibale et al. (2006a). Burkert et al. (2005) compared the lipase production by Geotrichum candidum in both 3-L stirred tank and airlift bioreactors. (2006b). was 25. Gordillo et al. Kar et al. (2005). Li et al.
The work of Yang et al. while lower speeds seemed to limit oxygen levels. 2007. rugosa lipase in the constitutive Pichia pastoris expression system. and an empirical model was fitted to experimental data. (2005) studied the lipase production in a 2-L stirred tank reactor at different agitation speeds and air flow rates. (2006) reported the lipase production in a 50-g packed-bed bioreactor at 40 °C and 50 mL min−1 of air. respectively. while a two-stage fermentation strategy. 500 g L−1 at the 800-L scale were obtained. The time to replace the volume. A maximum lipase activity was detected in the late stationary phase at 200 rpm and air flow rate of 0. on the other hand. The same trend verified for lipase production in submerged fermentation is valid for solid-state fermentation: most of the works are reporting the lipase production in tray bioreactors (conical flasks). 2006). or fluidized bioreactor. Immobilized cells showed high stability for repeated use.Food Bioprocess Technol 735. when the lipid source had been fully consumed. Nine repeated batches were carried out in flasks for 140 h and six repeated batches in a 5-L bioreactor. 2008. and the optimal composition of the medium were optimized. Several drawbacks have been found using a methanol non-limited fed-batch. Li et al. A lipase activity of approximately 14. Cavalcanti et al. The production was lower when compared to that obtained in a 100-g tray bioreactor.1 U mL−1 h−1 in batch fermentation to 17. intermittently agitated. Dutra et al. Alkan et al. Higher stirring rates resulted in mechanical and/or oxidative stress. We have not found reports focusing on the use of a rotating drum. could be manipulated to allow adequate growth rate for efficient lipase production. (2008) scaled up from 5. stirred reactor and bubble column. prevent the inhibition by substrate or metabolic products. The most pronounced effect of oxygen on lipase production was determined by stirring rate. A maximum enzyme activity (17.000 U mL−1 and a cell wet weight of ca. Vargas et al. An increase in the availability of oxygen at higher air flow rates led to faster lipid uptake and anticipation of enzyme release in culture medium. Different from SmF. In the previous cited work. SSF is mostly restricted to a packed-bed configuration. The lipase productivity increased from 3.to 800-L fed-batch bioreactors for the high cell density fermentation of C. mainly.000 U h−1 in a 2. (2007) compared different fed-batch cultivation strategies for the production of Rhizopus oryzae lipase from P. Zhao et al. and 430 U L−1 for shaken flasks. which shifted at 48 h by fine tuning the culture temperature and pH. Surribas et al.4 U gds−1) was obtained at 27 °C at an air flow rate of 0. using a few grams of substrate (Kempka et al. The fermentation conditions for both lab and pilot scale were optimized. was considered effective in pilot-scale fermentation. mainly making possible to conduct the process by long periods and improving the productivity compared to the batch process. (2005) investigated the lipase production by immobilized mycelium from R. simplicissimum in solid-state fermentation. The fed-batch processes are amply employed to minimize the effects of the cell metabolism control and. 2007. The lipase productivity reached 42.5-L bioreactor. the volume of the replaced medium. A maximum lipase activity of 1. (2005) used repeated fed-batch strategy to produce lipase from Acinetobacter radioresistens. Fed-Batch Processes The fed-batch processes are characterized by the addition of one or more nutrients to the bioreactor during the process. Benjamin and Pandey (1997) carried out experiments in batch and repeated-batch (fed-batch type) for lipase production using immobilized C. Alonso et al. Dissolved oxygen constant feeding. Falony et al. maintaining the products inside the bioreactor until the final of fermentation. 2008. rugosa cells in packed-bed bioreactor. Mala et al. 2008.500 U gds−1 was achieved after 12 h of fermentation.4 mL min−1 and allowing each cycle to run for 12 h. The exponential feeding combined with pH control succeeded in small-scale studies. (2007) reported the lipase production in a 1-kg tray bioreactor. the authors also verified that the pH control presented a high influence on lipase production. The influence of temperature and air flow rate on enzyme production was assessed by statistical experimental design. A temperature limited fed-batch has been proposed to solve both prob- . pastoris in a 20-L bioreactor.8 L min−1. A constant cell concentration was shown to be a pre-requisite to extend the number of repeated cycles. Repeated-Batch Processes The repeated-batch processes combines the advantages of fed-batch and batch processes. Oxygen limitation appeared at early cell dry weight and high cell death was observed. keeping the flow rate of the feed at 0. (2005) used a 30-g packed-bed bioreactor to improve productivity and scaling-up of lipase production using P. arrhizus in submerged fermentation using repeated-batch fermentations. where there are variations in bioreactor configuration. Higher lipase activities could be achieved at lower temperature levels and higher air flow rate values.9 U mL−1) was obtained when the fermentation was carried out in repeated-batch mode using a feed medium containing arabic gum and caprylic acid. A maximum lipase activity (26. Diaz et al.6 U mL−1 h−1 in repeated-batch fermentation.8 vvm. Azeredo et al. and adequate cell growth rate was critical for obtaining high lipase yield.
