There is a growing interest globally in producing ethanol from different lignocellulosic biomasses (agriculture residue, perennial crops, woody substance and municipal solid waste). These are mainly composed of cellulose, hemicellulose and lignin, and may serve as cheap and renewable feedstocks for bioethanol production. Cellulose is made up of uniform structure of β-1,4 linked glucose units and its biodegradability may vary in different biomasses depending on the strength of association of the cellulose with other plant compounds. The cost of enzymatic hydrolysis of biomass varies from 15 - 30% of the total cost of biofuel production. An important enzyme cellobiase or β-glucosidase (BGL) catalyzes the hydrolysis of alkyl, aryl- β-glucosides, diglucosides and oligosaccharides . This is a key enzyme for cellulose degradation and is widely used in the productionof fuel ethanol, prevent discoloration of fruit juices, cause enzymatic release of aromatic compounds from glucosidic precursors present in fruits and fermenting products (Shoseyov et al., 1990; Das et al., 2004; Zhang et al., 2006). BGL is a part of the cellulase complex. It is inhibited by glucose, has high susceptibility to thermal inactivation and also lower BGL concentration leads to catabolite repression due to accumulation of cellobiose (Ikram-ul-Haq et al., 2006). It is therefore desirable to have a higher extracellular BGL production. So, the focus is on various agro-industrial waste that can be utilized to produce BGL enzyme by microbial transformations inan eco-friendly and economic way. With the purpose of reducing the cost of the culture media we selected different wastes or substrates for the maximum extracellularproductionof BGL.
The results concerning fungi strains growth in basic medium containing olive oil (first step of screening) can be seen in Fig. 1. In the first 24 hours all strains exhibited similar growth; however after 48 hours the growth rate had become differentiated. The diameter and morphology of the colony (homogeneity, sporulation and absence of sectors) were taken as the parameters for selection. The growth rate of A. niger cont45, A. niger 11T64A5 and 3T5B8 strains was comparatively slower. Nine fungi strains (seven A. niger: 11T53B6, 11T25A5, 11T25A3, 11T53B8, 11T53B7, 3T5B8 sector, 11T53A14, Thermomyces lanuginosus IOC-4145 and A. niger cont32) were selected as lipase producers. In the second step that consisted of growing in medium containing Bromophenol blue dye at pH 4.6, all seven A. niger strains (11T53B6, 11T25A5, 11T25A3, 11T53B8, 11T53B7, 3T5B8 sector, 11T53A14) changed the medium color from blue to yellow, probably due to the productionof fatty acids by hydrolytic action of lipase. The other two strains achieved a good growth rate, without changing the medium color. In the third step all nine strains were tested insolidstatefermentation using wheat bran and olive oil 2% (w/w) as substrate in 250ml Erlenmeyer flasks containing 20g of the medium. The values of
According to the previous studies and present study on productionof CPC, it is concluded that using SSF is more suitable than SLF to produce of antibiotics by filamentous fungi. Some criteria were considered for this conclusion; increasing outcome, better control ofsolidstate fermenter conditions and decreasing the cost. Also, since the content of fermenter is important, it is worthy to find a suitable content. There is no direct relation between CPC production capacity of a strain in SSF and SLF conditions, and a strain's potential is the main factor of maximum CPC productionin SSF.
