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α-Galactosidase production by Aspergillus oryzae in solid-state fermentation
Bioresource Technology 98 (2007) 958–961
Short Communication
a-Galactosidase production by Aspergillus oryzae in solid-state fermentation S.K. Shankar, V.H. Mulimani
*
Department of Biochemistry, Gulbarga University, Gulbarga, 585106, Karnataka, India Received 6 December 2005; received in revised form 11 March 2006; accepted 23 March 2006 Available online 19 May 2006
Abstract Comparisons were made for a-galactosidase production using red gram plant waste (RGPW) with wheat bran (WB) and other locally available substrates using the fungus Aspergillus oryzae under solid-state fermentation (SSF). RGPW proved to be potential substrate for a-galactosidase production as it gave higher enzyme titers (3.4 U/g) compared to WB (2.7 U/g) and other substrates tested. Mixing WB with RGPW (1:1, w/w) resulted enhanced a-galactosidase yield. The volume of moistening agent in the ratio of 1:2 (w/v), pH 5.5 and 1 ml (1 · 106 spores) of inoculum volume and four days incubation were optimum for a-galactosidase production. Increase in substrate concentration (RGPW + WB) did not decrease enzyme yield in trays. 2006 Elsevier Ltd. All rights reserved. Keywords: a-Galactosidase; Solid state fermentation; Aspergillus oryzae; Red gram plant waste; Parameter optimization; Tray fermentation
1. Introduction a-Galactosidase (a-D-galactopyrinoside galactohydrolase EC 3.2.1.22) is widely distributed in microorganisms, plants and animals. It hydrolyses variety of simple a-Dgalactosides such as oligosaccharides and more complex polysaccharides (Dey and Pridham, 1972). a-Galactosidase has several applications. In processing of beet sugar, the enzyme aided raffinose degradation can be utilized to avoid raffinose inhibition of normal crystallization. Hydrolysis of raffinose and stachyose present in leguminous food and feed is another application area, as these oligosaccharides cause intestinal discomfort, flatulence and low feed utilization in monogastrites (Ulezlo and Zaprometova, 1982). In the pulp and paper industry, biobleaching can be improved by adding a-galactosidase in combination with xylanase. Furthermore, conversion of the cheap gelling agent, guar gum, to a more attractive locust bean gum like polysaccharide can be achieved by partial removal of some galactoside units from guar polysaccharide (Bulpin et al., 1990). Asper*
Corresponding author. Tel.: +91 8472 248819; fax: +91 8472 245632. E-mail address: v_h_mulimani@rediffmail.com (V.H. Mulimani).
0960-8524/$ - see front matter 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2006.03.013
gillus oryzae has been identified as a source of a-galactosidase (Cruz and Park, 1982). This organism has many advantages over many other microbial sources. It is widely accepted as source of enzyme used for food and feeds. A. oryzae has been accorded as GRAS (generally regarded as safe) status (Reichelt, 1983). SSF has gained importance for the production of microbial enzymes due to economical advantages over conventional submerged fermentation (Holker et al., 2004; Pandey et al., 1999) and due to the possibility of using cheap and abundant agro-industrial waste as substrate. It can be of special interest in those processes where the crude fermented product may be used directly as the enzyme source (Holker and Lenz, 2005; Krishna, 2005 and Pandey et al., 1999). There has been considerable interest to produce a-galactosidase in SSF processes. Among various groups of microorganisms used in SSF, the filamentous fungi are most exploited because of their ability to grow on complete solid substrate and production of wide range of extracellular enzymes (Krishna, 2005). The aim of this work was to optimize the production of a-galactosidase by A. oryzae under SSF conditions. To our knowledge, this is the first report of
S.K. Shankar, V.H. Mulimani / Bioresource Technology 98 (2007) 958–961
production of a-galactosidase from A. oryzae using red gram plant waste, which is an agricultural crop residue obtained after the postharvestment of red gram crop in India. 2. Methods 2.1. Microorganism, chemicals, solid substrates and inoculum
959
0.1 ml of suitably diluted enzyme + 0.8 ml of 0.2 M acetate buffer (pH 4.8) + 0.1 ml of 2 mM PNPG. It was incubated at 50 C for 15 min. The reaction was arrested by adding 3 ml of 0.2 M Na2CO3 solution. The absorbance was measured at 405 nm in a spectrophotometer (Elico Ltd., India). One unit of enzyme activity was defined as the amount of enzyme, which produced 1 lmol of paranitrophenol min 1 under assay conditions. a-Galactosidase production under SSF was expressed as U/g dry fermented mass. Each sample was tested in duplicate.
