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Production of pectinase from deseeded sunflower head by Aspergillus niger in submerged and solid-state conditions
Bioresource Technology 97 (2006) 2054–2058
Production of pectinase from deseeded sunXower head by Aspergillus niger in submerged and solid-state conditions Sarvamangala R. Patil a
a,b,¤
, A. Dayanand
b
Department of Microbiology, Vishweshwariah College of Applied Sciences, Gulbarga 585 102, India b PG Department of Microbiology, Gulbarga University, Gulbarga 585 106, India Received 16 May 2005; received in revised form 9 September 2005; accepted 16 September 2005 Available online 2 November 2005
Abstract Studies were carried out on the production of pectinases using deseeded sunXower head by Aspergillus niger DMF 27 and DMF 45 in submerged fermentation (SmF) and solid-state fermentation (SSF). Higher titres of endo- and exo-pectinases were observed when medium was supplemented with carbon (4% glucose for SmF and 6% sucrose for SSF) and nitrogen (ammonium sulphate, 0.3% for both SmF and SSF) sources. Green gram husk proved to be relatively a better supplement to attain higher yield of endo-pectinase (11.7 U/g) and exo-pectinase (30.0 U/g) in solid-state conditions. Maximum production of endo-pectinase (19.8 U/g) and exo-pectinase (45.9 U/g) by DMF 45 were recorded in SSF when compared to endo-pectinase (18.9 U/ml) and exo-pectinase (30.3 U/ml) by DMF 27 in SmF under optimum process conditions. © 2005 Elsevier Ltd. All rights reserved. Keywords: Pectinase; SunXower head; Aspergillus niger; Submerged; Solid-state fermentation
1. Introduction The northern part of the state of Karnataka, India is a semi arid tropical region, well known for the production of pulses (red gram, bengal gram and green gram) and oil seeds (sunXower), which are cultivated in about 1,80,000 and 30,000 ha area, respectively. In the past ten years, on an average 0.52 tons/ha of pulses and 0.79 tons/ha of oil seeds are being produced every year, generating 3.1 tons/ha of husk of pulses and 14.3 tons/ha deseeded sunXower head as agro wastes annually. Husk of pulses is partly used as cattle feed and deseeded dried sunXower head is burnt to ash after harvesting. Microbial pectinases have tremendous potential to oVer mankind. Most pectic enzyme preparations are used in the fruit processing industry and pectic enzymes alone account * Corresponding author. Address: Department of Microbiology, Vishweshwariah College of Applied Sciences, Gulbarga 585 102, India. Tel.: +91 8472 232391. E-mail address: [email protected] (S.R. Patil).
0960-8524/$ - see front matter © 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2005.09.015
for about one quarter of the world’s food enzyme production. Fungal pectinases are among the most important industrial enzymes and are of great signiWcance with wide range application in textile processing, degumming of plant bast Wbers, treatment of pectic wastewaters, papermaking and coVee and tea fermentations. Therefore, the biotechnological potential of pectinolytic enzymes from fungi has drawn a great deal of attention from various researchers worldwide. Various agro-industrial wastes such as wheat bran (Taragano et al., 1997), sugarcane bagasse (Solis-Pereyra et al., 1993, 1996), coVee pulp (Boccas et al., 1994), lemon peel (Larios et al., 1989) and apple pomace (Hours et al., 1988) have been explored for the microbial production of pectinase. Several authors have reported a wide range of pectin (15–25%) in sunXower head (Federico et al., 1988). This high content of pectin was a stimulant to explore sunXower head as a substrate for the microbial production of pectinase. Solid-state fermentation holds tremendous potential for the production of enzymes. Agro-industrial residues or wastes are generally considered as suitable substrates for
S.R. Patil, A. Dayanand / Bioresource Technology 97 (2006) 2054–2058