Effect of different nutrient sources on productionof enzymes The productionof enzyme and other commercially important products by filamentous fungi in submerged fermentation have long been established. In recent years studies on solidstatefermentation (SSF) have increased significantly (20). The choice of the kind offermentation depends on the physiological adaptation of the organism. Generally, in submerged cultivation the growth form of filamentous fungi varies between pelleted and filamentous, each form having its own characteristics, which can affect the rate of enzyme production by influencing the mass transfer rate (20). While in submerged fermentation (SmF), the fungus is exposed to hydrodynamic forces, in SSF, growth is restricted to the surface of the solid matrix. SSF is defined as the culture in which a microorganism grows on a moist insoluble solid material in the absence or near absence of free water (1). Our isolated strain of T. aurantiacus showed a very low CMCase and xylanase activities when it was grown on liquid medium (data not shown). However, this fungus was a good producer of these enzymes on SSF. When SSF was used, any substrate produced more activity than in SmF. This may be due SSF provides the fungus with an environment closer to its natural habitat (wood and decayed organic matter), which stimulates this strain to produce more hemicellulolytic enzymes. As is shown in Table 1, the T. aurantiacus produced xylanase and CMCase independently of the material used as nutrient
Lipases are hydrolytic enzymes that act in aqueous-organic interfaces, catalyzing the cleavage of ester bonds in triglycerides and producing glycerol and free fatty acids (Freire and Castilho, 2000). There has been a growing interest for lipases of microbial origin since the eighties. Lipases find an increasing range of applications due to the different catalytic reactions and their regio and enantio selectivity in detergents, foods, pharmaceuticals, fine chemicals, leathers and pulp and paper industries (Freire and Castilho, 2000). However, the development of low-cost processes for the productionof lipases create a greater industrial application for these enzymes. In this context, solid-statefermentation (SSF) is an interesting low-cost alternative for the productionof biomolecules. SolidStateFermentation has great potentials due to its simplicity of operation, low capital cost and high volume productivity (Akpan et al., 1999) and has gained renewed interest because of its potential to produce higher yields of fungal metabolites than submerged fermentation (Akpan and Adelaja 2004). In SSF, agroindustrial residues can be employed as culture medium. These low-cost and abundant raw materials contribute to reduce production costs (Freire and Castilho, 2000). Many agricultural products from cereals or legumes are cheap and readily available in the developing countries as sources of carbohydrates and proteins, so they could provide the required nutrients in the fermentation medium (Akpan et al. 1999). The use of agricultural products such as wheat bran solid medium for enzyme production has been well established, but it is scarce in the tropics. However, alternatives such as rice bran solid medium require supplements such as yeast extract and peptone; but these are expensive materials in the tropics (Akpan et al. 1999). The objective of our study was to investigate the synergistic effect of various agricultural residues (rice bran, palm kernel cake, soy bean, groundnut cake and starch) combined in different ratios (as sources of carbon, nitrogen and elemental supplements) as solid media to support the growth of Aspergillus niger F7-02 for lipase production.
the areas ofsolid waste management, biomass energy conservation and its application to produce secondary metabolites (Pandey, 1992).In recent years research interest in batch solid-statefermentation (SSF) has addressed the productionof many innovative and high value products, e.g. single-cell protein (SCP), protein enriched feed, ethanol, enzymes, mycotoxins, from starchy materials and a variety of wastes utilized by fungi (Pandey et al., 1999). The use of filamentous fungi for the productionof commercially important metabolites has increased rapidly over the past half century and the preference ofproductionof fungal enzymes in submerged fermentation (SmF) has been rapidly switched over to SSF.
PRODUCTIONOF LIPASES WITH Aspergillus niger AND Aspergillus fumigatus THROUGH SOLIDSTATEFERMENTATION: EVALUATION OF SUBSTRATE SPECIFICITY AND USE IN ESTERIFICATION AND ALCOHOLYSIS REACTIONS. Filamentous fungi were cultured under solidstatefermentationof soybean residues to produce lipases. Enzymes produced by Aspergillus niger esterified oleic and butyric acids in the presence of ethanol, while enzymes produced by Aspergillus fumigatus demonstrated no esterification activity toward lauric acid. In case of A. niger, direct lyophilization of fermented bran led to higher esterification activity. The esterification of oleic acid by enzymes of A. fumigatus was neither influenced by pH adjustment nor by the extraction process. Conversions to ethyl esters were higher after pH adjustment with lyophilized liquid extract of A. niger.
