Bioresource Technology 101 (2010) 8308–8314

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Bioresource Technology

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Simultaneous production of lipases and biosurfactants by submergedand solid-state bioprocesses

Luciane Maria Colla a, Juliana Rizzardi a, Marta Heidtmann Pinto b, Christian Oliveira Reinehr a,Telma Elita Bertolin a, Jorge Alberto Vieira Costa b,*

a Laboratory of Fermentations, Course of Food Engineering, College of Engineering and Architecture, University of Passo Fundo, Campus I, km 171, BR 285, P.O. Box 611,CEP 99001-970, Passo Fundo, RS, Brazilb Laboratory of Biochemical Engineering, School of Chemistry and Food Engineering, Federal University of Rio Grande, P.O. Box 474, CEP 96201-900, Rio Grande, RS, Brazil

a r t i c l e i n f o a b s t r a c t

Article history:Received 7 December 2009Received in revised form 20 May 2010Accepted 25 May 2010Available online 1 July 2010

Keywords:Aspergillus spp.BiosurfactantsLipasesSubmerged and solid-state bioprocesses

0960-8524/$ - see front matter � 2010 Elsevier Ltd. Adoi:10.1016/j.biortech.2010.05.086

* Corresponding author. Fax: +55 53 32338676.E-mail addresses: lmcolla@upf.br (L.M. Colla),

dqmjorge@furg.br (J.A.V. Costa).

Lipases and biosurfactants are compounds produced by microorganisms generally involved in the metab-olization of oil substrates. However, the relationship between the production of lipases and biosurfac-tants has not been established yet. Therefore, this study aimed to evaluate the correlation betweenproduction of lipases and biosurfactants by submerged (SmgB) and solid-state bioprocess (SSB) usingAspergillus spp., which were isolated from a soil contaminated by diesel oil. SSB had the highest produc-tion of lipases, with lipolytic activities of 25.22 U, while SmgB had 4.52 U. The production of biosurfac-tants was not observed in the SSB. In the SmgB, correlation coefficients of 91% and 87% were obtainedbetween lipolytic activity and oil in water and water in oil emulsifying activities, respectively. A correla-tion of 84% was obtained between lipolytic activity and reduction of surface tension in the culture med-ium. The surface tension decreased from 50 to 28 mN m�1 indicating that biosurfactants were producedin the culture medium.

� 2010 Elsevier Ltd. All rights reserved.

1. Introduction

Lipases (triacylglycerol-acylhidrolases) are enzymes capable ofcatalyzing a variety of reactions, such as the partial or completehydrolysis of triacylglycerols and reactions of esterification, transe-sterification and interesterification of lipids. Because of this, lipasesare applicable to a wide range of industrial sectors. In the chemicalindustry, they are used for the production of surfactants and deter-gents, to resolve the racemic mixtures and for the treatment of res-idues that are rich in oils and fats. In the health sector they are usedin medicines, diagnostics, cosmetics and antibiotics (Hasan et al.,2006).

In the food industry, lipases are used to synthesize emulsifierssuch as mono-and diglycerides (Kittikun et al., 2008) and for theproduction of lipids with high levels of polyunsaturated fatty acids(Reshma et al., 2008). They are also used for the development offlavors (Salah et al., 2007), the maturation of cheese (Dupuiset al., 1993) and sausage meat, among others. Furthermore, lipaseshave an important application in the field of bioenergy, particularlyfor the production of biodiesel (Park et al., 2006), which is an

ll rights reserved.

jorgealbertovc@terra.com.br,

expanding sector, given the worldwide concern with the use ofrenewable energy.

Biosurfactants are compounds that are produced by microorgan-isms and are chemically characterized as glycolipids, lipopeptides,fatty acids, phospholipids, neutral lipids or lipopolysaccharides(Desai and Banat, 1997).

