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

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

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, Brazil b 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 ARTICLE INFO Article history: Received 7 December 2009 Received in revised form 20 May 2010 Accepted 25 May 2010 Available online 1 July 2010 Keywords: Aspergillus spp. Biosurfactants Lipases Submerged and solid-state bioprocesses
ABSTRACT

Lipases and biosurfactants are compounds produced by microorganisms generally involved in the metabolization of oil substrates. However, the relationship between the production of lipases and biosurfactants has not been established yet. Therefore, this study aimed to evaluate the correlation between production of lipases and biosurfactants by submerged (SmgB) and solid-state bioprocess (SSB) using Aspergillus spp., which were isolated from a soil contaminated by diesel oil. SSB had the highest production of lipases, with lipolytic activities of 25.22 U, while SmgB had 4.52 U. The production of biosurfactants was not observed in the SSB. In the SmgB, correlation coefficients of 91% and 87% were obtained between lipolytic activity and oil in water and water in oil emulsifying activities, respectively. A correlation of 84% was obtained between lipolytic activity and reduction of surface tension in the culture medium. The surface tension decreased from 50 to 28 mN m-1 indicating that biosurfactants were produced in the culture medium. 2010 Elsevier Ltd. All rights reserved.

1. Introduction Lipases (triacylglycerol-acylhidrolases) are enzymes capable of catalyzing a variety of reactions, such as the partial or complete hydrolysis of triacylglycerols and reactions of esterification, transesterification and interesterification of lipids. Because of this, lipases are applicable to a wide range of industrial sectors. In the chemical industry, they are used for the production of surfactants and detergents, to resolve the racemic mixtures and for the treatment of residues that are rich in oils and fats. In the health sector they are used in medicines, diagnostics, cosmetics and antibiotics (Hasan et al., 2006).

In the food industry, lipases are used to synthesize emulsifiers such as mono-and diglycerides (Kittikun et al., 2008) and for the production of lipids with high levels of polyunsaturated fatty acids (Reshma et al., 2008). They are also used for the development of flavors (Salah et al., 2007), the maturation of cheese (Dupuis et al., 1993) and sausage meat, among others. Furthermore, lipases have an important application in the field of bioenergy, particularly for the production of biodiesel (Park et al., 2006), which is an expanding sector, given the worldwide concern with the use of renewable energy. Biosurfactants are compounds that are produced by microorganisms 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 emulsion formation, stabilization of aerated systems, modification of rheological properties, and for the improvement of the consistency and texture of products containing starch and fat (Nitschke and Costa, 2007). Biosurfactants may also have antimicrobial and anti-adhesive properties, so they are important in the control of pathogenic microorganisms on surfaces that are in contact with food, as they prevent the formation of biofilms (Nitschke and Costa, 2007). The main advantages of biosurfactants are their low toxicity and biodegradability, environmental acceptability, stability and functionality under various extreme conditions of pH and temperature (Desai and Banat, 1997). The possibility of producing them from agroindustrial waste was also cited as an advantage, with reports of production from waste such as molasses (Joshi et al., 2008), cheese whey (Rodrigues et al., 2006), lubricating oil and peanut cake (Thavasi et al., 2007). SmgB and SSB have been reported as methods for biotechnological production of lipases and biosurfactants. In the SSB, microorganisms grow on the surface of moist solids particles (Pandey, 2003), whereas in SmgB, microorganisms are suspended in a liquid medium containing the dissolved nutrients (Schmidell et al., 2001). SSB has advantages such as the use of low-cost substrates and simple equipments, low volumes of water, low energy demand and higher concentration of products obtained in comparison to SmgB. On the other hand, SmgB has the advantages of great homogeneity of the culture medium and maintenance of parameters such as temperature and pH. Moreover, is a bioprocess used worldwide, and more information about its engineering processes and controls are available. SmgB presents less oxygen transfer in liquid media, whereas heat transfer is the main problem related to SSB (Pandey et al., 2000).