A fungal strain of A. oryzae was used (Prashanth and Mulimani, 2005). It was grown on PDA slants and stored at 4 C in a refrigerator. PNPG (p-nitrophenyl-a-D-galactopyranoside) was purchased from Sigma chemicals (USA). Solid substrate viz. red gram plant waste (RGPW), wheat bran (WB), rice bran (RB), chickpea plant waste (CPPW), red gram flour (RGF), red gram husk (RGH), pine apple waste (PAW), apple waste (AW), orange waste (OW), groundnut cake (GC), sugarcane bagasse (SB), carob pod (CP) were obtained locally. Inoculum was prepared by suspending the spores from a PDA slant by adding 10 ml of sterile distilled water containing 0.1% Tween 80, which contained 1 · 106 spores/ml.
3. Results and discussion
2.2. Fermentation media
3.1. Screening the substrates for a-galactosidase production
Ten grams of finely chopped solid substrates, which passed through sieve of 1200 lm (for RGF particle size was 400 lm) were taken into 250 ml conical flasks. The substrates were moistened with mineral salt solution (1:2 w/v) containing (g l 1) K2HPO4, 6.3; KH2PO4, 1.8; NH4NO3, 1; MgSO4, 1; CaCl2, 0.1; FeSO4, 0.1; MnSO4, 0.1; NaMo7O24, 0.006. After autoclaving at 121 C for 20 min, and cooling to room temperature, the flasks were inoculated with 1 ml spore suspension and incubated at 30 C. For tray fermentation, enamel coated metallic trays (45 · 30 · 4 cm) containing 100–500 g of red gram plant waste + wheat bran (1:1, w/w), moistened with mineral salt solution as above, autoclaved and inoculated with 10% of inoculum (w/v). The contents of the trays were mixed before and after inoculation. The trays were covered with aluminium foil and incubated in a temperature control chamber at 30 C.
Table 1 shows the results on enzyme production by different substrates. Evidently RGPW served as the best substrate for a-galactosidase production by A. oryzae as it gave the highest enzyme titer (3.4 U/g). WB (2.7 U/g)
2.3. Enzyme extraction Enzyme extraction was carried out by mixing fermented mass with sodium acetate buffer (0.2 M, pH 4.8; 1:10, w/v) for 1 h on an orbital shaker at 200 rpm. Contents of the flasks were filtered through muslin cloth and the filtrate was centrifuged at 2070 · g for 10 min. The supernatant obtained was used for a-galactosidase assay. 2.4. Assay of a-galactosidase Enzyme activity was assayed according to the method of Dey and Pridham (1972). Reaction mixture contained
2.5. Effect of initial pH, inoculum volume and moistening agent on a-galactosidase production To investigate the effect of pH on a-galactosidase production, media pH were adjusted at different pH (2.8–7.2) with 0.1 M HCL and 0.1 M NaOH. To investigate the effect of inoculum size, 0.1–2 ml spore suspension (1 · 106 spores ml 1) was used. To study the effect of mineral salts, volume of moistening agent was varied from 5–50 ml. Fermentation was carried out at 30 C for 4 days.