2. Methods 2.1. Inoculum and substrate Two strains of A. niger DMF 27 and DMF 45, isolated and characterized in our laboratory from agro waste dumping pit soil were employed in submerged and solid-state fermentation, respectively. The cultures were maintained on potato dextrose agar at 4 °C by periodic sub culturing. The inoculum was prepared in pectin medium harvesting the spores from 96 h old cultures at 25 °C. The conidia were dispersed in 0.01% Tween 80 solution. Deseeded dried sunXower head was grinded and Wne powder was used as substrate for submerged fermentation. The particles of the same with mesh sieve size adjusted to 500 m were used for solid-state fermentation. The pectin content of deseeded sunXower head was determined by gravimetric method (Ranganna, 1979). 2.2. Medium and fermentation Medium for submerged fermentation contained (%) (NH4)2SO4, 0.1; MgSO4 · 7H2O, 0.5; KH2PO4, 0.5; FeSO4 · 7H2O, 0.0005 (Solis-Pereyra et al., 1996) and 8 g powder of sunXower head (particle size > 500 m). To study the eVect of supplementation of glucose and sucrose, they were added individually at 2–10% concentration in the medium. To study the eVect of supplementation of nitrogen source, ammonium phosphate and ammonium sulphate were added at 0.1–0.5% concentrations. Husk of pulses (bengal, green and red gram) in the range of 5–25% were also added independently as supplement. Fermentation duration was examined from 24 to 120 h. Medium was autoclaved for 15 min at 120 °C. Inoculum size and pH were 1 £ 105 spores/ ml and 5.0, respectively. Fermentation was carried in 250 ml Erlenmeyer Xask by taking 100 ml medium at 34 °C on rotatary shaker (300 rpm). For SSF, medium components were same as above. Initial substrate moisture was 65%. Carbon source, nitrogen source, husk of pulses and fermentation duration were assessed similar to submerged condition. Medium was autoclaved for 15 min at 120 °C. The pH and inoculum size of the medium were adjusted to 5.0 and 1 £ 107 spores/g,
respectively. Fermentation was carried out in 250 ml shallow glass bottle at 34 °C. 2.3. Enzyme assay Liquid samples obtained from submerged fermentation were Wltered using muslin cloth. For SSF, a known quantity of fermented material was mixed with equivalent amount of distilled water and mixed thoroughly on a magnetic stirrer for 20 min and Wltered. The Wltrates were stored at 4 °C for enzyme assay. Polygalacturonase activities were measured at 45 °C by Viscometry (Ghildyal et al., 1981) for endo-pectinase (endo-p) and by the release of reducing sugars (Miller, 1959) for exo-pectinase (exo-p). For endo-p, 1 ml of a suitably diluted sample was mixed with 18 ml of 2% pectin in 0.1 M acetate buVer with pH 4.5 and reduction in viscosity was followed with Ostwald Viscometer. One endo-pectinase unit (U) was deWned as the amount of enzyme that reduces the viscosity of the solution by 50% per minute under the conditions mentioned above. For exop, 0.3 ml of a suitably diluted sample was added to a solution containing 1 ml of 0.9% of substrate and 0.7 ml of 0.1 M acetate buVer with pH 4.5. Samples were incubated at 45 °C for 30 min. Reducing sugars were determined by dinitro salicylic acid (DNS) method using galacturonic acid as reference. One exo-p unit (U) was deWned as the quantity of enzyme that liberates one micromole of galacturonic acid per minute under the conditions mentioned above. 3. Results and discussion 3.1. Evaluation of fermentation period Production of pectinase was evaluated up to 120 h (Fig. 1). The pectin content of deseeded sunXower head was 21.36% (data not shown). A gradual increase in the production of pectinase over a period up to 72 h was observed in submerged and up to 96 h in solid-state conditions (Fig. 1). The production of both endo- and exo-pectinase was high, 18 Pectinase activity (U/ml and U/g)
the production of enzymes in SSF process (Pandey et al., 1999, 2000; Pandey, 2003). Many investigations were carried out (Blandino et al., 2002; Hours et al., 1988; Sebastian et al., 1996; Solis-Pereyra et al., 1996; Boccas et al., 1994) for the production of pectinases in solid-state conditions. Submerged fermentation has also been used for pectinases production (Aguilar and Huitron, 1990; Shivakumar and Krishnanand, 1995; Galiotou-Panayotou and Kapantai, 1993). The present study reports on the development and standardization of bioprocess for the production of pectinase by Aspergillus niger in submerged and solid-state conditions employing sunXower head as substrate.
2055
DMF27 endo
16
DMF45 endo
14
DMF27 exo
12
DMF45 exo
10 8 6 4 2 0 0
48 72 96 24 Fermentation period (h)
120
Fig. 1. Evaluation of fermentation period for the production of pectinase from sunXower head by A. niger in submerged and solid-state conditions.