In the food industry, biosurfactants can be used for emulsionformation, stabilization of aerated systems, modification of rheo-logical properties, and for the improvement of the consistencyand texture of products containing starch and fat (Nitschke andCosta, 2007). Biosurfactants may also have antimicrobial andanti-adhesive properties, so they are important in the control ofpathogenic microorganisms on surfaces that are in contact withfood, as they prevent the formation of biofilms (Nitschke and Costa,2007).

The main advantages of biosurfactants are their low toxicityand biodegradability, environmental acceptability, stability andfunctionality under various extreme conditions of pH and temper-ature (Desai and Banat, 1997). The possibility of producing themfrom agro-industrial waste was also cited as an advantage, with re-ports of production from waste such as molasses (Joshi et al.,2008), cheese whey (Rodrigues et al., 2006), lubricating oil andpeanut cake (Thavasi et al., 2007).

SmgB and SSB have been reported as methods for biotechno-logical production of lipases and biosurfactants. In the SSB,

L.M. Colla et al. / Bioresource Technology 101 (2010) 8308–8314 8309

microorganisms grow on the surface of moist solids particles(Pandey, 2003), whereas in SmgB, microorganisms are suspendedin a liquid medium containing the dissolved nutrients (Schmidellet al., 2001). SSB has advantages such as the use of low-cost sub-strates and simple equipments, low volumes of water, low energydemand and higher concentration of products obtained in com-parison to SmgB. On the other hand, SmgB has the advantagesof great homogeneity of the culture medium and maintenanceof parameters such as temperature and pH. Moreover, is a bio-process used worldwide, and more information about its engi-neering processes and controls are available. SmgB presents lessoxygen transfer in liquid media, whereas heat transfer is themain problem related to SSB (Pandey et al., 2000).

The synthesis of lipases and biosurfactants by microorganismsmay occur due to the need of microorganisms to metabolize com-pounds which are insoluble in water (Desai and Banat, 1997). Bio-surfactants are compounds produced by reactions of secondarymetabolism with functions of cell adhesion and motility, differen-tiation and accessibility to substrates and molecules of carbon andenergy storage (Van Hamme et al., 2006). Although biosurfactantscan be produced by organic synthesis using lipases (Paula et al.,2005), which catalyzing the esterification of fatty acids and sugars,the correlation between the production of lipases and biosurfac-tants within bioprocesses has not been yet established.

Therefore, this study aims to assess the relationship betweenthe production of lipases and biosurfactants in SmgB and SSB byAspergillus spp.

2. Methods

2.1. Microorganisms, maintenance and preparation of inoculum

Two strains of Aspergillus sp. (O-8 and O-4) were isolated from asoil contaminated by diesel oil. The strain Aspergillus sp. O-8 wasselected as good producer of lipases using SmgB (Colla et al.,in press) and the strain Aspergillus sp. O-4 was selected as goodproducer of lipases using SSB (Colla et al., 2009).

After being isolated, the organisms were kept in vials with po-tato-dextrose-agar (PDA), refrigerated at 4 �C and replicated every3 months. The preparation of inoculum for the bioprocess was car-ried out by the inoculation of the fungi in Petri dishes containingsolidified PDA medium, and incubated at 30 �C for 5 days.

2.2. Production of lipases and biosurfactants through SmgB

The production of lipases and biosurfactants through SmgB wasperformed using the fungus Aspergillus sp. O-8 and the conditionsoptimized by Colla (2009) for lipases production. The culture med-ium was prepared with 10% (w/v) of wheat bran, which was sub-jected to baking at 100 �C for 30 min in 50% of the total volumeof distilled water.

Then, the medium was filtered and added with 10% (v/v) of salinesolution, 45 g L�1 of yeast extract and 20 g L�1 of soybean oil as car-bon source. The saline solution contained 2 g L�1 of KH2PO4, 1 g L�1

MgSO4, and 10 mL L�1 of trace solution composed of 0.63 mg L�1 ofFeSO4�7H2O, 0.01 mg L�1 of MnSO4, and 0.62 mg L�1 of ZnSO4 (Bert-olin et al., 2001). The liquid medium was autoclaved at 121 �C for20 min and the pH of the medium was adjusted to 7.0 by using1.5 M of HCl or NaOH 1 M solutions.