Table 1 Production of a-galactosidase by A. oryzae in solid state fermentation using various substrates and their combinations with RGPW (1:1, w/w) Substrates (10 g)
a-Galactosidase activity (U/g)
Red gram plant waste Chick pea plant waste Red gram flour Red gram husk Wheat bran Rice bran Pine apple waste Apple waste Orange waste Groundnut cake Sugarcane bagasse Carob pod RGPW + WB RGPW + RB RGPW + PAW RGPW + CPPW RGPW + OW RGPW + RGH RGPW + AW RGPW + SB RGPW + GC
3.40 ± 0.13 0.68 ± 0.03 2.20 ± 0.36 0.660 ± 0.05 2.70 ± 0.16 0.45 ± 0.05 0.285 ± 0.01 0.300 ± 0.02 0.810 ± 0.04 0.179 ± 0.03 0.281 ± 0.03 0.237 ± 0.02 4.37 ± 0.11 2.83 ± 0.16 2.78 ± 0.05 2.17 ± 0.12 2.81 ± 0.05 3.01 ± 0.01 2.80 ± 0.02 2.71 ± 0.04 0.179 ± 0.03
Red gram plant waste (RGPW); Chickpea plant waste (CPPW); Red gram husk (RGH); Wheat bran (WB); Rice bran (RB); Pine apple waste (PAW); Apple waste (AW); Orange waste (OW); Ground nut cake (GC); Sugarcane bagasse (SB).
960
S.K. Shankar, V.H. Mulimani / Bioresource Technology 98 (2007) 958–961
and RGF (2.2 U/g) also served well for enzyme production but relatively low enzyme titers were observed. But other substrates such as CPPW, RGH, RB, PAW, AW, OW, GC, SB and CP were less effective for enzyme production (Table 1). a-Galactosidase production on WB was surprisingly less. This was in contrast to earlier reports, which described WB as a potential substrate for a-galactosidase production (Cruz and Park, 1982; Annunziato et al., 1986 and Wang et al., 2004). This is the first report of utilization of RGPW as solid substrate for a-galactosidase production by A. oryzae by SSF. Wong-Leung et al. (1993) used sugarcane waste and soybean waste for production of a-galactosidase by Monuscus, but they did not make comparative study with WB. In another study by Kotwal et al. (1998), soy flour and coconut cake gave higher enzyme titers compared to WB in SSF by a thermophilic fungus Humicola sp. Combination of RGPW (ratio 1:1, w/w) with WB, RB, AW, CPPW, OW, RGH, AW, SB resulted increase in enzyme production (Table 1). Maximum activity of 4.37 U/g was produced with RGPW + WB (1:1, w/w). In most of the combinations tested, the increase in enzyme production was observed. This indicated that RGPW served as a good substrate for a-galactosidase production. But, RGPW + GC and RGPW + SB did not enhance the enzyme yield. During the study of a-galactosidase from Penicillium sp. in SSF higher enzyme yield was obtained with WB combined with various substrates and the maximum production was observed when WB 89% + 12% soybean meal + 8% beet pulp was used (Wang et al., 2004). Similarly, the production of a-galactosidase by Aspergillus niger NCIM 839 increased by 32.62% with the inclusion of 4% guar flour and 1% lactose in a basal wheat bran medium in SSF (Srinivas et al., 1993). Annunziato et al. (1986) reported that the yield of a-galactosidase increased when soybean flour was supplemented with WB but no activity was found when rice was supplemented in a SSF by A. oryzae.
to increased competition for carbon source and nutrients, which could lead to exhaustion of nutrients and this imbalance would result in reduced enzyme production. Wang et al. (2004) reported similar findings with Penicillium sp. Fungi are well known to favor a moist environment for their growth. Initial moisture content is a critical factor for growth and enzyme production. Optimum moisture level has to be maintained for appropriate growth and enzyme production in SSF. From experimental results, it was found that 1:2, w/v of moistening agent was sufficient for maximum enzyme production (81% moisture content). Same trend was observed when RGPW + WB (1:1, w/w) was used (data not shown). It is well established that lower moisture tends to reduce stability and substrate swelling and higher moisture levels leads to particle agglumeration, gas transfer limitation and competition from bacteria (Gowthaman et al., 2001).