2056
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except for early hours, in solid-state condition (5.1 U/g and 17.1 U/g) when compared to submerged condition (4.5 U/ml and 16.0 U/ml). It is known that the period of fermentation depends upon the nature of medium, fermenting organism, concentration of nutrients and the process physiological conditions. Generally, the period of fermentation in synthetic medium by pectinolytic fungi vary from 48 to 72 h. The optimized fermentation period by several researchers (Aguilar and Huitron, 1990; Galiotou-Panayotou and Kapantai, 1993; Solis-Pereyra et al., 1993; Taragano et al., 1997) indicate a wide range in both submerged (40–120 h) and solidstate fermentations (90–120 h). Results obtained in this study were similar to those observed by Shivakumar and Krishnanand (1995), Solis-Pereyra et al. (1996) and Pandey (1991). 3.2. EVect of glucose and sucrose EVect of glucose and sucrose as source of carbon on the production of pectinase from sunXower head in both submerged and solid-state conditions was examined. The production of exo-pectinase was more inXuenced by both sugars than endo-pectinase in submerged as well as solid-state conditions (Figs. 2 and 3). However, eVect of sucrose was more
Pectinase activity (U/ml and U/g)
30
DMF27 endo DMF45 endo
25
DMF27 exo DMF45 exo
20 15 10 5 0 0
2
4 6 Glucose (%)
8
10
Fig. 2. EVect of glucose as carbon source on the production of pectinase from sunXower head by A. niger in submerged and solid-state conditions.
in solid-state condition when compared to submerged condition. Glucose (4–6%) showed increase in the production of pectinase in submerged condition whereas 6–8% sucrose gives better yield of pectinase in solid-state condition. An adequate supply of carbon as energy source is critical for optimum growth aVecting the growth of organism and its metabolism. At low concentration of carbon sources, an increasing substrate concentration usually favours product synthesis. The eVect of various synthetic carbon sources such as pectin, glucose, sucrose, fructose, galacturonic acid and many other such on the production of pectinase in both submerged and solid state conditions were studied by Taragano et al. (1997), Solis-Pereyra et al. (1996), Shivakumar and Krishnanand (1995), Boccas et al. (1994), Hours et al. (1988) and Budiatman and Lonsane (1987) indicating a wide range of concentrations. 3.3. EVect of nitrogen sources The eVect of ammonium phosphate and ammonium sulphate on the production of pectinase from sunXower head in both submerged and solid-state conditions is as shown in Figs. 4 and 5, respectively. Both showed better eVect on production of pectinase in solid-state condition when compared to submerged condition. Increase in the production of endo- and exo-pectinases was observed up to 0.3% concentration of both the nitrogenous compounds. However, the increase was very less with ammonium phosphate as compared to ammonium sulphate. The observations of Hours et al. (1988) suggested that lower levels of (NH4)2SO4 (0.16%), or K2HPO4 (0.1%) added to the medium did not inXuence enzyme yield. The eVect of inorganic nitrogen on the production of pectinase was also reported by Boccas et al. (1994), Aguilar and Huitron (1990), Galiotou-Panayotou and Kapantai (1993) and Larios et al. (1989). Our investigation revealed that both ammonium phosphate and ammonium sulphate did inXuence production of pectinase positively in both submerged and solid-state conditions. Although,
Pectinase activity (U/ml and U/g)
30 25 20 15 10 5
Pectinase activity (U/ml and U/g)
25 DMF27 endo DMF45 endo DMF27 exo DMF45 exo
35
DMF27 endo 20
DMF45 endo DMF27 exo
15
DMF45 exo
10 5 0
0 0
2
4
6
8
10
0
0.1 0.2 0.3 0.4 Ammonium phosphate (%)
0.5
Sucrose (%) Fig. 3. EVect of sucrose as carbon source on the production of pectinase from sunXower head by A. niger in submerged and solid-state conditions.
Fig. 4. EVect of ammonium phosphate as nitrogen source on the production of pectinase from sunXower head by A. niger in submerged and solidstate conditions.
S.R. Patil, A. Dayanand / Bioresource Technology 97 (2006) 2054–2058 30
35
DMF45 endo DMF27 exo
20
DMF45 exo
15 10 5 0 0
0.1 0.2 0.3 0.4 Ammonium sulphate (%)
Pectinase activity (U/ml and U/g)
Pectinase activity (U/ml and U/g)
DMF27 endo 25
2057
DMF27 endo DMF45 endo
30
DMF27 exo 25
DMF45 exo
20 15 10 5
0.5
0 0
Fig. 5. EVect of ammonium sulphate as nitrogen source on the production of pectinase from sunXower head by A. niger in submerged and solid-state conditions.