The production of biocompounds was performed in erlenmey-ers of 250 mL with initial volume of 100 mL. The inoculation ofthe media was performed using spores of the fungus Aspergillussp. O-8 contained in a circular area of 20 mm (diameter) of PDA.After inoculated, the cultures were incubated for 6 days at 30 �Cwith agitation of 120 min�1.

Two replicates were accomplished for each culture time. Lipaseactivity and biosurfactant production were determined by analysisof cell free supernatant samples every 24 h.

2.3. Production of lipases and biosurfactants through SSB

The production of lipases and biosurfactants through SSB wasperformed using the fungus Aspergillus sp. O-4 and the conditionsoptimized by Colla (2009) for lipases production. The culturemedium was prepared with 85.7% (w/w) of soybean meal and14.3% (w/w) of rice husk, used for increasing the porosity of themedia and for the oxygen transfer. The medium was added by71% (v/w) of saline solution (2 g L�1 of KH2PO4, 1 g L�1 of MgSO4,and 10 mL L�1 of trace solution, composed of 0.63 mg L�1 of FeS-O4�7H2O, 0.01 mg L�1 of MnSO4, and 0.62 mg L�1 of ZnSO4 anddistilled water until the volume of 1 L) to provide the necessarymicronutrients (Bertolin et al., 2001). Olive oil (2% w/w) andsodium nitrate (2% w/w) were added as carbon and nitrogensources, respectively. The culture medium was autoclaved at121 �C for 20 min. The pH was adjusted to 4.5 by adding solutionof H2SO4 1.5 M, and moisture was adjusted to 60% by adding steriledistilled water.

The experiments were performed in erlenmeyers of 300 mLcontaining 50 g of medium and after being inoculated with2.5 mL of a suspension of spores for achieving an initial concentra-tion of spores in the culture medium of 2 � 106 spores g�1. Afterinoculated, the cultures were incubated for 12 days at 30 �C. Tworeplicates were accomplished for each culture time. Lipase activityand biosurfactant production were determined by analysis of fer-mented bran every 48 h.

2.4. Analytical determinations

2.4.1. Collection of extractsSmgB medium was centrifuged to separate the cells, and the ex-

tracts were used for the analytical determinations.The SSB bran was subject to extraction in order to determine

the lipolytic and emulsifying activities. For the determination ofthe lipolytic activity, 1 g of fermented bran was extracted with10 mL of 0.2 M pH 7.0 phosphate buffer solution in an agitatedwater bath at 160 min�1 for 30 min at 37 �C, with subsequent fil-tration for the separation of solids. For the determination of emul-sifying activity, 5 g of fermented bran were extracted with 30 mL ofdistilled water at 90 �C followed by extraction in water bath at50 �C for 30 min, with subsequent filtration and centrifugationfor the separation of solids and spores.

2.4.2. Estimate of cell growthThe dry cell biomass concentration in the culture medium of

SmgB was determined after harvesting the cells by centrifugationand then drying at 70 �C to a constant mass (Raza et al., 2007).

The fermented bran obtained through SSB was submitted to theestimation of proteins by Kjeldhal method (AOAC, 1995) in order toestimate the cell growth.

2.4.3. Determination of lipolytic activityThe lipolytic activity was determined by the method recom-

mended by Burkert et al. (2004), which is based on titration withNaOH 0.01 M of the fatty acids released by the action of lipase en-zyme, contained in the enzyme extract, on the triacylglycerols ofolive oil emulsified in Arabic gum.

One unit of lipolytic activity was defined as the amount of en-zyme that releases 1 lmol of fatty acid per min per mL of enzymeextract (1 U = 1 lmol min�1 mL�1) under the test conditions.

Table 1Lipolytic activity (U), oil in water (O/W) and water in oil (W/O) emulsifying activities(UE), and fungal growth (g L�1 and % proteins) obtained using SmgB (Aspergillus sp. O-8) and SSB (Aspergillus sp. O-4).