The synthesis of lipases and biosurfactants by microorganisms may occur due to the need of microorganisms to metabolize compounds which are insoluble in water (Desai and Banat, 1997). Biosurfactants are compounds produced by reactions of secondary metabolism with functions of cell adhesion and motility, differentiation and accessibility to substrates and molecules of carbon and energy storage (Van Hamme et al., 2006). Although biosurfactants can 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 biosurfactants within bioprocesses has not been yet established. Therefore, this study aims to assess the relationship between the production of lipases and biosurfactants in SmgB and SSB by Aspergillus spp. 2. Methods 2.1. Microorganisms, maintenance and preparation of inoculum Two strains of Aspergillus sp. (O-8 and O-4) were isolated from a soil contaminated by diesel oil. The strain Aspergillus sp. O-8 was selected as good producer of lipases using SmgB (Colla et al., in press) and the strain Aspergillus sp. O-4 was selected as good producer of lipases using SSB (Colla et al., 2009). After being isolated, the organisms were kept in vials with potato-dextrose-agar (PDA), refrigerated at 4 C and replicated every 3 months. The preparation of inoculum for the bioprocess was carried out by the inoculation of the fungi in Petri dishes containing solidified 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 was performed using the fungus Aspergillus sp. O-8 and the conditions optimized by Colla (2009) for lipases production. The culture medium was prepared with 10% (w/v) of wheat bran, which was subjected to baking at 100 C for 30 min in 50% of the total volume of distilled water. Then, the medium was filtered and added with 10% (v/v) of saline solution, 45 g L-1 of yeast extract and 20 g L-1 of soybean oil as carbon 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 of FeSO4_7H2O, 0.01 mg L-1 of MnSO4, and 0.62 mg L-1 of ZnSO4 (Bertolin et al., 2001). The liquid medium was autoclaved at 121 C for 20 min and the pH of the medium was adjusted to 7.0 by using 1.5 M of HCl or NaOH 1 M solutions.

The production of biocompounds was performed in erlenmeyers of 250 mL with initial volume of 100 mL. The inoculation of the media was performed using spores of the fungus Aspergillus sp. O-8 contained in a circular area of 20 mm (diameter) of PDA. After inoculated, the cultures were incubated for 6 days at 30 C with agitation of 120 min-1. Two replicates were accomplished for each culture time. Lipase activity and biosurfactant production were determined by analysis of cell free supernatant samples every 24 h. 2.3. Production of lipases and biosurfactants through SSB The production of lipases and biosurfactants through SSB was performed using the fungus Aspergillus sp. O-4 and the conditions optimized by Colla (2009) for lipases production. The culture medium was prepared with 85.7% (w/w) of soybean meal and 14.3% (w/w) of rice husk, used for increasing the porosity of the media and for the oxygen transfer. The medium was added by 71% (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 FeSO4 _7H2O, 0.01 mg L-1 of MnSO4, and 0.62 mg L-1 of ZnSO4 and distilled water until the volume of 1 L) to provide the necessary micronutrients (Bertolin et al., 2001). Olive oil (2% w/w) and sodium nitrate (2% w/w) were added as carbon and nitrogen sources, respectively. The culture medium was autoclaved at 121 C for 20 min. The pH was adjusted to 4.5 by adding solution of H2SO4 1.5 M, and moisture was adjusted to 60% by adding sterile distilled water. The experiments were performed in erlenmeyers of 300 mL containing 50 g of medium and after being inoculated with 2.5 mL of a suspension of spores for achieving an initial concentration of spores in the culture medium of 2 _ 106 spores g-1. After inoculated, the cultures were incubated for 12 days at 30 C. Two replicates were accomplished for each culture time. Lipase activity and biosurfactant production were determined by analysis of fermented bran every 48 h. 2.4. Analytical determinations 2.4.1. Collection of extracts SmgB medium was centrifuged to separate the cells, and the extracts 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 of the lipolytic activity, 1 g of fermented bran was extracted with 10 mL of 0.2 M pH 7.0 phosphate buffer solution in an agitated water bath at 160 min-1 for 30 min at 37 C, with subsequent filtration for the separation of solids. For the determination of emulsifying activity, 5 g