Galiotou-Panayotou and Kapantai (1993) has made a similar observation, inhibitory eVects were also recorded with ammonium nitrate and potassium nitrate.
5 10 15 20 Green gram husk (%)
25
Fig. 7. InXuence of green gram husk as supplement on the production of pectinase from sunXower head by A. niger in submerged and solid-state conditions.
30
3.4. InXuence of husk of pulses The inXuence of husk of pulses (bengal, green and red gram) on the production of pectinase from sunXower head in both submerged and solid-state conditions is as shown in Figs. 6–8. Husk of green gram seemed to be relatively a better supplement for the production of endo- and exo-pectinase in both submerged and solid-state conditions when compared to other husks. Both, green gram and red gram husks showed a linear increase in the production of endoand exo-pectinase up to 20% concentration in both submerged and solid-state conditions. However, bengal gram husk reduced the production of endo- and exo-pectinase after the concentration of 10%, in both submerged and solid state conditions. Addition of any natural substance as 25
DMF27 endo
Pectinase activity (U/ml and U/g)
DMF45 endo DMF27 exo
20
DMF45 exo 15
10
5
Pectinase activity (U/ml and U/g)
DMF27 endo DMF45 endo
25
DMF27 exo 20
DMF45 exo
15 10 5 0 0
5
10 15 20 Red gram husk (%)
25
Fig. 8. InXuence of red gram husk as supplement on the production of pectinase from sunXower head by A. niger in submerged and solid-state conditions.
supplement to the main substrate for the production of desired end product is not uncommon in fermentation practices, especially in solid-state conditions. Husk of pulses, chieXy available in the region, as other agro wastes in addition to sunXower head, are known to be rich in not only nitrogen but also mineral contents. Therefore, an attempt was made in the present study to use husk of pulses as supplement to the substrate sunXower head with an aim to enhance the yield of pectinase. Among all the husk of pulses, the inXuence of green gram husk was considerably more to give better yield of endo-pectinase and exo-pectinase, particularly in solid-state condition. 3.5. Standardized bioprocess
0 0
5 10 15 20 Bengal gram husk (%)
25
Fig. 6. InXuence of bengal gram husk as supplement on the production of pectinase from sunXower head by A. niger in submerged and solid-state conditions.
The development strategies that are employed in Wnding the substrate and microorganisms for economic value and their potentials in the fermentation process are most critical and important. The successful approach to achieve this
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S.R. Patil, A. Dayanand / Bioresource Technology 97 (2006) 2054–2058
objective is to understand the multifacet interactions of the substrate and organism in the prevailing conditions. Several researchers have reported the production of a wide range of fungal pectinases from variety of substrates under optimum conditions. Hours et al. (1988) recorded 1062–1300 U/g pectinase production from apple pomace with an adequate addition of organic nitrogen using Aspergillus foetidus. Blandino et al. (2002) recorded 20–25 U/g of pectinase from wheat employing Aspergillus awamori. Galiotou-Panayotou and Kapantai (1993) observed the production of 14.5 U/ml pectinase production by A. niger on supplemented citrus pectin. However, no literature as per our survey, indicated the production of fungal pectinase from sunXower head except, the pectinase production by yeast, Cryptococcus albidus from olive vegetation waters enriched with sunXower calathide meal (Federico et al., 1988). An attempt was made here for the production of pectinases from the substrate sunXower head in both submerged and solid-state conditions employing DMF 27 and DMF 45 strains of A. niger respectively. Standardized bioprocess with all optimized conditions resulted maximum production of both endo-pectinase (19.8 U/g) and exo-pectinase (45.9 U/g) by A. niger DMF 45 in SSF compared to endo-pectinase (18.9 U/ml and exopectinase (30.6 U/ml) by A. niger DMF 27 in SmF (data not shown). Similar to these observations, Solis-Pereyra et al. (1993) while investigating eVects of diVerent carbon sources on the synthesis of pectinase by A. niger in submerged and solid-state fermentations, recorded 18.8 and 4.9 times higher endo- and exo-pectinase productivities, respectively. 4. Conclusions Results of this study indicated that deseeded sunXower head could be an attractive and promising substrate especially in solid-state fermentation for the production of pectinases by A. niger DMF 45. It would be worth to look into the complex nutritive value of both sunXower head and green gram husk for their further exploration to produce fungal pectinase. Acknowledgements We thank the Department of Microbiology, Gulbarga University for providing necessary facilities for the present work. References Aguilar, G., Huitron, C., 1990. Constitutive exo pectinase produced by Aspergillus sp. CH-Y-1043 on diVerent carbon source. Biotechnol. Lett. 12 (9), 655–660.