Time (d) LA (U) EAO/W (UE) EAW/O (UE) X (g/L)

SmgB (Aspergillus sp. O-8)0 2.82 ± 0.13 0.90 ± 0.02 1.73 ± 0.35 1.79 ± 0.181 3.21 ± 0.09 0.96 ± 0.05 22.52 ± 5.23 2.57 ± 0.082 3.48 ± 0.18 0.61 ± 0.07 30.56 ± 0.21 4.49 ± 0.033 3.79 ± 0.07 1.76 ± 0.25 42.67 ± 6.65 3.06 ± 0.024 4.52 ± 0.04 2.95 ± 0.18 14.40 ± 2.90 4.19 ± 1.545 3.56 ± 0.08 0.32 ± 0.12 21.26 ± 0.91 3.64 ± 0.596 3.71 ± 0.04 1.19 ± 0.15 21.88 ± 9.07 3.13 ± 1.13

Proteins (%)

SSB (Aspergillus sp. O-4)0 16.96 ± 0.19 0.52 ± 0.20 0.19 ± 0.19 13.34 ± 0.012 20.68 ± 0.21 0.43 ± 0.07 0.95 ± 1.64 14.76 ± 0.434 25.07 ± 0.49 1.79 ± 0.05 0.79 ± 0.45 16.76 ± 0.396 24.13 ± 0.37 1.92 ± 0.13 0.96 ± 1.38 18.37 ± 1.658 21.91 ± 0.19 2.01 ± 0.05 1.19 ± 0.32 19.52 ± 0.51

10 14.20 ± 0.10 1.18 ± 0.26 2.85 ± 1.72 20.21 ± 0.0712 11.58 ± 0.41 0.68 ± 0.01 0.00 ± 0.00 15.74 ± 1.62

LA: lipolytic activity; EAO/W: oil in water emulsifying activity and EAW/O: water inoil emulsifying activity.

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2.4.4. Determination of emulsifying activityThe oil in water (O/W) and water in oil (W/O) emulsifying activ-

ities were determined by the method recommended by Pinto et al.(2009), using 3.5 mL of extract and 2 mL of soybean oil. The mix-ture was agitated in a vortex agitator at 700 min�1 for 1 min. After60 min, the absorbance of the emulsified O/W mixture was readthrough a spectrophotometer at 610 nm. The O/W emulsifyingactivity was obtained by Eq. (1). After 24 h, readings of the W/Oemulsion height that was formed and its total height were per-formed, being the W/O emulsifying activity determined accordingto Eq. (2). Two blanks were accomplished, the first using water in-stead of the sample and the second using not fermented culturemedia.

EAO=W ¼ ðABSsample � ABSblankÞ � D ð1Þ

EAW=O ¼ ðEsample � EblankÞ � D ð2Þ

where EA is emulsifying activity (UE); O/W is oil in water; W/O iswater in oil; ABS is absorbance; E is percentage of height of emulsi-fied layer (mm) divided by total height of the liquid column (mm)and D is dilution of sample in water.

2.4.5. Determination of surface tensionThe extracts obtained during the SmgB were used for the deter-

mination of the surface tension with a tensiometer (Kruss Proces-sor Tensiometer K-6, Germany), according to the Du-Nuoy’s ringmethod (Joshi et al., 2008; Costa et al., 2006).

2.5. Statistical analysis of data

The influence of incubation time (0, 2, 4 and 6 days) and modeof cultivation (SSB or SmgB) on lipolytic activity, W/O emulsifyingactivity and O/W emulsifying activity was assessed by analysis ofvariance (ANOVA) and Tukey’s test at a 5% significance level wasused to compare the means.

Linear and non-linear regression models were used to assess thecorrelations between lipolytic activity and W/O and O/W emulsify-ing activity and between lipolytic activity and surface tension inextracts of submerged fermentation. The correlation (r) and deter-mination (r2) coefficients of the fitted models were calculated andthe analysis of residues of the regressions was carried out.