of fermented bran were extracted with 30 mL of distilled water at 90 C followed by extraction in water bath at 50 C for 30 min, with subsequent filtration and centrifugation for the separation of solids and spores. 2.4.2. Estimate of cell growth The dry cell biomass concentration in the culture medium of SmgB was determined after harvesting the cells by centrifugation and then drying at 70 C to a constant mass (Raza et al., 2007). The fermented bran obtained through SSB was submitted to the estimation of proteins by Kjeldhal method (AOAC, 1995) in order to estimate the cell growth. 2.4.3. Determination of lipolytic activity The lipolytic activity was determined by the method recommended by Burkert et al. (2004), which is based on titration with NaOH 0.01 M of the fatty acids released by the action of lipase enzyme, contained in the enzyme extract, on the triacylglycerols of olive oil emulsified in Arabic gum. One unit of lipolytic activity was defined as the amount of enzyme that releases 1 mol of fatty acid per min per mL of enzyme extract (1 U = 1 mol min-1 mL-1) under the test conditions. 2.4.4. Determination of emulsifying activity The oil in water (O/W) and water in oil (W/O) emulsifying activities were determined by the method recommended by Pinto et al. (2009), using 3.5 mL of extract and 2 mL of soybean oil. The mixture was agitated in a vortex agitator at 700 min-1 for 1 min. After 60 min, the absorbance of the emulsified O/W mixture was read through a spectrophotometer at 610 nm. The O/W emulsifying activity was obtained by Eq. (1). After 24 h, readings of the W/O emulsion height that was formed and its total height were performed, being the W/O emulsifying activity determined according to Eq. (2). Two blanks were accomplished, the first using water instead of the sample and the second using not fermented culture media. EAO/W ABSsample -- ABSblank _ D EAW/O Esample -- Eblank _ D 2 where EA is emulsifying activity (UE); O/W is oil in water; W/O is water in oil; ABS is absorbance; E is percentage of height of emulsified 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 tension

The extracts obtained during the SmgB were used for the determination of the surface tension with a tensiometer (Kruss Processor Tensiometer K-6, Germany), according to the Du-Nuoys ring method (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 mode of cultivation (SSB or SmgB) on lipolytic activity, W/O emulsifying activity and O/W emulsifying activity was assessed by analysis of variance (ANOVA) and Tukeys test at a 5% significance level was used to compare the means. Linear and non-linear regression models were used to assess the correlations between lipolytic activity and W/O and O/W emulsifying activity and between lipolytic activity and surface tension in extracts of submerged fermentation. The correlation (r) and determination (r2) coefficients of the fitted models were calculated and the 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 in SmgB and SSB Table 1 presents the results of lipolytic activity, O/W and W/O emulsifying activities, and fungal growth from SmgB (Aspergillus sp. O-8) and SSB (Aspergillus sp. O-4). A comparison of the results of lipolytic activity obtained in the SmgB 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 (Aspergillus sp. O4), the maximum lipolytic activity was 25.07 U. In both cases, the maximum lipase production occurred after 4 days of cultivation. The highest lipolytic activities obtained in the SSB may be related to the characteristics of that mode of cultivation, if compared to the submerged one. In the SSB, the final product is concentrated (Schmidell et al., 2001) and fungi have the appropriate characteristics for the bioprocess such as: tolerance to low water activities and production of enzymes by the hyphae (Pandey, 2003). Although lipolytic activity in the SSB was higher than that of the SmgB, higher activities (57 U) were found in earlier stages of lipase production optimization by the SSB with the fungus Aspergillus sp. O4 (Colla, 2009). This fact can be attributed to the loss of the funguss ability to produce lipases. According to Makhsumkhanov et al. (2003) it may be related with depletion of the maintenance resources during storage. Furthermore, there may be an accumulation of certain metabolites, which decreases the cultures productivity (Makhsumkhanov et al., 2003).