Blandino, A., Iqbalsyah, T., Pandiella, S.S., Cantero, D., Webb, C., 2002. Polygalacturonase production by Aspergillus awamori on wheat in solid-state fermentation. Appl. Microbiol. Biotechnol. 58, 164–169. Boccas, F., Roussos, S., Gutierrez, M., Serrano, L., Viniegra, G.G., 1994. Production of pectinase from coVee pulp in solid-state fermentation system: selection of wild fungal isolate of high potency by a simple three-step screening technique. J. Food Sci. Technol. 31 (1), 22–26. Budiatman, S., Lonsane, B.K., 1987. Cassava Wbrous waste residue: a substitute to wheat bran in solid-state fermentation. Biotechnol. Lett. 9 (8), 597–600. Federico, F., Gianfranco, M., Maurizio, S., Maurizio, P., 1988. Pectic enzyme production by Cryptococcus albidus var. albidus on olive vegetation waters enriched with sunXower calathide meal. Biol. Wastes 25, 291–301. Galiotou-Panayotou, M.P.R., Kapantai, M., 1993. Enhanced polygalacturonase production by Aspergillus niger NRRL-364 grown on supplemented citrus pectin. Lett. Appl. Microbiol. 17, 145–148. Ghildyal, N.P., Ramakrishna, S.V., Devi, P.N., Lonsane, B.K., Asthane, H.N., 1981. Large-scale production of pectolytic enzyme by solid-state fermentation. J. Food Sci. Technol. 18, 248–251. Hours, R.A., Voget, C.E., Ertola, R.J., 1988. Some factors aVecting pectinase production from apple pomace in solid-state-cultures. Biol. Wastes 24, 147–157. Larios, G., Garcia, J.M., Huitron, C., 1989. Endo-polygalacturonase production from untreated lemon peel by Aspergillus sp. CH-Y-1043. Biotechnol. Lett. 11, 729–734. Miller, G.L., 1959. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal. Chem. 31, 426–428. Pandey, A., 1991. EVect of particle size of substrate on enzyme production in solid-state fermentation. Bioresour. Technol. 37, 169–172. Pandey, A., Selvakumar, P., Carlos, R.S., Poonam, N., 1999. Solid-state fermentation for the production of industrial enzymes. Current Sci. 77 (1), 149–162. Pandey, A., Soccol, C.R., Mitchell, D., 2000. New developments in solidstate fermentation—Part I: bioprocesses and applications. Process Biochem. 35, 1153–1169. Pandey, A., 2003. Sold-state fermentation. Biochem. Eng. J. 13, 81–84. Ranganna, S., 1979. Manual of Analysis of Fruit and Vegetables Products. Tata McGraw-Hill Publ., Co. Ltd., New Delhi, p. 634. Sebastian, F.C., Jorge, A.A., Roque, A.H., 1996. Pectinase production proWle of Aspergillus foetidus in solid-state cultures at diVerent acidities. Biotechnol. Lett. 18 (3), 251–256. Shivakumar, P.D., Krishnanand, 1995. Anaerobic degradation of pectin by mixed consortia and optimization of fermentation parameters for higher pectinase activity. Lett. Appl. Microbiol. 20, 117– 119. Solis-Pereyra, S., Favela-Torres, E., Viniegra-Gonzalez, G., GutierrezRojas, M., 1993. EVect of diVerent carbon sources on the synthesis of pectinase by Aspergillus niger in submerged and solid state fermentation. Appl. Microbiol. Biotechnol. 39, 36–41. Solis-Pereyra, S., Favela-Torres, E., Gutierrez, Rojas, M., Roussos, S., Saucedo Castaneda, G., Gunasekaran, P., Viniegra-Gonzalez, G., 1996. Production of pectinases by Aspergillus niger in solid-state fermentation at high initial glucose concentrations. World J. Microbiol. Biotechnol. 12, 257–260. Taragano, V., Sanchez, V.E., Pilosof, A.M.R., 1997. Combined eVect of water activity depression and glucose addition on pectinase and protease production by Aspergillus niger. Biotechnol. Lett. 19 (3), 233–236.