the maximum cell concentration at 16 h was 30. Stepwise feeding was carried out to simulate an exponential feeding and to investigate the effects of specific growth rate on cell growth and lipase production. Lipase productivity in continuous cultures increased by 50% compared to data obtained from batch fermentation and was dependent on the dilution rate applied. The highest final cell concentration obtained was 90 g L−1 when the set point of specific growth rate was 0. three growth regimes were recognized and the production of several enzymes from T. Gordillo et al. This combined strategy resulted in lower productivities when compared to a methanol non-limited fed-batch.7 U mL−1 at 179. such as carbon dioxide evolution rate by mass spectrometry. rugosa using pure or carbon source mixtures. Results obtained were compared to previous data from batch and fed-batch cultures for the purpose of selecting the best process strategies for the lipase production with C. Under steady-state conditions. At the time of the initial consumption of glucose in the batch-phase culture. and lipase activity was 96 U mL−1 after 35 h. due to build-up of over-supplied oleic acid.Food Bioprocess Technol lems. Continuous Processes Montesinos et al. Jensen et al. Mathematical Modeling of Lipase Production Some basic steps are required to scale-up lipase production processes. For the intermittent feeding.5 h. The obtained profile of the feed rate was applied to the feed forward control fermentation. A constant specific growth rate strategy afforded higher final lipase activity (117 U mL−1) at low specific growth rates. Fed-batch cultures (intermittent and stepwise feeding) were carried out in a 7. cylindracea NRRL Y17506 to produce extracellular lipase from oleic acid as a carbon source. (1998a) studied the lipase production by C. lipase activity was suppressed. A temperature-limited fed-batch was applied thereafter to solve oxygen transfer limitations.3fold purer final product was obtained mainly due to cell death reduction. rugosa in a 6-L bioreactor using two different fed-batch operational strategies to maximize lipase activity: constant substrate feeding rate and specific growth rate control. adjusting the substrate feeding rate. In the growth phase.200 mg L−1). and a 1. Finally. whereas production of protease was the highest in regime II (600 mg L−1 <NH4Cl<1.2 g L−1 dry cell. Ikeda et al. The two-step lipase production comprising a growth phase in fed-batch mode and a production phase. However. However.08 h−1 in relation to different ammonium concentrations in the feed medium. which is approximately 40 times higher than the production level obtained in flask culture.5-L bioreactor to improve cell concentration and lipase activity. Ito et al. (2002) studied the production of extracellular enzymes by the fungus Thermomyces lanuginosus in chemostat cultures at a dilution rate of 0. a methanol non-limited fed-batch resulted in better productivities. The authors found that during nitrogen limitation. lipase production by C. The highest lipase activity obtained in flask culture was 3 U mL−1 after 48 h of fermentation. the nutrient supply was automatically initiated by means of monitoring the respiratory rate change. High specific growth rate decreased extracellular lipase production in the latter part of fed-batch cultures. The best lipase yields were obtained in fed-batch fermentation using oleic acid. With a constant specific growth rate strategy. (2001) investigated the lipase production by a two-step fed-batch culture (2 L bioreactor) of an organic solventtolerant bacterium. (2004) developed a fed-batch fermentation process to enable the production of large amounts of recombinant human lysosomal acid lipase in Schizosaccharomyces. a feedback control strategy has been developed based on the estimation of state variables (cells and specific growth rate) from the measurement of indirect variables. The first one has been widely discussed in the literature and includes the choice of a suitable microorgan- . An on–off controller was then used to maintain the specific growth rate at the desired value.02 h−1 and the highest lipase activity was 23. rugosa was enhanced 10-fold compared to a batch operation.and intracellular lipases in continuous cultures of C. (2003) investigated the production of extra. rugosa. Finally. the cultivation was extended for a longer time. 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. A constant substrate feeding rate strategy showed that a maximum lipase activity (55 U mL−1) was reached at low substrate feeding rates. the cells were grown up to 50 g dry cell weight and the lipase expression was 16 U mL−1. Maximum yields relative to consumed substrate were obtained with oleic acid at low dilution rates.5 h of fermentation. The range and the production of carbohydrate hydrolyzing enzymes and lipase increased from regime I (NH4Cl≤ 600 mg L−1) to regime III (NH4Cl≥1.200 mg L−1). lanuginosus was recorded under different nutrient limitations ranging from nitrogen to carbon/energy limitations. a medium with low concentration of salt was used to overcome cell death problems. the final cell concentration was 52 g L−1 and the lipase activity was 6. was carried out. Kim and Hou (2006) cultivated C.3 U mL−1 after 138. whereas lipase tends to accumulate inside the cell at higher rates of substrate addition. in which lipase was induced by the addition of 5% stearic acid. In the second fed-batch strategy studied.