mentation than in submerged fermentation (Castilho et al., 2000). Most studies on lipolytic enzymes production with bacteria, fungi and yeasts have been performed in sub- merged fermentation; however, there are only few reports on lipase synthesis insolidstatefermentation. In recent years, considerable research has been carried out using ag- ricultural wastes, which are renewable and abundantly available to produce value-added products. For example babassu oil cake (Gombert et al., 1999), olive cake and sugar cane bagasse (Cordova et al., 1998), gingelly oil cake (Kamini et al., 1998), wheat bran (Mahadik et al., 2002), rice bran (Rao et al., 1993), Jatropha curcas seed cake (Mahanta et al., 2008), niger seed oil cake (Imandi et al., 2010a), and palm kernel cake (Imandi et al., 2010b) have been used as the substrates for lipase production.
Microorganism: The study was carried out with the strain of Trichoderma harzianum No 53 obtained from the National Institute for Research on Plant Health (INISAV, Cuba). Inoculum: Inoculum was prepared by growing the culture in Czapek medium at 30 o C for 48 h under static conditions. Mycelia (10%, wet wt. basis), so obtained, were used as inoculum. Substrate: Sugar cane molasses (density 1.43 g/ml, sugar concentration 53%) was mixed with bagasse pith in 1:1 (w/w) ratio. This amount was calculated to prevent percolation to achieve a final concentration of 37% of total sugars based on dry bagasse pith (21% based on initial dry total weight). Bagasse pith was previously sieved through a 3-mm sieve. Initial pH and
As is evident from Table 1, although there are reports on cultivation of bacteria, yeast as well as fungi on bagasse, filamentous fungi have most widely been studied. Valino et al. (1997) used strains of Acinetobacter calcoaceticus and Cephalosporium sp.for studying interactions between microbiote of sugar cane bagasse. The results showed that a better adaptation of bagasse microbiote insolidfermentation, being able to more efficiently overcome any harmful effect compared to the mixture of bacteria and fungi each one separately. A strain of Aspergillus niger was used for biomass estimation on real and model supports (amberlite IRA-900 and bagasse) in SSF (Cordova-Lopez et al.1996). Zadrazil and Puniya (1995) employed white-rot fungus strains to study the effect of particle size of bagasse in SSF for the productionof animal feed with a view to enhancing the nutritive value of the bagasse. They used Pleurotus sp. P7 and P1, P. eryngii, Agrocybe aegarita A1, and Kuehneromyces mutabilis. The P. eryngii improved the digestibility of all the four experimental fractions of the bagasse. Importance of particle size in fungal SSF has also been emphasised by Pandey (1991 a). Gupte and Madamwar (1997 a, b) carried out co- culturing of A. ellipticus and A. fumigatus for the productionof cellulolytic enzymes. Cellulases and β -glucosidase were also produced by a strain of A. niger (Ray et al. 1993). Machado et al. (1996) screened 44 basidiomycete strains for their ability to produce ligninolytic enzymes. Among the tested strains, 12 and 7 failed to produce detectable peroxidases and phenol- oxidases, respectively. Eleven showed good production, and belonged to the genera Lentinus, Melanoporia, Peniophora, Tramets, Trichaptum and Trogia.
on those factors that increase the contact surface between the phases like: additional aeration generated by forced step of sterile air, agitation and moisture level of the substrate (Gervais and Molin, 2003). In a column bioreactor, the substrate humidity changes during fermentation owing to the saturated air passing through the medium, consequently, it was very important to define optimal conditions for these parameters in order to attain high growth and sporulation (Vandenberghe et al., 2004; Prado et al., 2005). Bacterial performance often occurs at high moisture levels. However, studies in SSF involving strains of Bacillus for the productionof enzymes, as have been observed as in aerobic SSF fungal studies, that increasing water content beyond the optimal level resulted in slowed growth and limited product formation, suggesting that the diffusion of gaseous oxygen into the larger liquid phase (within the interparticle spaces) was not adequate to support effective microbial respiration (Babu and Satyanarayana, 1996; Mamo and Gessesse, 1999). The observed results suggested that the efficiency of nutrients and oxygen transfer processes was sufficient to allow a good diffusion of solutes and gas, and promote the same cellular growth in the differents conditions: aerated columns, no aerated columns and flasks.