Production of pectinase from deseeded sunXower head by Aspergillus niger in submerged and solid-state conditions Sarvamangala R. Patil a
a,b,¤
, A. Dayanand
b
Department of Microbiology, Vishweshwariah College of Applied Sciences, Gulbarga 585 102, India b PG Department of Microbiology, Gulbarga University, Gulbarga 585 106, India Received 16 May 2005; received in revised form 9 September 2005; accepted 16 September 2005 Available online 2 November 2005
Abstract Studies were carried out on the production of pectinases using deseeded sunXower head by Aspergillus niger DMF 27 and DMF 45 in submerged fermentation (SmF) and solid-state fermentation (SSF). Higher titres of endo- and exo-pectinases were observed when medium was supplemented with carbon (4% glucose for SmF and 6% sucrose for SSF) and nitrogen (ammonium sulphate, 0.3% for both SmF and SSF) sources. Green gram husk proved to be relatively a better supplement to attain higher yield of endo-pectinase (11.7 U/g) and exo-pectinase (30.0 U/g) in solid-state conditions. Maximum production of endo-pectinase (19.8 U/g) and exo-pectinase (45.9 U/g) by DMF 45 were recorded in SSF when compared to endo-pectinase (18.9 U/ml) and exo-pectinase (30.3 U/ml) by DMF 27 in SmF under optimum process conditions. © 2005 Elsevier Ltd. All rights reserved. Keywords: Pectinase; SunXower head; Aspergillus niger; Submerged; Solid-state fermentation
1. Introduction The northern part of the state of Karnataka, India is a semi arid tropical region, well known for the production of pulses (red gram, bengal gram and green gram) and oil seeds (sunXower), which are cultivated in about 1,80,000 and 30,000 ha area, respectively. In the past ten years, on an average 0.52 tons/ha of pulses and 0.79 tons/ha of oil seeds are being produced every year, generating 3.1 tons/ha of husk of pulses and 14.3 tons/ha deseeded sunXower head as agro wastes annually. Husk of pulses is partly used as cattle feed and deseeded dried sunXower head is burnt to ash after harvesting. Microbial pectinases have tremendous potential to oVer mankind. Most pectic enzyme preparations are used in the fruit processing industry and pectic enzymes alone account * Corresponding author. Address: Department of Microbiology, Vishweshwariah College of Applied Sciences, Gulbarga 585 102, India. Tel.: +91 8472 232391. E-mail address: [email protected] (S.R. Patil).
0960-8524/$ - see front matter © 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2005.09.015
for about one quarter of the world’s food enzyme production. Fungal pectinases are among the most important industrial enzymes and are of great signiWcance with wide range application in textile processing, degumming of plant bast Wbers, treatment of pectic wastewaters, papermaking and coVee and tea fermentations. Therefore, the biotechnological potential of pectinolytic enzymes from fungi has drawn a great deal of attention from various researchers worldwide. Various agro-industrial wastes such as wheat bran (Taragano et al., 1997), sugarcane bagasse (Solis-Pereyra et al., 1993, 1996), coVee pulp (Boccas et al., 1994), lemon peel (Larios et al., 1989) and apple pomace (Hours et al., 1988) have been explored for the microbial production of pectinase. Several authors have reported a wide range of pectin (15–25%) in sunXower head (Federico et al., 1988). This high content of pectin was a stimulant to explore sunXower head as a substrate for the microbial production of pectinase. Solid-state fermentation holds tremendous potential for the production of enzymes. Agro-industrial residues or wastes are generally considered as suitable substrates for
S.R. Patil, A. Dayanand / Bioresource Technology 97 (2006) 2054–2058
