Biotechnology Letters 25: 12251229, 2003. 2003 Kluwer Academic Publishers. Printed in the Netherlands.

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A novel application of solid state culture: production of lipases by Yarrowia lipolytica

A. Domnguez, M. Costas, M.A. Longo & A. Sanromn
Department of Chemical Engineering, University of Vigo, 36200 Vigo, Spain Author for correspondence (Fax: +34 986 812382; E-mail: sanroman@uvigo.es)
Received 5 February 2003; Revisions requested 28 March 2003/6 May 2003; Revisions received 17 April 2003/20 May 2003; Accepted 20 May 2003

Key words: barley bran, lipase, nut, oil, solid state culture, Yarrowia lipolytica

Abstract An extracellular lipase was produced in solid state cultures of Yarrowia lipolytica CECT 1240 using nylon sponge and several food and agroindustrial wastes (barley bran and triturated nut) as, respectively, inert support and support substrate. The highest activity was obtained with triturated nut (23 kU l1 ) followed by sunower oil-soaked barley bran (21 kU l1 ). The activities were 5 fold greater those obtained in the control cultures with just inert support.

Introduction One of the limitations for extensive industrial use of microbial lipases is their cost, which is determined by the production yields, downstream processing requirements and enzyme stability (Kanwar et al. 2002). Therefore, it is of interest to increase the productivity of the fermentation processes by optimisation of culture conditions. Since the raw materials employed in the culture medium contribute to the total production costs (Castilho et al. 2000), the reduction in the substrate cost would be a suitable strategy to increase the productivity of the process. Solid state culture is generally dened as the growth of microorganisms on solid materials in the absence or near absence of free water (Pandey 1992). In the last ten years, there has been a vast change in the solid state culture with signicant developments. Comparative studies between submerged and solid state cultures claim higher yields, more concentrated enzyme product (with potential downstream recovery advantage) and other advantages for products usually obtained in submerged cultures (Moo-Young et al. 1983, Pandey et al. 1999). Recently, several reports have been published indicating the application of this culture in upgrading agroindustrial wastes and in the production of ne

chemicals and enzymes. The utilisation of by-products and wastes from food and agricultural industries has several advantages over submerged cultures such as superior productivity, simpler techniques, reduced energy requirements, low wastewater output, improved product recovery and the reduction in production costs, since they supply the microorganisms with some nutritive substances (Pandey et al. 2000). The main drawback of this type of cultivation is scale-up. This is largely due to several problems encountered in the heat transfer and homogeneity of the cultures. However, research attention was directed towards the development of designs such as mixed solid state bioreactors (Nagel et al. 2001) and rotating drum bioreactors (Stuart et al. 1999) that overcome these problems. Most studies on lipolytic enzymes production by bacteria, fungi and yeasts have been performed in submerged cultures, however, there are only a few reports on lipase synthesis in solid state cultures. In recent years increasing attention has been paid to the conversion of processing industry wastes in lipase by solid state culture. There are several reports dealing with extracellular lipase production by fungus such as Rhizhopus sp., Aspergillus sp., Penicillium sp. on different solid substrates (Kamini et al. 1998, Christen et al. 1995, Gombert et al. 1999, Cordova et al. 1998,