Regarding the W/O emulsifying activity, the biosurfactant produced by the fungus Aspergillus sp. O-8 in the SmgB had an emulsification rate of 42.7 UE using soybean oil as the lipid phase. Kumar et al. (2007) obtained W/O emulsification rates of 70 UE for the exopolysaccharide produced by Planococcus maitriensis. Abouseoud et al. (2008) obtained emulsification rates of 4555 UE using diesel oil, kerosene, heptane and sunflower oil as oily phases; the biosurfactants produced by Pseudomonas fluorescens were identified as rhamnolipids. Pinto et al. (2009) obtained emulsifying activities W/O of 17.9, 20.50, 23.47 and 24.8 UE, using Corynebacterium aquaticum (culture 1), C. aquaticum and Bacillus sp. (culture 2), Corynebacterium sp., Bacillus cereus and Bacillus mycoides (culture 3) and Bacillus subtilis (culture 4), respectively. The W/O emulsifying activities obtained in these studies were similar to those obtained in our study. Thus, it can be concluded that good W/O emulsifying activities were obtained for the biosurfactant produced by Aspergillus sp. O-8 in SmgB. The W/O emulsions formed with the extracts from the SmgB were stable after 24 h, with a maximum emulsifying activity of 42.67 UE (accounting for 42% of emulsified layer). The emulsions obtained with the SSB extracts were unstable. The O/W and W/O emulsifying activities may give some indications about the kind of biosurfactants produced in bioprocesses. The O/W emulsions are stabilized by watersoluble emulsifiers, while W/O emulsions are stabilized by oil-soluble emulsifiers (Fennema, 2007). Therefore, low concentrations of oilsoluble emulsifiers were produced in the SSB, with a hydrophobic compound as the predominant group. The inability of the fungus Aspergillus sp. O-4 to produce lipases could be related with the poor results obtained by this isolate for producing compounds with surface active characteristics, resulting in low emulsifying activities when compared with the emulsifying activities demonstrated by the fungus Aspergillus sp. O-8 (Table 1). With regards to the O/W emulsifying activities, higher values were obtained by Aspergillus sp. O-8 (2.95 UE) in the SmgB. This result was lower than those reported by Martins et al. (2008), who obtained maximum O/W emulsifying activities in SSB with the fungus Phealemonium sp. and Aspergillus fumigatus, 7.67 and 9.10 UE g-1, respectively. The lower activities obtained in our study when compared to those of Martins et al. (2008) could be due to the kind of bioprocess employed. In our study, the SmgB was used, while SSB was used by Martins et al. (2008). The SSB is known to result in more concentrated fermentation products, for providing the best conditions to obtain biocompounds with filamentous fungi (Pandey, 2003; Schmidell et al., 2001). It is therefore recommended that further experiments using the fungus Aspergillus sp. O-8 in SSB must be carried out. LA: lipolytic activity; EAO/W: oil in water emulsifying activity and EAW/O: water in oil emulsifying activity.

The bacterial biosurfactants produced by Pinto et al. (2009) presented O/W emulsifying activities of 132160 UE, which are much higher than the emulsifying activities obtained in our study. This fact might indicate that both fungi used in our study produce more biosurfactants with ability to stabilize W/O emulsions, especially Aspergillus O-8 in SmgB, which presented higher W/O emulsifying activities. 3.1.1. Statistical analysis of the influence of time and method of cultivation on the production of lipases and on the O/W and W/O emulsifying activities The SSB had maximum lipolytic activity and emulsifying activity in fermentation times that were less than 6 days. Thus, in order to compare the two cultivation methods used, statistical comparisons were made between lipolytic activity and emulsifying activity in the initial, 2, 4 and 6 days of incubation. The analysis of variance of lipolytic activity, O/W emulsifying activity and W/O emulsifying activity as a function of incubation time and mode of cultivation showed that the interaction between the variables was significant (p < 0.001) for the three studied responses, and it should be assessed at the expense of individual factors. Table 2 shows the homogeneous groups of means according to Tukeys test, obtained for the lipase activity and W/O and O/W emulsifying activity according to the mode of cultivation (SSB or SmgB) and incubation time. It was found that higher lipolytic activities were obtained in SSB regardless of the incubation time measured, and they were significantly 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 significantly 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 true for the SSB, with maximum lipolytic activities of 25.07 U at 4 days of bioprocess, significantly higher (p = 0.000175) than those obtained at 2 and 6 days of SSB. Regarding the W/O emulsifying activity obtained at 2, 4 and 6 days of SmgB, they were significantly higher (p < 0.05) than those 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 days SmgB (see Table 2), which was significantly higher than the activities obtained at 4 (p < 0.001) and 6 days (p < 0.001) of SmgB. The lowest O/W emulsifying activities were obtained at 0 and 2 days of SSB, with no significant difference of O/W emulsifying activities obtained for the SmgB at 2 days (p > 0.05). The maximum emulsifying activity obtained, 2.95 EU (see Table 2), was significantly higher (p < 0.05) than the others obtained in SmgB at 4 days of bioprocess.