2. Methods 2.1. Inoculum and substrate Two strains of A. niger DMF 27 and DMF 45, isolated and characterized in our laboratory from agro waste dumping pit soil were employed in submerged and solid-state fermentation, respectively. The cultures were maintained on potato dextrose agar at 4 °C by periodic sub culturing. The inoculum was prepared in pectin medium harvesting the spores from 96 h old cultures at 25 °C. The conidia were dispersed in 0.01% Tween 80 solution. Deseeded dried sunXower head was grinded and Wne powder was used as substrate for submerged fermentation. The particles of the same with mesh sieve size adjusted to 500 m were used for solid-state fermentation. The pectin content of deseeded sunXower head was determined by gravimetric method (Ranganna, 1979). 2.2. Medium and fermentation Medium for submerged fermentation contained (%) (NH4)2SO4, 0.1; MgSO4 · 7H2O, 0.5; KH2PO4, 0.5; FeSO4 · 7H2O, 0.0005 (Solis-Pereyra et al., 1996) and 8 g powder of sunXower head (particle size > 500 m). To study the eVect of supplementation of glucose and sucrose, they were added individually at 2–10% concentration in the medium. To study the eVect of supplementation of nitrogen source, ammonium phosphate and ammonium sulphate were added at 0.1–0.5% concentrations. Husk of pulses (bengal, green and red gram) in the range of 5–25% were also added independently as supplement. Fermentation duration was examined from 24 to 120 h. Medium was autoclaved for 15 min at 120 °C. Inoculum size and pH were 1 £ 105 spores/ ml and 5.0, respectively. Fermentation was carried in 250 ml Erlenmeyer Xask by taking 100 ml medium at 34 °C on rotatary shaker (300 rpm). For SSF, medium components were same as above. Initial substrate moisture was 65%. Carbon source, nitrogen source, husk of pulses and fermentation duration were assessed similar to submerged condition. Medium was autoclaved for 15 min at 120 °C. The pH and inoculum size of the medium were adjusted to 5.0 and 1 £ 107 spores/g,
respectively. Fermentation was carried out in 250 ml shallow glass bottle at 34 °C. 2.3. Enzyme assay Liquid samples obtained from submerged fermentation were Wltered using muslin cloth. For SSF, a known quantity of fermented material was mixed with equivalent amount of distilled water and mixed thoroughly on a magnetic stirrer for 20 min and Wltered. The Wltrates were stored at 4 °C for enzyme assay. Polygalacturonase activities were measured at 45 °C by Viscometry (Ghildyal et al., 1981) for endo-pectinase (endo-p) and by the release of reducing sugars (Miller, 1959) for exo-pectinase (exo-p). For endo-p, 1 ml of a suitably diluted sample was mixed with 18 ml of 2% pectin in 0.1 M acetate buVer with pH 4.5 and reduction in viscosity was followed with Ostwald Viscometer. One endo-pectinase unit (U) was deWned as the amount of enzyme that reduces the viscosity of the solution by 50% per minute under the conditions mentioned above. For exop, 0.3 ml of a suitably diluted sample was added to a solution containing 1 ml of 0.9% of substrate and 0.7 ml of 0.1 M acetate buVer with pH 4.5. Samples were incubated at 45 °C for 30 min. Reducing sugars were determined by dinitro salicylic acid (DNS) method using galacturonic acid as reference. One exo-p unit (U) was deWned as the quantity of enzyme that liberates one micromole of galacturonic acid per minute under the conditions mentioned above. 3. Results and discussion 3.1. Evaluation of fermentation period Production of pectinase was evaluated up to 120 h (Fig. 1). The pectin content of deseeded sunXower head was 21.36% (data not shown). A gradual increase in the production of pectinase over a period up to 72 h was observed in submerged and up to 96 h in solid-state conditions (Fig. 1). The production of both endo- and exo-pectinase was high, 18 Pectinase activity (U/ml and U/g)
the production of enzymes in SSF process (Pandey et al., 1999, 2000; Pandey, 2003). Many investigations were carried out (Blandino et al., 2002; Hours et al., 1988; Sebastian et al., 1996; Solis-Pereyra et al., 1996; Boccas et al., 1994) for the production of pectinases in solid-state conditions. Submerged fermentation has also been used for pectinases production (Aguilar and Huitron, 1990; Shivakumar and Krishnanand, 1995; Galiotou-Panayotou and Kapantai, 1993). The present study reports on the development and standardization of bioprocess for the production of pectinase by Aspergillus niger in submerged and solid-state conditions employing sunXower head as substrate.
2055
DMF27 endo
16
DMF45 endo
14
DMF27 exo
12
DMF45 exo
10 8 6 4 2 0 0
48 72 96 24 Fermentation period (h)
120
Fig. 1. Evaluation of fermentation period for the production of pectinase from sunXower head by A. niger in submerged and solid-state conditions.