3. Results and discussion

3.1. Production of lipases and O/W and W/O emulsifying activities inSmgB and SSB

Table 1 presents the results of lipolytic activity, O/W and W/Oemulsifying activities, and fungal growth from SmgB (Aspergillussp. O-8) and SSB (Aspergillus sp. O-4).

A comparison of the results of lipolytic activity obtained in theSmgB and SSB (Table 1) showed that the SmgB (Aspergillus sp. O-8)had a maximum lipolytic activity of 4.52 U, while in the SSB (Asper-gillus sp. O4), the maximum lipolytic activity was 25.07 U. In bothcases, the maximum lipase production occurred after 4 days ofcultivation.

The highest lipolytic activities obtained in the SSB may be re-lated to the characteristics of that mode of cultivation, if comparedto the submerged one. In the SSB, the final product is concentrated(Schmidell et al., 2001) and fungi have the appropriate characteris-tics for the bioprocess such as: tolerance to low water activitiesand production of enzymes by the hyphae (Pandey, 2003).

Although lipolytic activity in the SSB was higher than that of theSmgB, higher activities (57 U) were found in earlier stages of lipaseproduction optimization by the SSB with the fungus Aspergillus sp.

O-4 (Colla, 2009). This fact can be attributed to the loss of the fun-gus’s ability to produce lipases. According to Makhsumkhanovet al. (2003) it may be related with depletion of the maintenanceresources during storage. Furthermore, there may be an accumula-tion of certain metabolites, which decreases the culture’s produc-tivity (Makhsumkhanov et al., 2003).

Regarding the W/O emulsifying activity, the biosurfactant pro-duced by the fungus Aspergillus sp. O-8 in the SmgB had an emul-sification rate of 42.7 UE using soybean oil as the lipid phase.Kumar et al. (2007) obtained W/O emulsification rates of 70 UEfor the exopolysaccharide produced by Planococcus maitriensis.Abouseoud et al. (2008) obtained emulsification rates of 45–55UE using diesel oil, kerosene, heptane and sunflower oil as oilyphases; the biosurfactants produced by Pseudomonas fluorescenswere identified as rhamnolipids. Pinto et al. (2009) obtained emul-sifying activities W/O of 17.9, 20.50, 23.47 and 24.8 UE, using Cory-nebacterium aquaticum (culture 1), C. aquaticum and Bacillus sp.(culture 2), Corynebacterium sp., Bacillus cereus and Bacillus myco-ides (culture 3) and Bacillus subtilis (culture 4), respectively.

The W/O emulsifying activities obtained in these studies weresimilar to those obtained in our study. Thus, it can be concludedthat good W/O emulsifying activities were obtained for the biosur-factant produced by Aspergillus sp. O-8 in SmgB.

The W/O emulsions formed with the extracts from the SmgBwere stable after 24 h, with a maximum emulsifying activity of42.67 UE (accounting for 42% of emulsified layer). The emulsionsobtained with the SSB extracts were unstable. The O/W and W/Oemulsifying activities may give some indications about the kindof biosurfactants produced in bioprocesses. The O/W emulsionsare stabilized by water-soluble emulsifiers, while W/O emulsionsare stabilized by oil-soluble emulsifiers (Fennema, 2007). There-fore, low concentrations of oil-soluble emulsifiers were producedin the SSB, with a hydrophobic compound as the predominantgroup. The inability of the fungus Aspergillus sp. O-4 to produce li-pases could be related with the poor results obtained by this iso-late for producing compounds with surface active characteristics,resulting in low emulsifying activities when compared with theemulsifying activities demonstrated by the fungus Aspergillus sp.O-8 (Table 1).