3.2. Effect of initial pH and volume of moistening agent on a-galactosidase production
RGPW proved to be a potential substrate for a-galactosidase production in SSF. Combination of WB (1:1, w/w) with RGPW enhanced a-galactosidase activity. Optimum pH for a-galactosidase production was towards acidic pH range (pH 5.5) that prevented bacterial contaminations. The process of a-galactosidase production in laboratory scale may have the potential to scale-up.
The fungus could adapt to broad range of pH from 2.5 to 8.0. The optimum pH of the medium for a-galactosidase production by A. oryzae was 5.5 (data not shown). It has been reported that many kinds of fungi have more acidic pH optima during submerged culture (Wang et al., 2004). It could be speculated that solid substrate contributed to a better buffering capacity, and filamentous fungi have reasonably good growth over a broad range of pH 2–9, with an optimal range of 3.8–6.0. This typical pH versatility of fungi can be beneficially exploited to prevent or minimize bacterial contamination, especially choosing a lower pH (Krishna, 2005). There was a gradual increase in the enzyme synthesis with increase in inoculum volume up to 1 ml, but thereafter, a decline was observed (data not shown). Higher inoculum size did not increase enzyme production; this might be due
3.3. Cultivation in trays From tray experiment, it was found that increase in concentration of solid substrate (100–400 g) i.e. RGPW + WB (1:1, w/w) did not decrease the a-galactosidase yield. The activity for 100, 200, 300, 400 and 500 g was 4.5, 4.2, 4.01, 4.08 and 3.08 U/g, respectively (data not shown). Decrease in enzyme production might be due to increased bed height of the substrate in the tray that affected the aeration. Similar observation was made by Kotwal et al. (1998) using WB based media by thermophilic fungus Humicola sp. Maximum activity of a-galactosidase was obtained on 4th day of incubation in trays (4.34 U/g). Srinivas et al. (1994) reported optimum incubation period of 4– 5 days for a-galactosidase production by A. niger in SSF whereas Kotwal et al. (1998) reported 6 days for higher enzyme production by Humicola sp. 4. Conclusion
Acknowledgement Shankar S.K. acknowledges the award of a University Junior Research Fellowship by the Gulbarga University, Gulbarga. References Annunziato, M.E., Mohaney, R.R., Mudgett, R.E., 1986. Production of a-galactosidase from Aspergillus oryzae grown in solid state culture. J. Food Sci. 51, 1370–1371.
S.K. Shankar, V.H. Mulimani / Bioresource Technology 98 (2007) 958–961 Bulpin, P.V., Gildley, M.J., Jeffcoat, R., Underwood, D.R., 1990. Development of a biotechnological process for the modification of galactomannan polymers with plant a-galactosidase. Carbohyd. Polym. 12, 155–168. Cruz, R., Park, Y.K., 1982. Production of fungal a-galactosidase and its application to the hydrolysis of galactooligosaccharides in soybean milk. J. Food Sci. 47, 1973–1975. Dey, P.M., Pridham, J.B., 1972. Biochemistry of a-galactosidase. Adv. Enzymol. 36, 911–930. Gowthaman, M.K., Krishna, C., Moo-Young, M., 2001. Fungal solid state fermentation—An overview. In: Khachatourians, G.G., Arora, D.K. (Eds.), Applied Mycology and Biotechnology, Agriculture and Food Production. Elsevier Science, The Netherlands, pp. 305– 352. Holker, U., Hofer, M., Lenz, J., 2004. Biotechnological advantages of laboratory-scale solid-state fermentation with fungi (Mini review). Appl. Microbiol. Biotechnol. 64, 175–186. Holker, U., Lenz, J., 2005. Solid-state fermentation—are there any biotechnological advantage? Curr. Opin. Microbiol. 8, 201– 306. Kotwal, S.M., Gote, M.M., Sainkar, S.R., Khan, M.I., Khire, J.M., 1998. Production of a-galactosidase by thermophilic fungus Humicola sp. in solid state fermentation and its application in soymilk hydrolysis. Proc. Biochem. 33, 337–343. Krishna, C., 2005. Solid-state fermentation system—An overview. Critic. Rev. Biotechnol. 25, 1–30.
