Lipase Production by Surface-Adhesion Fermentation (SAF) of Filamentous Fungi from Coahuila's Semi-Desert.

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1 Lipase Production by Surface-Adhesion Fermentation (SAF) of Filamentous Fungi from Coahuila's Semi-Desert. Juan Carlos Cano, Nancy D. Uresti, Anna Ilina, Jose L. Martínez * Biotechnology Department of Chemistry Faculty, Autonomous University of Coahuila. Blvd. V. Carranza e Ing. J. C. Valdés, Col. República, Saltillo, Coahuila. CP 25280, Mexico. ( josemartinez@uadec.edu.mx) ABSTRACT Different lipases are produced on industrial scale for application in different industries due to the fact that they are extracellular enzymes, secreted by both fungi and bacteria. Two principal fermentation systems to produce lipases are solid state fermentation (SSF) and submerged fermentation (SmF). The surface-adhesion fermentation (SAF) is a recently system to produce metabolite, apply the fungal biofilms that are less known than bacterial biofilms, but they can be used for enzyme production. The objective of this work was the lipase production by Aspergillus strain applying surface-adhesion fermentation (SAF) using fibrous network of Opuntia ficus-indica as support matrix. In the present study were used three strains of Aspergillus niger (7A1, 12 'and 14) of different sources of Coahuila semi-desert of Mexico, and assayed to produce lipases by FAS (Mata, 2010) using dried leaves of Opuntia ficus-indica. The biofilms were formed on fibrous network of Opuntia and applied the mineral grow media (MGM) (Coca, 2008). Every 24 h, were evaluated: protein, sugar and lipase production. The particle Opuntia with formed biofilms were applied to SAF to produce lipase at least 5 times. Some physiological characteristics were evaluate. During the production process of biofilm was able to obtain an enzyme level at IU L-1 at 48 hours, in all species of fungi. Similar levels were obtained on SSF, but biomass yields were lower in SAF than in SSF. Was demonstrated that fibrous material of Opuntia ficus-indica permit the fungi growth on the support surface and creating a strongly adherent biofilm. These systems present a perspective to reuse the biofilm for produce enzymes. In addition, high levels of activity lipase were detected. This is the first report on the use of fibrous network material of Opuntia ficus-indica, a structural material, as an immobilizing matrix for the entrapment of fungal biomass and its application in lipase production. It was also observed that the lipase production was higher with whey alone as well as with olive oil as an inducer. Based on the results it can be suggested that the application of surface-adhesion fermentation (SAF) is a new process to use in the enzyme industry. INTRODUCTION Lipases (triacylglycerol acyl hydrolase; E.C ) are hydrolytic enzymes that act in aqueous-organic interfaces, catalyzing the cleavage of ester bonds in triglycerides and producing glycerol and free fatty acids. However, in environments with low water availability, lipases are able to catalyze esterification,

2 interesterification and transesterification reactions, being thus very versatile biocatalysts (1). Due to the different reactions lipases are able to catalyze and due to their regio- and enantio-selectivity, lipases find an increasing range of applications, such as in the detergent, food, pharmaceutical, fine chemicals, leather and pulp and paper industries (2, 3). Greater industrial application of these enzymes would depend on the development of low-cost processes for their production. In this context, surface-adhesion fermentation (SAF) appears as an interesting low-cost alternative for the production of theses biomolecules (4). In SAF, agroindustrial solid residues can be employed as support for biofilm formation. Application of low-cost and abundant raw materials (for example, the fibrous nopal network) contributes to reduce production costs. The concept of a biofilm presumes a population or a community of microorganisms living attached to a surface, which may be applied in repeated cycles of enzyme production. Biofilms can be developed on biotic as well as abiotic surfaces, from single specie or as a community derived from several species (4). Filamentous fungi are naturally adapted to growth on surfaces and under these conditions show a particular physiological behavior different to that in submerged culture as well as solid state culture (5). Solid support provides the fungi with environmental conditions similar to their natural habitat. In contrast to SSF, is not characterized by the presence of low moisture contents, which contribute to overheating and deficient oxygen supply. In the present paper we report the lipases production by three strains of Aspergillus niger (denominated as 7, 12 and 14) on surface-adhesion fermentation (SAF) using fibrous material of Opuntia ficus-indica (nopal). MATERIALS AND METHODS Fungal strains and inoculum formation Aspergillus niger strains used in this work were isolated previously from the samples obtained from semidesert of Coahuila State (Mexico). Spores were obtained through strains propagation for 5 days at 30 ºC on potato dextrose agar, were scraped into a 0.01% Tween 80 solution, and counted in a Neubauer chamber. These suspensions were used as the inoculums. Support treatment The fibrous material of Opuntia ficus-indica was cut into small irregular pieces of about 2 x 2 cm 2, washed in boiling distilled water for 30 min and left for 1 h in distilled water. The water was changed three times to thoroughly clean the pieces. Washed pieces were oven-dried at 70 C for 48 h to get to a constant weight (5 g), cooled and kept in desiccators until subsequent use (Fig. 1). Fig. 1. Opuntia ficus-indica fibrous networks used as support for lipase production by Aspergillus niger strains during surface adhesion fermentation (SAF).