With regards to the O/W emulsifying activities, higher valueswere obtained by Aspergillus sp. O-8 (2.95 UE) in the SmgB. This re-sult was lower than those reported by Martins et al. (2008), who

L.M. Colla et al. / Bioresource Technology 101 (2010) 8308–8314 8311

obtained maximum O/W emulsifying activities in SSB with the fun-gus Phealemonium sp. and Aspergillus fumigatus, 7.67 and9.10 UE g�1, respectively. The lower activities obtained in ourstudy when compared to those of Martins et al. (2008) could bedue to the kind of bioprocess employed. In our study, the SmgBwas used, while SSB was used by Martins et al. (2008). The SSB isknown to result in more concentrated fermentation products, forproviding the best conditions to obtain biocompounds with fila-mentous fungi (Pandey, 2003; Schmidell et al., 2001). It is thereforerecommended that further experiments using the fungus Aspergil-lus sp. O-8 in SSB must be carried out.

The bacterial biosurfactants produced by Pinto et al. (2009) pre-sented O/W emulsifying activities of 132–160 UE, which are muchhigher than the emulsifying activities obtained in our study. Thisfact might indicate that both fungi used in our study produce morebiosurfactants with ability to stabilize W/O emulsions, especiallyAspergillus O-8 in SmgB, which presented higher W/O emulsifyingactivities.

3.1.1. Statistical analysis of the influence of time and method ofcultivation on the production of lipases and on the O/W and W/Oemulsifying activities

The SSB had maximum lipolytic activity and emulsifying activ-ity in fermentation times that were less than 6 days. Thus, in orderto compare the two cultivation methods used, statistical compari-sons were made between lipolytic activity and emulsifying activityin the initial, 2, 4 and 6 days of incubation.

The analysis of variance of lipolytic activity, O/W emulsifyingactivity and W/O emulsifying activity as a function of incubationtime and mode of cultivation showed that the interaction betweenthe variables was significant (p < 0.001) for the three studied re-sponses, and it should be assessed at the expense of individual fac-tors. Table 2 shows the homogeneous groups of means accordingto Tukey’s test, obtained for the lipase activity and W/O and O/Wemulsifying activity according to the mode of cultivation (SSB orSmgB) and incubation time.

It was found that higher lipolytic activities were obtained in SSBregardless of the incubation time measured, and they were signif-icantly higher than the lipolytic activity obtained in the SmgB(p < 0.05, see Table 2).

The lipolytic activity at 4 days of the SmgB (4.52 U) was signif-icantly higher than those obtained at 2 days of the SmgB(p = 0.0065) and 6 days of the SmgB (p = 0.043). The same was truefor the SSB, with maximum lipolytic activities of 25.07 U at 4 daysof bioprocess, significantly higher (p = 0.000175) than those ob-tained at 2 and 6 days of SSB.

Regarding the W/O emulsifying activity obtained at 2, 4 and6 days of SmgB, they were significantly higher (p < 0.05) than those

Table 2Homogeneous groups (alfa = 0.050) for Tukey’s test for the influence of the culturemode (SSB or SmgB) and incubation time on lipolytic activity (LA) and water in oil(W/O) and oil in water (O/W) emulsifying activities(EA).

Culturemode

Incubation time (d) LA (U)* EA W/O (UE)* EA O/W (UE)*

SmgB 0 2.82a 1.73a 0.90bc

SmgB 2 3.48ab 30.56c 0.61ab

SmgB 4 4.52c 14.40b 2.95e

SmgB 6 3.71b 21.88b 1.19c

SSB 0 16.96d 0.19a 0.52a

SSB 2 20.68e 0.95a 0.43a

SSB 4 25.07g 0.79a 1.79d

SSB 6 24.13f 0.96a 1.92d

LA: Lipolytic activity; EA W/O: water in oil emulsifying activity and EA O/W: oil inwater emulsifying activity.* Within each column means with the same superscript were not significantlydifferent by the Tukey test at p = 0.05.

obtained in all times of SSB and in the initial time of SmgB (Table 2).The maximum W/O emulsifying activity was obtained at 2 daysSmgB (see Table 2), which was significantly higher than the activi-ties obtained at 4 (p < 0.001) and 6 days (p < 0.001) of SmgB.

The lowest O/W emulsifying activities were obtained at 0 and2 days of SSB, with no significant difference of O/W emulsifyingactivities obtained for the SmgB at 2 days (p > 0.05). The maximumemulsifying activity obtained, 2.95 EU (see Table 2), was signifi-cantly higher (p < 0.05) than the others obtained in SmgB at 4 daysof bioprocess.