3.2. Correlation of production of lipases with the emulsifying activities and the surface tension of the extracts obtained from SmgB Considering the best results of emulsifying activity shown by the SmgB, its correlation with the production of lipases and surface tension of extracts was evaluated. Table 2 Homogeneous groups (alfa = 0.050) for Tukeys test for the influence of the culture mode (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). Culture mode Incubation time LA: Lipolytic activity; EA W/O: water in oil emulsifying activity and EA O/W: oil in water emulsifying activity. * Within each column means with the same superscript were not significantly different by the Tukey test at p = 0.05. Fig. 1. Correlation of the lipolytic activity (LA) with the oil in water (O/W) and water in oil (W/O) emulsifying activities (EA) and with the surface tension (ST) using the fungus Aspergillus sp. O-8 in SmgB. L.M. Colla et al. / Bioresource Technology 101 (2010) 83088314 8311 Fig. 1 shows the correlation between the production of lipases and the emulsifying activity, and between the production of lipases and the surface tension. The O/W emulsifying activity had a polynomial second-order correlation with the lipolytic activity (Fig. 1a), with a correlation coefficient of 91%. A moderate linear relationship (r = 87%) was established between the lipase activity and the W/O emulsifying activity (Fig. 1b). Figs. 24 present the plots of observed versus predicted values and residuals versus predicted values for the relationships between the O/W emulsifying activity and lipolytic activity (Fig. 2a and b), the W/O emulsifying activity and lipolytic activity (Fig. 3a and b) and the surface tension and lipolytic activity (Fig. 4a and b), respectively. In Figs. 2a, 3a and 4a, it was found that the points were aligned when compared with the adjusted straight line. Furthermore, it appears that most of the studied points are within the limits of the dashed lines, which corresponds to the area of the graph where there is a 95% probability of passing the real line of the graphs in question. Figs. 2b, 3b and 4b allowed the verification of the random distribution of residues around zero, which is a requirement to obtain adequate models. The polar and apolar portions that constitute molecules with surfactant properties are synthesized from the metabolism of lipids and carbohydrates (Desai and Banat, 1997). Mono- and diglycerides are biosurfactants that could be formed by the action of lipases on the hydrolysis of triglycerides which form the inducer (soybean oil).