1226 Miranda et al. 1999). Few researchers, though, have investigated the synthesis of lipases by yeasts using this mode of culture. Amongst them, Rao et al. (1993) determined that the C/N ratio of the medium is an important parameter for lipase production by the yeast Candida rugosa in solid state culture. Several factors may affect extracellular lipase production, such as pH, temperature, aeration and medium composition. Besides, the presence of triacylglycerols or fatty acids has been reported to increase lipolytic enzyme secretion by a number of microorganisms (Marek & Bednarski 1996). In solid state cultures, the type of substrate could be used to enhance the production of enzymes, as many food and agroindustrial wastes are rich in fatty acids, triacylglycerols and/or sugars. The use of cheap raw materials would diminish the operating costs of the process. Moreover, total capital investment for lipase production has been reported to be signicantly lower in solid state cultures than in submerged cultures (Castilho et al. 2000). The yeast, Yarrowia lipolytica, is an important lipase producer and the inuence of several factors on lipolytic enzyme secretion has been assessed (Corzo & Revah 1999, Destain et al. 1997). However, there are no reports on the production of lipases by this yeast in solid state cultures. In this work, lipase production by Yarrowia lipolytica in solid state culture has been investigated, using food-agroindustrial wastes (triturated nut and barley bran) as support substrates. The inuence of several lipidic compounds on lipolytic enzyme secretion was also assessed. NaCl solution to a previously prepared agar plate, and 1 ml of this suspension was added to the liquid medium. The asks were incubated in an orbital shaker at 150 rpm and 30 C, for 48 h. These cultures of Yarrowia lipolytica were used to inoculate (2% v/v) the production medium in solid state cultures. Solid substrate was prepared (Stredansky & Conti 1999) by impregnating 5 g carrier (nylon sponge, barley bran, nut) with 15 ml culture medium. Nut was ground in a blender in order to obtain a regular particle size in all cultures, this substrate is termed triturated nut in the present work. Prior to use, all the supports were autoclaved at 121 C for 20 min. Solid state cultures were carried out in Erlenmeyer asks (250 ml) and incubated statically at 30 C and 90% humidity to avoid evaporation. They were capped with cellulose stoppers which permit passive aeration. Samples were obtained by extruding the ask contents with a syringe. Experiments were done in duplicate and samples were analysed in triplicate. The values in the gures correspond to mean values with a deviation less than 15%. Analytical methods Reducing sugars: They were measured by the dinitrosalicylic acid method using glucose as a standard. Lipase assay: Lipolytic activity was measured using tributyrin as substrate (Huge-Jensen et al. 1987). The reaction mixture was 0.114 M tributyrin in 5 mM Tris/HCl buffer pH 7, containing 5 mM CaCl2 and 2% (w/v) gum arabic and emulsied in a homogenizer at 9500 rpm for 7 min. The culture sample (0.2 ml) was added to 10 ml substrate, at an initial pH of 7 and 30 C, under agitation. The assay solution was kept at pH 7 by addition of 10 mM NaOH in a pHstat. The rate of NaOH addition was recorded, and the enzyme activity was calculated from the slope of the straight lines obtained. Randomly chosen measurements were performed in triplicate; they differed by less than 5%. One lipase activity unit was dened as the amount of enzyme that produced 1 mol butyric acid per min under standard assay conditions. The activities were expressed in kU l1 . pH: pH measurements on 0.05 ml culture samples were made by using a micro pH electrode.

Materials and methods Microorganism and growth In order to obtain the inoculum for lipase production, Yarrowia lipolytica CECT 1240 (ATCC 18942) was grown in 250 ml Erlenmeyer asks containing 150 ml basal medium (Corzo & Revah 1999) containing (per litre): 20 g glucose, 2 g urea, 1 g KH2 PO4 , 0.5 g MgSO4 7H2 O, 0.1 g CaCl2 , 0.1 g NaCl, 0.5 mg H3 BO3 , 0.2 mg FeCl3 4H2 O, 0.4 mg ZnSO4 7H2 O, 0.4 mg MnSO4 H2 O, 0.1 mg KI, 0.04 mg CuSO4 5H2 O, 200 g thiamin, 8 g biotin. The medium without urea and vitamins was sterilised at 121 C for 20 min. After cooling, the vitamins and urea, previously sterilised by microltration (0.22 m), were added to the basal medium. A cell suspension was obtained by addition of 10 ml 0.9%

1227 Results In solid state fermentation the microorganism not only grows at or near the surface of the solid material but also penetrates deep into the intercellular and intracellular spaces of the support, showing a close contact or association with it. For this reason, food and agricultural wastes can be employed as support providing part of the nutrients to the microorganism, making the whole process much more economical. As it has already been mentioned in the Introduction, fatty acids and acylglycerols induce microbial lipase production in submerged cultures. The materials that can be employed in solid state culture may be either inert (synthetic materials) or non-inert (organic materials). The former act only as an attachment whereas the latter also function as a source of nutrients such as sugars and oils, on account of which they are called support substrates. The aim of this paper is to evaluate the potential of solid state cultures using several materials (triturated nut and barley bran) as support substrate for lipase production in solid state conditions by the yeast Yarrowia lipolytica. Moreover, the effect of supplementing the culture medium with several likely inducers (mainly oils) has been assessed. First, two experiments were carried out using nylon sponge and barley bran as support. The superiority of barley bran over nylon sponge for lipase production in solid state cultures of Y. lipolytica was demonstrated, since maximum lipase activities of 12 kU l1 and 4.5 kU l1 , respectively, were obtained. In cultures with barley bran the lipase began on the third day increasing up to a value around 12 kU l1 on the eleventh day. Moreover, glucose was consumed at 2.5 g l1 day, down to a residual concentration around 5 g l1 . This suggests that the yeast is able to metabolise some of the carbohydrates contained in the support. Lipids are generally essential for inducing a high lipase yield (Sharma et al. 2001). Therefore, the addition of different lipid materials (corn, sunower and olive oils) to the culture medium was carried out. As it is shown in Figure 1, the evolution of lipase production was different depending on the lipid material used. The highest lipase activity was obtained when sunower oil-soaked barley bran was utilised. Lipase began on the fourth day (8 kU l1 ), and from there onwards it increased up to a maximum value of 21 kU l1 in the last day of the culture. Since addition of exogenous oils appeared to be required for increasing lipase secretion in solid state cultures on barley bran, a triacylglycerol-rich waste

Fig. 1. Lipase production in solid state cultures of Y. lipolytica grown on oil-soaked barley bran: , corn oil, , sunower oil and , olive oil and on , triturated nut.