It was thus, seen that maximum xylanase synthesis by the Bacillus sp was observed on wheat bran medium (16.13 U/ml) when YEP was added under SSF, while corn cob supported maximum xylanase synthesis (9.88 U/ml), also under SSF, when supplementation was done with MSS (Figs. 5 and 6). In both these conditions, interestingly, addition of xylose exhibited repression. This suggested xylan degradation, stimulating xylanase synthesis and xylose production, took place in both these cases initiated by the complex C sources of wheat bran and corn cob. Agricultural residues have been reported to induce xylanase synthesis efficiently (Bakir et al., 2001; Santos et al., 2003; Gosh and Nanda, 1991), and additional xylose perhaps caused substrate inhibition. Alternately, feed back inhibition and catabolite repression by xylose cannot also be ruled out. Catabolite repression on synthesis of xylanase has been reported earlier (Kelley et al., 1989), while according to Liu et al. xylose did not cause any catabolite repression in the yeast, Trichosporon cutaneum (Liu et al., 1999). As xylose is converted to xylitol and then to xylulose during catabolism (Ethier et al., 1994) and as xylitol and xylulose do not induce xylanase synthesis (Liu et al., 1999), therefore stimulatory
Laccases (benzenediol:oxygen oxidoreductases, EC 220.127.116.11) are glycosylated polyphenol oxidases that contain four copper ions per molecule. These enzymes catalyse the one-electron oxidation of a wide variety of organic and inorganic substrates, including mono-, di- and polyphenols, methoxyphenols, aromatic amines, and ascorbate, with concomitant four-electron reduction of oxygen to water (Leonowicz et al., 2001). Laccases are produced by the majority of white-rot fungi described to-date as well as by other types of fungi and plants, insects and some bacteria. Fungal laccases are believed to be involved in the degradation of lignin, the removal of potentially toxic phenols arising during lignin degradation, the fruit body development, pigment production and antimicrobial activity (Eggert, 1997).
Polygalacturonases are enzymes with the biotechnological potential for use in fruit juice clarification and for the enhancement of filtration efficiency. The aim of this work was to assess the productionof polygalacturonase by the fungus Neosartorya glabra by means ofsolid-state and submerged fermentation using fruit peel residues as the carbon source, and also apply the enzyme in the clarification and decrease in viscosity of passion fruit and apple juices. The highest polygalacturonase (4.52 U/g/h) production was obtained by means of submerged fermentationin Vogel´s medium (1964) containing orange peel – Bahia variety (Citrus sinensis), at a concentration of 1.5% (w/v, dried mass) at 30-35°C for 72 h. The polygalacturonase of the crude extract presented optimal activity at 60°C and pH 5.5. The enzyme retained around 90% of the initial activity after 180 minutes at 40°C, and 50% of the initial activity after 150 minutes at 50°C. The enzyme was shown to be stable at acid pH values (3.0-6.5) after 120 minutes at 25 o C. All these favourable enzymatic properties make the polygalacturonase
Evidently A. niger was capable to produce glucoamylase insolid-statefermentation using a potato processing residue as substrate and the best yield was after 48 h (13.46 U mL -1 for assay number 1 and 15.82 U mL -1 for assay number 2). In the beginning as the enzyme was produced, the free glucose concentration grew up to 48 h and then its concentration as well as the glucoamylase activity started to fall. The enzyme production by the microorganism is repressed due to the glucose high concentration (Rajoka and Yasmeen, 2005) known as "glucose repression", which involves complex interactions between DNA binding repressors, their cognate elements and components of the transcriptional machinery in yeast (Griggs and Johnston, 1991; Keleher et al., 1992; Hu et al., 1995; Treitel and Carlson, 1995; De Vit et al., 1997; Park et al., 1999). Nevertheless, when the glucose concentration decreases, the microorganism starts to produce glucoamylase again and then to hydrolyze the starch to produce more glucose. This modulation (initial repression and subsequent desrepression) was also observed for Saccharomyces diastaticus (Kim et al., 2004).