3.2. Correlation of production of lipases with the emulsifying activitiesand the surface tension of the extracts obtained from SmgB

Considering the best results of emulsifying activity shown bythe SmgB, its correlation with the production of lipases and surfacetension of extracts was evaluated.

Fig. 1. Correlation of the lipolytic activity (LA) with the oil in water (O/W) andwater in oil (W/O) emulsifying activities (EA) and with the surface tension (ST)using the fungus Aspergillus sp. O-8 in SmgB.

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Fig. 1 shows the correlation between the production of lipasesand the emulsifying activity, and between the production of lipasesand the surface tension.

The O/W emulsifying activity had a polynomial second-ordercorrelation with the lipolytic activity (Fig. 1a), with a correlationcoefficient of 91%. A moderate linear relationship (r = 87%) wasestablished between the lipase activity and the W/O emulsifyingactivity (Fig. 1b).

Figs. 2–4 present the plots of observed versus predicted valuesand residuals versus predicted values for the relationships be-tween the O/W emulsifying activity and lipolytic activity (Fig. 2aand b), the W/O emulsifying activity and lipolytic activity (Fig. 3aand b) and the surface tension and lipolytic activity (Fig. 4a andb), respectively. In Figs. 2a, 3a and 4a, it was found that the pointswere aligned when compared with the adjusted straight line. Fur-thermore, it appears that most of the studied points are within thelimits of the dashed lines, which corresponds to the area of thegraph where there is a 95% probability of passing the real line ofthe graphs in question.

Figs. 2b, 3b and 4b allowed the verification of the random dis-tribution of residues around zero, which is a requirement to obtainadequate models.

The polar and apolar portions that constitute molecules withsurfactant properties are synthesized from the metabolism of lip-ids and carbohydrates (Desai and Banat, 1997). Mono- and diglyce-rides are biosurfactants that could be formed by the action oflipases on the hydrolysis of triglycerides which form the inducer(soybean oil). The fact that these compounds have structures withpolar and apolar portions in the same molecule confer them tohave surface active characteristics.

Another product released by the hydrolysis of triglycerides bylipases is glycerol which can be esterified to free fatty acids (Berg

Fig. 2. (a) Observed versus predicted values and (b) residuals versus predictedvalues of correlation between the oil in water (O/W) emulsifying activity (EA) andlipolytic activity (LA) using the fungus Aspergillus sp. O-8 in SmgB.

et al., 2006) in order to form sugar esters (low molecular weightglycolipids that could explain the reduced surface activity of ex-tracts), similarly to what occurs in reactions of organic synthesisof biosurfactants with lipases.

Furthermore, the fatty acids released by the microorganismmay be used as substrate to form the lipid fraction of biosurfactant,while the fatty acids are metabolized by b-oxidation and reused forthe synthesis of bioemulsifiers, as reported by Kitamoto et al.(2002). According to these authors, when alcohols or long chainfatty acids (12–18 carbons) are used as substrates, the fatty acidsin biosurfactant (glycolipids-mannosylerythritol lipids) havechains of 2, 4 or 6 carbons. Even or odd fatty acids as substratesgenerate biosurfactants with even or odd chains, respectively; i.e.the fatty acids in biosurfactants are intermediates for the degrada-tion of acids via b-oxidation of substrates. The addition of an inhib-itor to the fatty acids synthesis had insignificant effect on theproduction of biosurfactants, while the addition of a b-oxidationinhibitor effectively inhibited that production (Kitamoto et al.,2002).

Table 3 shows the results of surface tension of the extracts ob-tained by the SmgB (Aspergillus sp. O-8) over time, noting that thesurface tension of the fermented medium was reduced by the bio-surfactant production until a minimum of 28.8 mN m�1, and thesurface tension of the medium in the initial time was 50 mN m�1.The increased surface tension in the extracts obtained during6 days of fermentation may be due to the degradation of biosurfac-tant molecules.