(2008) proposed an unstructured kinetic model to simulate the experimental data. rugosa batch and continuous lipase production. and substrate amounts and applied to the production of C. of how the manipulated variables affect the performance. which was found to be 8. α and β. respectively. Ghadge et al. the whole model was validated. Oleic acid accumulation observed during batch fermentation can be predicted using a model involving growth-associated lipase production and olive oil hydrolysis. the use of the same values of the parameters. such as bubble diameter and bubble rise velocity. In this section. based on back propagation algorithm. When compared to experimental data. the analysis of technical and economical viability of the process is required. based on the ANN model. the extent of deactivation was different in the two columns. The fed-batch process mode matched the simulation results. The extent of lipase deactivation in both columns was found to increase with an increase in hydrodynamic and interfacial flow parameters. However. 0. However. Haider et al. The flow parameters were estimated using an in-house computational fluid dynamic code based on the k-ε approach. Further. Becker and Markl (2000) modeled olive oil degradation by the thermophilic lipolytic strain Bacillus thermoteovorans IHI-91 in chemostat and batch culture. A maximum (7.99.15 and 0. rugosa is growth-associated. The second step is related to the choice of the bioreactor configuration for the process development and the study. The HNM can be used in process monitoring using on-line measurements of CO2 and substrate feed rate to infer enzyme activities and also substrate and biomass concentrations. The third step deals with the development and validation of mathematical models as a tool for scale-up. . (1997) proposed a simple structured mathematical model coupled with a methodology to estimate state variables and parameters and applied to C. and optimization. and continuous operations with a 10-fold increase in productivity relative to batch operation. Lipase solution was subjected to hydrodynamic flow parameters in 0. the highest predicted lipase activity was reached in fed-batch cultures with a prefixed substrate feeding in order to maintain constant—at their optimal values—the relation substrate/biomass or the specific growth rate. Finally. the HNM exhibited higher accuracy.8% higher than the maximum values predicted by a statistical regression-based optimization technique response surface methodology. The experimental data used corresponded to fed-batch operation with constant substrate feed rate. rugosa lipase in batch. The rate of deactivation was found to be depending on the average turbulent normal stress and interfacial flow parameters. and how these models have been used as a tool for process scale-up or process improvement.Food Bioprocess Technol ism and substrates for lipase production. (2007) proposed an efficient hybrid neural phenomenological model (HNM) for the lipase production process by C. it is focused on the main aspects regarding the mathematical models for lipase production reported in the last 10 years. The logistic model. (1998a) developed a simple structured mathematical model coupled with a methodology to estimate biomass amounts. Montesinos et al. For estimation of the best strategy to improve lipase productivity. an oscillatory behavior.69 U mL−1) of lipolytic activity at 72 h of culture was obtained. using the ANN–GA method. and the modified Luedeking–Piret model were found to be suitable to efficiently predict cell mass. Process modeling was carried out using Advanced Continuous Simulation Language. was predicted using the latter model. with high correlation coefficient (R2). The maximum enzyme productivity was predicted in continuous culture at moderate dilution rates and substrate feeding of 8 g L−1. Simulations confirmed that this accumulation was the cause for sudden growth cessation occurring in batch fermentations with higher olive oil starting concentrations. and 0. (2008) modeled and optimized the lipase production by a soil microorganism using artificial neural network (ANN) and genetic algorithm (GA) techniques. and glucose consumption. Rajendran et al. it was found that the lipase production by C. lipase production. The ANN model.100 g L−1 MgSO4. to obtain a general understanding of the underlying principles and limitations of the process and to quantify its stoichiometry. The rate of deactivation was found to follow a first-order kinetics. fed-batch. Boareto et al. as observed in some chemostat experiments. showing satisfactory results. respectively. rugosa. resulted in the following values of the media constituents: 9. ANNs were trained to represent the aqueous and intracellular lipase activity and were further associated with a reduced version of the mechanistic model of the proposed HNM.009 g L−1 FeSO4. Gordillo et al.991 mL L−1 oil. process control. (2005) investigated the effect of hydrodynamic flow parameters and interfacial flow parameters on the activity of lipase in a bubble column reactor. From the estimated values of the Luedeking–Piret model parameters. specific growth rate. Chemostat data were successfully described using the Monod chemostat model extended by terms for maintenance requirements and wall growth. An attempt was made to develop rational correlations for the extent of deactivation as well as for the deactivation constant. Optimization using GA. showed to be highly accurate in predicting the system with a correlation coefficient value close to 0. at laboratory scale. different simulations of batch. and continuous cultures were performed. the Luedeking–Piret model.385 m bubble column reactors over a wide range of superficial gas velocity. Once model parameters were determined. fed-batch.