In recent years, a considerable interest has been shown in using agricultural products and their residues as alternative sources of carbon such for citric acid production by A. niger (Soccol, 2001; Kolicheski, 1995; Soccol et al., 1999; Vandenberghe et al., 2000c). A variety of agro- industrial residues and by-products have been investigated with SSF techniques for their potential to be used as substrates for citric acid production (Vandenberghe et al., 2000a). A cost reduction on citric acid production can be achieved by using less expensive substrates, such as apple pomace, carrot and orange waste, cassava bagasse, coffee husk, corncob, kiwifruit peel, mussel processing wastes, okara (soy residue), rice and wheat bran (Vandenberghe et al., 1999; Garg and Hang, 1995; Aravantinos-Zafiris et al., 1994; Khare et al., 1995; Hang and Woodams, 1998; Vandenberghe, 2000). These residues are very well adapted to solid-state cultures due to their cellulosic and starchy nature. There has been an increasing trend towards efficient utilization of these residues, besides being a form of reducing environmental concerns (Soccol and Vandenberghe, 2003).
Nevertheless, when this temperature (50ºC) was applied to estimate the thermostability of endo-PG, an enzyme inactivation of approximately 50% was observed after 120 minutes of treatment (Figure 3). On the other hand, after the same time of treatment at 25, 30 and 40ºC, almost all enzyme activity was preserved. The total inactivation of endo-PG was quickly noticed after about 15 minutes at 60 and 70ºC, as reported by Naidu and Panda (2003) for polygalacturonase produced by A. niger in submerged process. This study corroborated the conclusions reported by Daniel (1996), who suggested the low stability of enzymes at temperatures higher than the ideal for the microbial growth.
displayed in Figure 4, where the horizontal axis represents the samples collected after the fermentation process. Knowing that the fermented material of each module was split into three samples, Sample 1 was collected in the axial position closest to bottom of the bed. Moisture content increased from the bottom to the top of the bed, while the enzyme activity suffered some variation in the first module, remained stable in the following two modules and steeply decreased in the last one. A similar trend was also observed by other authors (1, 7, 14, 18, 29), although the drop in the upper module was more intense than any other presented in the literature.
The effect of different carbon sources on pectinase synthesis by fungi in SSF have been studied and it is generally agreed that the optimum medium for the enhanced productionofextracellular pectinase is that containing pectic materials as an inducer (5,8,17). According to this, the present results corroborate that the culture medium with high levels of pectin, such as orange bagasse, raised the highest exo-Pg activity (11). Data of Fig. 2 showed that, on the contrary of obtained for exo-Pg, the productionof endo-Pg was not significantly affected by temperature and composition of the culture medium (Figs. 2B and D). The enzyme production by Monascus sp at 45ºC was 1.8 U/mL after 72 h, when wheat bran was the carbon source (Fig. 2A). At 50ºC, the maximum activity value was 1.6 U/mL in 20 h in the same conditions (Fig. 2B). In Aspergillus sp culture, at 45ºC, activity of 1.9 U/mL was detected after 72 h with wheat bran as substrate (Fig. 2C). The maximum activity obtained infermentation at 50ºC was 1.8 U/mL when a mixture of wheat bran and orange bagasse was the substrate (Fig. 2D).
in the solid substrate and temperature control. As the water content is limited, its control is essential to optimize the solid-statefermentation. The ideal water content forms an aqueous film on the surface, which facilitates the dissolution and transfer of nutrients and oxygen (GERVAIS & MOLIN 2003). In the present study, it has been observed that starting from water activity between 0.95 and 0.98, there was a drop inproduction for both studied enzymes. That could be related with fungal inhibition, marked by the extrapolation of the ideal water level for the development of the selected lineage, which could be influencing the metabolic route responsible for enzyme production.