According to Pyaza et al. (2006), reduction of the mediumsurface tension is a good indication of biosurfactant production,though not related to the emulsification ability of the biosurfac-tant produced. Biosurfactants that are capable of reducing sur-face tension, such as glycolipids or lipopeptides, generally have

Fig. 3. (a) Observed versus predicted values and (b) residuals versus predictedvalues of correlation between the water in oil (W/O) emulsifying activity (EA) andlipolytic activity (LA) using the fungus Aspergillus sp. O-8 in SmgB.

Fig. 4. (a) Observed versus predicted values and (b) residuals versus predictedvalues of correlation between the surface tension (ST) and lipolytic activity (LA)using the fungus Aspergillus sp. O-8 in SmgB.

L.M. Colla et al. / Bioresource Technology 101 (2010) 8308–8314 8313

low molecular weight structures, while high molecular weightbiosurfactants, such as amphypatic polysaccharides, proteins,lipopolysaccharide, lipoproteins or complex mixtures of thesebiopolymers, are associated with the ability of emulsification(Rosenberg and Ron, 1999).

The highest emulsifying activities and reduction of surface ten-sion in the extracts of the SmgB were obtained in the stationaryphase of the microorganism growth, which occurred after 2 daysof fermentation (Table 1). This pattern of biosurfactant productionis not associated with the growth of microorganisms, being one ofthe possible ways of biosurfactant synthesis, as reported by Ronand Rosemberg (2001). As the nutrient limitation begins, thegrowth speed decreases, but the carbon remains transported intothe cells and used for the lipids biosynthesis; the final productsformed under such circumstances can be lipids, polysaccharides,

Table 3Surface tension (mN m�1) of the extracts fromSmgB by the fungus Aspergillus sp. O-8.

Bioprocesstime (d)

Surface tension(mN m�1)

0 50.0 ± 3.141 31.2 ± 0.082 30.3 ± 0.183 30.8 ± 0.154 28.8 ± 0.475 29.3 ± 0.736 52.3 ± 0.62

storage polymers such as poly-hydroxybutyrate or antibiotics.The limiting nutrients that may lead to those conditions are thenitrogen, magnesium, iron and phosphorus (Desai and Banat,1997). Moreover, in the solid-state bioprocess, the emulsifyingactivity peaks were obtained during the microorganism growth,which explains the low yields obtained.

Regarding the relationship between the lipolytic activity andthe surface tension in the extracts of SmgB, an inverse linear corre-lation was obtained (r = 84%) considering the data obtained be-tween 1 and 4 days of incubation time (Fig. 2c). As the action oflipases occurs on the carbon source used as inducer, the productionof compounds capable of reducing the surface tension, such asmono- and diglycerides, may occur. Thus, the higher the lipolyticactivity, the lower the surface tension. In addition, lipases maybe involved in the synthesis of biosurfactants, which reduce sur-face tension. Therefore, the correlation between lipolytic activityand surface tension was observed only after 24 h of culture con-firming that biosurfactants have to be present to reduce the surfacetension.

4. Conclusion

The production of lipases has advantages when performed bySSB as highest enzymatic activities obtained. However, the simul-taneous production of biosurfactants in such culture was not ob-served due to the apparent loss of the ability of the fungus toproduce the biocompounds. The maximum lipolytic activity ob-tained in SmgB by the fungus Aspergillus sp. O-8 was 4.52 U, andmaximum O/W and W/O emulsifying activities were 2.95 and 42UE, respectively. The reduction of surface tension in the media ran-ged from 50 to 28 mN m�1, which confirmed the production of bio-surfactant by Aspergillus sp. O-8.

The results obtained in this study are relevant as they show thesimultaneous production of two biocompounds with broad indus-trial applications (lipases and biosurfactants) in a single bioproc-ess. Moreover, further work on scale-up must be carried out inorder to evaluate the economics of the process.

Acknowledgements

The authors are pleased to acknowledge the National Councilfor Scientific and Technological Development (CNPq) for the finan-cial support.

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