The fact that these compounds have structures with polar and apolar portions in the same molecule confer them to have surface active characteristics. Another product released by the hydrolysis of triglycerides by lipases is glycerol which can be esterified to free fatty acids (Berg et al., 2006) in order to form sugar esters (low molecular weight glycolipids that could explain the reduced surface activity of extracts), similarly to what occurs in reactions of organic synthesis of biosurfactants with lipases. Furthermore, the fatty acids released by the microorganism may be used as substrate to form the lipid fraction of biosurfactant, while the fatty acids are metabolized by b-oxidation and reused for the synthesis of bioemulsifiers, as reported by Kitamoto et al. (2002). According to these authors, when alcohols or long chain fatty acids (1218 carbons) are used as substrates, the fatty acids in biosurfactant (glycolipids-mannosylerythritol lipids) have chains of 2, 4 or 6 carbons. Even or odd fatty acids as substrates generate biosurfactants with even or odd chains, respectively; i.e. the fatty acids in biosurfactants are intermediates for the degradation of acids via b-oxidation of substrates. The addition of an inhibitor to the fatty acids synthesis had insignificant effect on the production of biosurfactants, while the addition of a b-oxidation inhibitor effectively inhibited that production (Kitamoto et al., 2002). Table 3 shows the results of surface tension of the extracts obtained by the SmgB (Aspergillus sp. O-8) over time, noting that the surface tension of the fermented medium was reduced by the biosurfactant production until a minimum of 28.8 mN m-1, and the surface tension of the medium in the initial time was 50 mN m-1. The increased surface tension in the extracts obtained during 6 days of fermentation may be due to the degradation of biosurfactant molecules. According to Pyaza et al. (2006), reduction of the medium surface tension is a good indication of biosurfactant production, though not related to the emulsification ability of the biosurfactant produced. Biosurfactants that are capable of reducing surface tension, such as glycolipids or lipopeptides, generally have low molecular weight structures, while high molecular weight biosurfactants, such as amphypatic polysaccharides, proteins, lipopolysaccharide, lipoproteins or complex mixtures of these biopolymers, are associated with the ability of emulsification (Rosenberg and Ron, 1999). Fig. 2. (a) Observed versus predicted values and (b) residuals versus predicted values of correlation between the oil in water (O/W) emulsifying activity (EA) and lipolytic activity (LA) using the fungus Aspergillus sp. O-8 in SmgB. Fig. 3. (a) Observed versus predicted values and (b) residuals versus predicted values of correlation between the water in oil (W/O)

emulsifying activity (EA) and lipolytic activity (LA) using the fungus Aspergillus sp. O-8 in SmgB. 8312 L.M. Colla et al. / Bioresource Technology 101 (2010) 83088314 The highest emulsifying activities and reduction of surface tension in the extracts of the SmgB were obtained in the stationary phase of the microorganism growth, which occurred after 2 days of fermentation (Table 1). This pattern of biosurfactant production is not associated with the growth of microorganisms, being one of the possible ways of biosurfactant synthesis, as reported by Ron and Rosemberg (2001). As the nutrient limitation begins, the growth speed decreases, but the carbon remains transported into the cells and used for the lipids biosynthesis; the final products formed under such circumstances can be lipids, polysaccharides, storage polymers such as poly-hydroxybutyrate or antibiotics. The limiting nutrients that may lead to those conditions are the nitrogen, magnesium, iron and phosphorus (Desai and Banat, 1997). Moreover, in the solid-state bioprocess, the emulsifying activity peaks were obtained during the microorganism growth, which explains the low yields obtained. Regarding the relationship between the lipolytic activity and the surface tension in the extracts of SmgB, an inverse linear correlation was obtained (r = 84%) considering the data obtained between 1 and 4 days of incubation time (Fig. 2c). As the action of lipases occurs on the carbon source used as inducer, the production of compounds capable of reducing the surface tension, such as mono- and diglycerides, may occur. Thus, the higher the lipolytic activity, the lower the surface tension. In addition, lipases may be involved in the synthesis of biosurfactants, which reduce surface tension. Therefore, the correlation between lipolytic activity and surface tension was observed only after 24 h of culture confirming that biosurfactants have to be present to reduce the surface tension. 4. Conclusion The production of lipases has advantages when performed by SSB as highest enzymatic activities obtained. However, the simultaneous production of biosurfactants in such culture was not observed due to the apparent loss of the ability of the fungus to produce the biocompounds. The maximum lipolytic activity obtained in SmgB by the fungus Aspergillus sp. O-8 was 4.52 U, and maximum O/W and W/O emulsifying activities were 2.95 and 42 UE, respectively. The reduction of surface tension in the media ranged from 50 to 28 mN m-1, which confirmed the production of biosurfactant by Aspergillus sp. O-8. The results obtained in this study are relevant as they show the simultaneous production of two biocompounds with broad industrial applications (lipases and biosurfactants) in a single bioprocess. Moreover, further work on scale-up must be carried out in order to evaluate the economics of the process.

Acknowledgements The authors are pleased to acknowledge the National Council for Scientific and Technological Development (CNPq) for the financial support.