Fig. 2. Lipase production rate in solid state cultures of Y. lipolytica obtained in each culture condition ( on oil-soaked barley bran).

(triturated nut) was assayed as support substrate. Nut has an oil content of 6575% and a sugar content of 68%. In these cultures, reducing sugars concentration progressively diminished down to 8 g l1 on the sixth day; from there onwards it remained around 10 g l1 . Lipase production began on the fourth day (13 kU l1 ), then sharply increased up to a maximum value of 23 kU l1 (equivalent to 69 U g1 of dry matter) on the eleventh day (Figure 1). This value was almost 5-fold higher than that found in nylon sponge cultures and around 92% higher than that obtained in barley bran cultures. In all cultures, the evolution of pH was similar. The pH dropped from 6 to a value of 4.55. This behaviour could be attributed to the release of fatty acids in the presence of lipase.

1228 Figure 2 shows the lipase production rate obtained in each culture condition. The production rate was slower in nylon sponge (0.36 g l1 d1 ) and in barley bran cultures (0.93 g l1 d1 ), whereas in nut and in sunower oil-soaked barley bran cultures it was faster (average rate of about 1.76 g l1 d1 ). It is noteworthy that the supports showing a similar behaviour (sunower oil-soaked barley bran, triturated nut) have a higher content in polyunsaturated fatty acids, which could be responsible for the high lipase activities obtained. Besides, visual assessment indicated that cell growth was higher in these cultures. This could mean that lipase production is closely related to biomass, as it has been argued by other researchers (Okeke & Okolo 1990, Corzo & Revah 1999). As previously indicated, some researchers have studied lipase synthesis in solid state cultures of several microorganisms. A comparison between their results and those obtained here for Y. lipolytica would be necessary in order to determine if solid state culture could be developed into a promising process for lipase production. In the present work, a maximum activity of 69 U g1 dry matter (23 kU l1 ) was obtained when the yeast was grown on triturated nut, determined as the best support. This data compare favourably with those reported for solid state cultures of other microorganisms. Rhizopus rhizopodiformis and Rhizomucor pusillus reached lipase activities up to 80 U g1 dry matter and 20 U g1 dry matter, respectively, using a mixture of olive oil cake and sugar cane bagasse as support (Gombert et al. 1999). Lipase activity around 30 U g1 dry matter was secreted by Penicillium restrictum using babassu oil cake (Cordova et al. 1998). Rhizopus delemar when grown in a solid state culture with a polymeric resin produced a lipase which was produced and simultaneously adsorbed on the support: 96 U g1 initial dry matter were obtained when dextrin was used as the carbon source against only 68 and 58 U g1 for maltose and glucose, respectively (Christen et al. 1995). The highest lipase activity (364 U g1 dry substrate) was obtained in solid state cultures of Aspergillus niger on gingelly oil cake (Kamini et al. 1998). Moreover, the highest lipase activity obtained in the present work (23 kU l1 medium) was similar to that reported for submerged cultures of Yarrowia lipolytica (Corzo & Revah 1999), in which 32 kU l1 were detected after optimisation of the culture conditions. Therefore, solid state culture on agroindustrial wastes appears as a promising and economical method for lipase production by this microorganism: good enzyme yields can be obtained, using a cheap raw material and with low capital investment. Besides, the results obtained in this work conrm the need of lipidic materials in the culture medium in order to increase lipase activity levels. This agrees with the results reported by Corzo & Revah (1999) for submerged cultures of Yarrowia lipolytica 681, in which the addition of olive oil or tributyrin enhanced extracellular lipolytic production. A similar behaviour was found for other microorganisms, such as Hendersonula toruloidea, Rhizopus delemar, Alternaria brassicicola and Candida rugosa (Espinosa et al. 1990, Berto et al. 1997, Obradors et al. 1993, Odibo et al. 1995). The potential of food and agroindustrial wastes for lipase production in solid state cultures of Y. lipolytica has been demonstrated, since they led to much higher activities than nylon sponge. In addition to this, by employing these wastes the initial amount of the added glucose could be reduced. Therefore, the utilisation of such wastes both promoted high enzymatic levels and contributed to diminish production costs. Acknowledgements This work was nanced by the Spanish Ministry of Science and Technology and European FEDER (Project PPQ2001-3361). References
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