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Pandey, A., Selvakumar, P., Soccal, C.R., Nigam, P., 1999. Solid state fermentation for the production of industrial enzymes. Curr. Sci. 77, 149–169. Prashanth, S.J., Mulimani, V.H., 2005. Soymilk oligosaccharide hydrolysis by Aspergillus oryzae a-galactosidase immobilized in calcium alginate. Proc. Biochem. 40, 1199–1205. Reichelt, J.R., 1983. In: Godfrey, Reichalt, J. (Eds.), Industrial Enzymology. Nature Press, New York, p. 138. Srinivas, M.R.S., Nagin, C., Lonsane, B.K., 1994. Use of placket-burman design for rapid screening of several nitrogen sources, growth product promoters, minerals and enzyme inducers for the production of agalactosidase by Aspergillus niger MRSS 234 in solid state fermentation system. Bioprocess. Eng. 10, 139–144. Srinivas, M.R.S., Sriram, P., Lonsane, B.R., 1993. Growth kinetics, agalactosidase biosynthesis and concomitant production of invertase by Aspergillus niger NCIM 839 in solid state fermentation system. Chem. Mikrobiol. Technol. Lebensm. 15, 41–46. Ulezlo, V., Zaprometova, O.M., 1982. Microbial a-galactosidase (A review). Appl Biochem. Microbol. 18, 3–15. Wang, C.L., Li, D.F., Lu, Wang, W.Q., Lai, C.H., 2004. Influence of cultivation conditions on the a-galactosidase biosynthesis from a novel strain of Penicillium sp. in solid-state fermentation. Lett. Appl. Microbiol. 39, 369–375. Wong-Leung, Y.L., Fong, W.F., Lam, W.L., 1993. Production of agalactosidase by Monascus grown on soybean and sugarcane wastes. World J. Microbiol. Biotechnol. 9, 529–533.
Short Communication
a-Galactosidase production by Aspergillus oryzae in solid-state fermentation S.K. Shankar, V.H. Mulimani
*
Department of Biochemistry, Gulbarga University, Gulbarga, 585106, Karnataka, India Received 6 December 2005; received in revised form 11 March 2006; accepted 23 March 2006 Available online 19 May 2006
Abstract Comparisons were made for a-galactosidase production using red gram plant waste (RGPW) with wheat bran (WB) and other locally available substrates using the fungus Aspergillus oryzae under solid-state fermentation (SSF). RGPW proved to be potential substrate for a-galactosidase production as it gave higher enzyme titers (3.4 U/g) compared to WB (2.7 U/g) and other substrates tested. Mixing WB with RGPW (1:1, w/w) resulted enhanced a-galactosidase yield. The volume of moistening agent in the ratio of 1:2 (w/v), pH 5.5 and 1 ml (1 · 106 spores) of inoculum volume and four days incubation were optimum for a-galactosidase production. Increase in substrate concentration (RGPW + WB) did not decrease enzyme yield in trays. 2006 Elsevier Ltd. All rights reserved. Keywords: a-Galactosidase; Solid state fermentation; Aspergillus oryzae; Red gram plant waste; Parameter optimization; Tray fermentation
1. Introduction a-Galactosidase (a-D-galactopyrinoside galactohydrolase EC 3.2.1.22) is widely distributed in microorganisms, plants and animals. It hydrolyses variety of simple a-Dgalactosides such as oligosaccharides and more complex polysaccharides (Dey and Pridham, 1972). a-Galactosidase has several applications. In processing of beet sugar, the enzyme aided raffinose degradation can be utilized to avoid raffinose inhibition of normal crystallization. Hydrolysis of raffinose and stachyose present in leguminous food and feed is another application area, as these oligosaccharides cause intestinal discomfort, flatulence and low feed utilization in monogastrites (Ulezlo and Zaprometova, 1982). In the pulp and paper industry, biobleaching can be improved by adding a-galactosidase in combination with xylanase. Furthermore, conversion of the cheap gelling agent, guar gum, to a more attractive locust bean gum like polysaccharide can be achieved by partial removal of some galactoside units from guar polysaccharide (Bulpin et al., 1990). Asper*
Corresponding author. Tel.: +91 8472 248819; fax: +91 8472 245632. E-mail address: v_h_mulimani@rediffmail.com (V.H. Mulimani).