3 SAF Growth Media The mineral growth medium (MGM) contained (in g/l): NaH 2 PO , KH 2 PO 4 2.0, MgSO 4 7H 2 O 0.3, CaCl , and (NH 4 ) 2 SO 4 1% (w/v) as a nitrogen source, as well as sucrose at 4% (w/v) as a carbon source. As inducer the olive oil at 2% (V/V) was applied. The initial ph was adjusted to 6. Biofilm formation and submerged adhesion fermentation (SAF) The biofilms were formed on the treated Opuntia ficus-indica particles during SAF. The SAF was carried out on Erlenmeyer flasks of 125 ml capacity with 30 ml of MGM. Flasks were inoculated with 10 6 spores/ml and maintained for 72 h at 30ºC under low agitation (120 rpm). Each 24 h, 1 ml of supernatants were taken to assay extracellular lipase activity. The solid state fermentation (SSF) was carried out as control under similar assay condition: 5 g of Opuntia ficus-indica particles was inoculated with 10 6 spores/g and its moisture content was adjusted to 70% using MGM and olive oil as inducer. After 72 h of fermentation, MGM was added to the flasks with fermented solids, which were incubated in a rotary shaker (200 rpm) at 35 C for 20 min. The extract was obtained by manually pressing the material, followed by centrifugation (3000 rpm for 2 min). Lipase activity assay Lipase activity was assayed using p-nitrophenil propionate (pnpp) as substrate. Reaction mixture contained 0.2 ml of enzymatic extract, 2.5 ml of 25 mm sodium phosphate buffer (ph 7.0) and 20 µl of 50 mm pnpp solution. Reaction was carried out at 37 o C. The p-nitrophenol released as hydrolysis product, was measured spectrophotometrically at 348 nm each 10 min for 30 min as total time. Activity unit was defined as amount of enzyme needed to hydrolyze 1 μmol of pnpp per minute releasing 1 μmol of p-nitrophenol under assay conditions. Backgrounds were defined using boiled enzyme extracts. RESULTS AND DISCUSSION Lipase activity during SA fermentation The lipase activity levels detected during SA fermentation are presented in the Figure 2. The process was carried out in MGM media with external inducer (olive oil) addition. According to obtained results (Figures 2 and 3), Aspergillus niger strains (denominated as 7, 12 and 14) are able to form a biofilm strongly attached to support (Opuntia ficus-indica) as well as to produce extracellular lipase. Figure 3 demonstrates micro-photo of Aspergillus niger (strain 12) biofilm formed on the Opuntia ficusindica particles after different fermentation period. The maximum lipase activity level detected during the formation of biofilms was around of 19 IU/mL for the tree fungi (Fig. 2). However, time for maximum activity level was different for each Aspergillus niger strain (Fig. 2). In the case of strains 12 and 14, for followed 72 h of fermentation the activity slightly decreased.