A hybrid neural model (HNM) for the on-line monitoring of lipase production by Candida rugosa. and the use of mathematical models as a tool for process optimization. Peixoto.. Ellaiah... 91. The results discussed in this review clearly demonstrate that SSF. A. M. S. T. H. A. (2004). (2008). 45(4). F. 136. Sequential parametric optimization of lipase production by a mutant strain Rhizopus sp. on the optimization of the medium composition and operational variables. Benjamin. B. A. F. we believe that the use of engineering lipases will predominate in the near future since the production of engineering lipases will allow attainment of enzymes with new remarkable characteristics for a specific application. & Coelho. (2002). 183–192. J.1007/BF02686016. undoubtedly.1007/ s11274-006-9229-y. doi:10. Z. Maugeri. However. C. doi:10.. P. P.. L..1002/jctb. Alonso.. Srinivas. BTNT-2. A. substrate. Optimization of extracellular lipase production by Geotrichum sp. & Dogru. A. M. Enhancement of lipase production during repeated batch culture using immobilized Candida rugosa. (2005). G. doi:10. Modeling of olive oil degradation and oleic acid inhibition during chemostat and batch cultivations of Baccilus thermoleovorans IHI-91. 23.. Uyar. M. R. This review showed that many researchers worldwide direct their activities to the screening of new lipase-producing microorganisms and. Biotechnology and Bioengineering. All these efforts are justified by the great versatility of lipase applications. R. G. 319–327. L. Maldonado. R. H. 54. The use of agroindustrial residues as substrates for lipase production favors. Jr. O. 22(1). doi:10. Gomes. M. Of course.1016/ S0032-9592(96)00102-1. Optimizing lipases and related enzymes for efficient application. & Freire. 630–637. S. H. 77–84. & Rodrigues. in some cases. Considering the difficulties in handing the large volume bioreactors related to the lipase production by SSF. or fluidized bed bioreactors. F. M.0. E.. D. Sant’Anna. & Ramana. E. B.1007/ s00284-006-0425-7. U. B. systematic studies should be carried out to check whether the global production cost is lowered.1590/S0104-66322005000100002. mainly using submerged fermentation such as the screening of high lipase producers.. (2000). Oliveira. Azeredo. M. V. it could be more practical to use SSF for the production at small scale. other bioreactor configurations are investigated. Almeida. M. S. 82. doi:10.. & Pereira-Meirelles. M. 361–365. M. I. K. (2001)... T. (1997). Though the screening of microorganism producers of lipases has afforded satisfactory results to the present. (2007). M. M. N. Becker. M.. Alkan. This would allow the time domain to be partitioned in N-subintervals for which the feeding rate could be optimized.Food Bioprocess Technol Conclusions Critical analysis of current literature shows that microbial lipases are one of the most produced enzymes. E. pH. the difficulties imposed by the use of residues in the purification of enzyme produced make its use unfeasible. S. & Valdman. 32(5). P. doi:10. Valero. different process operation modes. Brazilian Archives of Biology and Technology. Process Biochemistry. (2005). Rocha-Leão.. Coutinho. T. There has been much development on lipase production by bioprocesses. based on our experience. P. P. F. (2007). Amaral. doi:10. (2005). oxygen. decreasing considerably the gradients. In all cases cited in this review. yields good results in terms of process productivity. including the downstream step.1016/S0960-8524(03)00152-4. 433–437. Brazilian Journal of Chemical Engineering. subsequently. 437–440.... 339–344. Improvement of lipase production at different stirring speeds and oxygen levels. An interesting alternative could be the use of an optimization tool such as the dynamic optimization or optimum control.. Journal of the Brazilian Sciences of Chemical Engineering. doi:10. & Pandey. 9–18. Boareto. Isolation and characterization of three distinct forms of lipases from Candida rugosa produced in solid-state fermentation.. P. 257–273. 44(2). P. F. SSF is difficult to scale-up due to existing gradients (mainly in packed-bed bioreactor) in temperature. S. Applied Biochemistry and Biotechnology. the feeding rate was not optimized.. F. F. J. Castilho. 200410548. J. successful substitution of synthetic medium by agroindustrial residues. The application of a dynamic optimization tool makes it possible to find the optimal profile of feeding rate that maximizes the production or productivity.. 1100–1106.. (2007). I. as long as the cultivation volume is kept to a very small scale.. Trends in Biotechnology.1016/S0167-7799 (02)02046-2..1002/1097-0290(20001220) 70:6<630::AID-BIT4>3. F. doi:10. intermittently agitated. using factorial design. & Krishna..CO.1678. 213–221. scale-up of process. 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