0960-8524/$ - see front matter 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2006.03.013
gillus oryzae has been identified as a source of a-galactosidase (Cruz and Park, 1982). This organism has many advantages over many other microbial sources. It is widely accepted as source of enzyme used for food and feeds. A. oryzae has been accorded as GRAS (generally regarded as safe) status (Reichelt, 1983). SSF has gained importance for the production of microbial enzymes due to economical advantages over conventional submerged fermentation (Holker et al., 2004; Pandey et al., 1999) and due to the possibility of using cheap and abundant agro-industrial waste as substrate. It can be of special interest in those processes where the crude fermented product may be used directly as the enzyme source (Holker and Lenz, 2005; Krishna, 2005 and Pandey et al., 1999). There has been considerable interest to produce a-galactosidase in SSF processes. Among various groups of microorganisms used in SSF, the filamentous fungi are most exploited because of their ability to grow on complete solid substrate and production of wide range of extracellular enzymes (Krishna, 2005). The aim of this work was to optimize the production of a-galactosidase by A. oryzae under SSF conditions. To our knowledge, this is the first report of
S.K. Shankar, V.H. Mulimani / Bioresource Technology 98 (2007) 958–961
production of a-galactosidase from A. oryzae using red gram plant waste, which is an agricultural crop residue obtained after the postharvestment of red gram crop in India. 2. Methods 2.1. Microorganism, chemicals, solid substrates and inoculum
959
0.1 ml of suitably diluted enzyme + 0.8 ml of 0.2 M acetate buffer (pH 4.8) + 0.1 ml of 2 mM PNPG. It was incubated at 50 C for 15 min. The reaction was arrested by adding 3 ml of 0.2 M Na2CO3 solution. The absorbance was measured at 405 nm in a spectrophotometer (Elico Ltd., India). One unit of enzyme activity was defined as the amount of enzyme, which produced 1 lmol of paranitrophenol min 1 under assay conditions. a-Galactosidase production under SSF was expressed as U/g dry fermented mass. Each sample was tested in duplicate.
A fungal strain of A. oryzae was used (Prashanth and Mulimani, 2005). It was grown on PDA slants and stored at 4 C in a refrigerator. PNPG (p-nitrophenyl-a-D-galactopyranoside) was purchased from Sigma chemicals (USA). Solid substrate viz. red gram plant waste (RGPW), wheat bran (WB), rice bran (RB), chickpea plant waste (CPPW), red gram flour (RGF), red gram husk (RGH), pine apple waste (PAW), apple waste (AW), orange waste (OW), groundnut cake (GC), sugarcane bagasse (SB), carob pod (CP) were obtained locally. Inoculum was prepared by suspending the spores from a PDA slant by adding 10 ml of sterile distilled water containing 0.1% Tween 80, which contained 1 · 106 spores/ml.
3. Results and discussion
2.2. Fermentation media
3.1. Screening the substrates for a-galactosidase production
Ten grams of finely chopped solid substrates, which passed through sieve of 1200 lm (for RGF particle size was 400 lm) were taken into 250 ml conical flasks. The substrates were moistened with mineral salt solution (1:2 w/v) containing (g l 1) K2HPO4, 6.3; KH2PO4, 1.8; NH4NO3, 1; MgSO4, 1; CaCl2, 0.1; FeSO4, 0.1; MnSO4, 0.1; NaMo7O24, 0.006. After autoclaving at 121 C for 20 min, and cooling to room temperature, the flasks were inoculated with 1 ml spore suspension and incubated at 30 C. For tray fermentation, enamel coated metallic trays (45 · 30 · 4 cm) containing 100–500 g of red gram plant waste + wheat bran (1:1, w/w), moistened with mineral salt solution as above, autoclaved and inoculated with 10% of inoculum (w/v). The contents of the trays were mixed before and after inoculation. The trays were covered with aluminium foil and incubated in a temperature control chamber at 30 C.