4 Fig. 2. Lipase production by Aspergillun niger strains (7, 12 and 14) on MGM media with sucrose as carbon source and olive oil as inducer at 24, 48 and 72 h of biofilm formation. Figure 3 demonstrates that the fungi (strain 12 as well as strains 12 and 14) adapted to growing on surfaces of Opuntia ficus-indica applied as support. This phenomenon is similar to observe in the case of SSF and fungi immobilization. Fig. 3. Micro-photo of Aspergillus niger biofilm formed on Opuntia ficus-indica: left,- 24 h; center,- 48 h; right,- 72 h of SAF. However, the lipase activity under SAF conditions was higher than detected on solid state fermentation (SSF) in similar media with olive oil as inducer. The activity in all cases (strains 7, 12 and 14) achieved the levels a a of 6.2 IU/mL. These data show an a advantage of SA fermentation in comparison with SSF enzyme a b c production. In the present study Opuntia ficus-indica fibrous networks were used as support for fungi adhesion. This support as well as all solid supports applied in previous reports (4, 5) has a common feature: their basic macromolecular structure. Similar to substrates, usually applied for SSF, Opuntia ficus-indica fibrous materials are composite and heterogeneous products from agriculture or by-products of agro-industry. Their structural macromolecules may simply provide an inert matrix (as reported for sugarcane bagasse, inert fibers, and resins) within which the carbon and other energy sources (sugars, lipids, and organic acids) are adsorbed (4). Thus, the nopal networks represent macromolecules matrix for microorganism adhesion also providing the carbon and energy source.

5 Natural adsorption on solid supports is an immobilization technique that it has been used with different microorganisms and some filamentous fungi. Once spores are adsorbed to the support, the fungi can grow forming a film (4, 5). In the present study, biofilms of Aspergillus niger were formed over all support (Opuntia ficus-indica networks) (Figs. 1 and 3). Biofilms formation and maintenance are dynamic processes that involve complex interactions of physical and biological nature (4, 5). Although fungal biofilms have been less characterized than bacterial biofilms (4), in the present study we demonstrate that they can be used for enzyme production by surface adhesion fermentation. This method differs to SSF due to liquid phase presence, and is similar to SSF due to fungus adhesion to the support. In the previous study we demonstrated considerable capacity of fungus Mucor griseocyanus to attach on Opuntia imbricata (coyonoxtle) applied in SAF (6). In this study we observed that Aspergillus niger strains formed biofilms strongly adhered to network fibrous of Opuntia ficus-indica. The obtained results may be important to define perspectives of industrial applications of this agro-industrial by-product as support for fungi biofilm formation, as well as to consider the Aspergillus niger strains isolated from semi-desert of Coahuila as potential source for lipase production. CONCLUSION The fibrous residue of cactus Opuntia ficus-indica has certain perspectives as support applied in SAF fermentation for production of fungal lipases. The SAF allows for the levels of lipase activity higher than in SSF. Aspergillus niger strains isolated from Coahuila semi-desert, form strongly adherent biofilms on fibrous network of Opuntia ficus-indica. This is the first report of this fibrous network material application as a structural matrix for fungal biomass immobilization and lipase production. These systems present perspectives due to possibility to reuse the biofilms in various cycles of enzyme production that may help to reduce cost of fermentation and product recovery processes. References [1] Couturier L., Taupin D., Yvergnaux F Lipase-catalyzed chemoselective aminolysis of various aminoalcohols with fatty acids. J. Mol. Catal. B: Enzym., 56 (1), [2] Mohanasrinivasan V., Dhrisya P., Dipinsha K. P., Manu Unnithan C., Megha Viswanath K. and Subathra Devi C A comparative study of the lipase yield by solid state and submerged fermentations using fungal species from biopharmaceutical oil waste. African J. Biotechnol., 8 (1), [3] Vardanega R., Remonatto D., Arbter F., Polloni A., Rigo E., Ninow J. L., Treichel H., Oliveira D. and Di Luccio M A Systematic Study on Extraction of Lipase Obtained by Solid State Fermentation of Soybean Meal by a Newly Isolated Strain of Penicillium spp. World J. Chem, 3 (2), [4] Gutiérrez-Correa M. and Villena G. K Surface adhesion fermentation: a new fermentation category. Rev. Per. Biol, 10 (2): [5] Villena G. K. and Gutiérrez-Correa M Production of lignocellulolytic enzymes by Aspergillus niger biofilms at variable water activities. Electr. J. Biotech, 10 (1),

6 [6] Martínez-Hernández J.L., Mata-Gómez M.A., Aguilar-González C.N. and Ilyina A A process to produce penicillin G acylase by surface-adhesion fermentation using Mucor griseocyanus to obtain 6-aminopenicillanic acid by penicillin G hydrolysis. Appl. Biochem. Biotech., 160 (7),