Table 1 shows the results on enzyme production by different substrates. Evidently RGPW served as the best substrate for a-galactosidase production by A. oryzae as it gave the highest enzyme titer (3.4 U/g). WB (2.7 U/g)
2.3. Enzyme extraction Enzyme extraction was carried out by mixing fermented mass with sodium acetate buffer (0.2 M, pH 4.8; 1:10, w/v) for 1 h on an orbital shaker at 200 rpm. Contents of the flasks were filtered through muslin cloth and the filtrate was centrifuged at 2070 · g for 10 min. The supernatant obtained was used for a-galactosidase assay. 2.4. Assay of a-galactosidase Enzyme activity was assayed according to the method of Dey and Pridham (1972). Reaction mixture contained
2.5. Effect of initial pH, inoculum volume and moistening agent on a-galactosidase production To investigate the effect of pH on a-galactosidase production, media pH were adjusted at different pH (2.8–7.2) with 0.1 M HCL and 0.1 M NaOH. To investigate the effect of inoculum size, 0.1–2 ml spore suspension (1 · 106 spores ml 1) was used. To study the effect of mineral salts, volume of moistening agent was varied from 5–50 ml. Fermentation was carried out at 30 C for 4 days.
Table 1 Production of a-galactosidase by A. oryzae in solid state fermentation using various substrates and their combinations with RGPW (1:1, w/w) Substrates (10 g)
a-Galactosidase activity (U/g)
Red gram plant waste Chick pea plant waste Red gram flour Red gram husk Wheat bran Rice bran Pine apple waste Apple waste Orange waste Groundnut cake Sugarcane bagasse Carob pod RGPW + WB RGPW + RB RGPW + PAW RGPW + CPPW RGPW + OW RGPW + RGH RGPW + AW RGPW + SB RGPW + GC
3.40 ± 0.13 0.68 ± 0.03 2.20 ± 0.36 0.660 ± 0.05 2.70 ± 0.16 0.45 ± 0.05 0.285 ± 0.01 0.300 ± 0.02 0.810 ± 0.04 0.179 ± 0.03 0.281 ± 0.03 0.237 ± 0.02 4.37 ± 0.11 2.83 ± 0.16 2.78 ± 0.05 2.17 ± 0.12 2.81 ± 0.05 3.01 ± 0.01 2.80 ± 0.02 2.71 ± 0.04 0.179 ± 0.03
Red gram plant waste (RGPW); Chickpea plant waste (CPPW); Red gram husk (RGH); Wheat bran (WB); Rice bran (RB); Pine apple waste (PAW); Apple waste (AW); Orange waste (OW); Ground nut cake (GC); Sugarcane bagasse (SB).
960
S.K. Shankar, V.H. Mulimani / Bioresource Technology 98 (2007) 958–961
and RGF (2.2 U/g) also served well for enzyme production but relatively low enzyme titers were observed. But other substrates such as CPPW, RGH, RB, PAW, AW, OW, GC, SB and CP were less effective for enzyme production (Table 1). a-Galactosidase production on WB was surprisingly less. This was in contrast to earlier reports, which described WB as a potential substrate for a-galactosidase production (Cruz and Park, 1982; Annunziato et al., 1986 and Wang et al., 2004). This is the first report of utilization of RGPW as solid substrate for a-galactosidase production by A. oryzae by SSF. Wong-Leung et al. (1993) used sugarcane waste and soybean waste for production of a-galactosidase by Monuscus, but they did not make comparative study with WB. In another study by Kotwal et al. (1998), soy flour and coconut cake gave higher enzyme titers compared to WB in SSF by a thermophilic fungus Humicola sp. Combination of RGPW (ratio 1:1, w/w) with WB, RB, AW, CPPW, OW, RGH, AW, SB resulted increase in enzyme production (Table 1). Maximum activity of 4.37 U/g was produced with RGPW + WB (1:1, w/w). In most of the combinations tested, the increase in enzyme production was observed. This indicated that RGPW served as a good substrate for a-galactosidase production. But, RGPW + GC and RGPW + SB did not enhance the enzyme yield. During the study of a-galactosidase from Penicillium sp. in SSF higher enzyme yield was obtained with WB combined with various substrates and the maximum production was observed when WB 89% + 12% soybean meal + 8% beet pulp was used (Wang et al., 2004). Similarly, the production of a-galactosidase by Aspergillus niger NCIM 839 increased by 32.62% with the inclusion of 4% guar flour and 1% lactose in a basal wheat bran medium in SSF (Srinivas et al., 1993). Annunziato et al. (1986) reported that the yield of a-galactosidase increased when soybean flour was supplemented with WB but no activity was found when rice was supplemented in a SSF by A. oryzae.
to increased competition for carbon source and nutrients, which could lead to exhaustion of nutrients and this imbalance would result in reduced enzyme production. Wang et al. (2004) reported similar findings with Penicillium sp. Fungi are well known to favor a moist environment for their growth. Initial moisture content is a critical factor for growth and enzyme production. Optimum moisture level has to be maintained for appropriate growth and enzyme production in SSF. From experimental results, it was found that 1:2, w/v of moistening agent was sufficient for maximum enzyme production (81% moisture content). Same trend was observed when RGPW + WB (1:1, w/w) was used (data not shown). It is well established that lower moisture tends to reduce stability and substrate swelling and higher moisture levels leads to particle agglumeration, gas transfer limitation and competition from bacteria (Gowthaman et al., 2001).
3.2. Effect of initial pH and volume of moistening agent on a-galactosidase production
RGPW proved to be a potential substrate for a-galactosidase production in SSF. Combination of WB (1:1, w/w) with RGPW enhanced a-galactosidase activity. Optimum pH for a-galactosidase production was towards acidic pH range (pH 5.5) that prevented bacterial contaminations. The process of a-galactosidase production in laboratory scale may have the potential to scale-up.
The fungus could adapt to broad range of pH from 2.5 to 8.0. The optimum pH of the medium for a-galactosidase production by A. oryzae was 5.5 (data not shown). It has been reported that many kinds of fungi have more acidic pH optima during submerged culture (Wang et al., 2004). It could be speculated that solid substrate contributed to a better buffering capacity, and filamentous fungi have reasonably good growth over a broad range of pH 2–9, with an optimal range of 3.8–6.0. This typical pH versatility of fungi can be beneficially exploited to prevent or minimize bacterial contamination, especially choosing a lower pH (Krishna, 2005). There was a gradual increase in the enzyme synthesis with increase in inoculum volume up to 1 ml, but thereafter, a decline was observed (data not shown). Higher inoculum size did not increase enzyme production; this might be due
3.3. Cultivation in trays From tray experiment, it was found that increase in concentration of solid substrate (100–400 g) i.e. RGPW + WB (1:1, w/w) did not decrease the a-galactosidase yield. The activity for 100, 200, 300, 400 and 500 g was 4.5, 4.2, 4.01, 4.08 and 3.08 U/g, respectively (data not shown). Decrease in enzyme production might be due to increased bed height of the substrate in the tray that affected the aeration. Similar observation was made by Kotwal et al. (1998) using WB based media by thermophilic fungus Humicola sp. Maximum activity of a-galactosidase was obtained on 4th day of incubation in trays (4.34 U/g). Srinivas et al. (1994) reported optimum incubation period of 4– 5 days for a-galactosidase production by A. niger in SSF whereas Kotwal et al. (1998) reported 6 days for higher enzyme production by Humicola sp. 4. Conclusion
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