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except for early hours, in solid-state condition (5.1 U/g and 17.1 U/g) when compared to submerged condition (4.5 U/ml and 16.0 U/ml). It is known that the period of fermentation depends upon the nature of medium, fermenting organism, concentration of nutrients and the process physiological conditions. Generally, the period of fermentation in synthetic medium by pectinolytic fungi vary from 48 to 72 h. The optimized fermentation period by several researchers (Aguilar and Huitron, 1990; Galiotou-Panayotou and Kapantai, 1993; Solis-Pereyra et al., 1993; Taragano et al., 1997) indicate a wide range in both submerged (40–120 h) and solidstate fermentations (90–120 h). Results obtained in this study were similar to those observed by Shivakumar and Krishnanand (1995), Solis-Pereyra et al. (1996) and Pandey (1991). 3.2. EVect of glucose and sucrose EVect of glucose and sucrose as source of carbon on the production of pectinase from sunXower head in both submerged and solid-state conditions was examined. The production of exo-pectinase was more inXuenced by both sugars than endo-pectinase in submerged as well as solid-state conditions (Figs. 2 and 3). However, eVect of sucrose was more
Pectinase activity (U/ml and U/g)
30
DMF27 endo DMF45 endo
25
DMF27 exo DMF45 exo
20 15 10 5 0 0
2
4 6 Glucose (%)
8
10
Fig. 2. EVect of glucose as carbon source on the production of pectinase from sunXower head by A. niger in submerged and solid-state conditions.
in solid-state condition when compared to submerged condition. Glucose (4–6%) showed increase in the production of pectinase in submerged condition whereas 6–8% sucrose gives better yield of pectinase in solid-state condition. An adequate supply of carbon as energy source is critical for optimum growth aVecting the growth of organism and its metabolism. At low concentration of carbon sources, an increasing substrate concentration usually favours product synthesis. The eVect of various synthetic carbon sources such as pectin, glucose, sucrose, fructose, galacturonic acid and many other such on the production of pectinase in both submerged and solid state conditions were studied by Taragano et al. (1997), Solis-Pereyra et al. (1996), Shivakumar and Krishnanand (1995), Boccas et al. (1994), Hours et al. (1988) and Budiatman and Lonsane (1987) indicating a wide range of concentrations. 3.3. EVect of nitrogen sources The eVect of ammonium phosphate and ammonium sulphate on the production of pectinase from sunXower head in both submerged and solid-state conditions is as shown in Figs. 4 and 5, respectively. Both showed better eVect on production of pectinase in solid-state condition when compared to submerged condition. Increase in the production of endo- and exo-pectinases was observed up to 0.3% concentration of both the nitrogenous compounds. However, the increase was very less with ammonium phosphate as compared to ammonium sulphate. The observations of Hours et al. (1988) suggested that lower levels of (NH4)2SO4 (0.16%), or K2HPO4 (0.1%) added to the medium did not inXuence enzyme yield. The eVect of inorganic nitrogen on the production of pectinase was also reported by Boccas et al. (1994), Aguilar and Huitron (1990), Galiotou-Panayotou and Kapantai (1993) and Larios et al. (1989). Our investigation revealed that both ammonium phosphate and ammonium sulphate did inXuence production of pectinase positively in both submerged and solid-state conditions. Although,
Pectinase activity (U/ml and U/g)
30 25 20 15 10 5
Pectinase activity (U/ml and U/g)
25 DMF27 endo DMF45 endo DMF27 exo DMF45 exo
35
DMF27 endo 20
DMF45 endo DMF27 exo
15
DMF45 exo
10 5 0
0 0
2
4
6
8
10
0
0.1 0.2 0.3 0.4 Ammonium phosphate (%)
0.5
Sucrose (%) Fig. 3. EVect of sucrose as carbon source on the production of pectinase from sunXower head by A. niger in submerged and solid-state conditions.
Fig. 4. EVect of ammonium phosphate as nitrogen source on the production of pectinase from sunXower head by A. niger in submerged and solidstate conditions.
S.R. Patil, A. Dayanand / Bioresource Technology 97 (2006) 2054–2058 30
35
DMF45 endo DMF27 exo
20
DMF45 exo
15 10 5 0 0
0.1 0.2 0.3 0.4 Ammonium sulphate (%)
Pectinase activity (U/ml and U/g)
Pectinase activity (U/ml and U/g)
DMF27 endo 25
2057
DMF27 endo DMF45 endo
30
DMF27 exo 25
DMF45 exo
20 15 10 5
0.5
0 0
Fig. 5. EVect of ammonium sulphate as nitrogen source on the production of pectinase from sunXower head by A. niger in submerged and solid-state conditions.
Galiotou-Panayotou and Kapantai (1993) has made a similar observation, inhibitory eVects were also recorded with ammonium nitrate and potassium nitrate.
5 10 15 20 Green gram husk (%)
25
Fig. 7. InXuence of green gram husk as supplement on the production of pectinase from sunXower head by A. niger in submerged and solid-state conditions.
30
3.4. InXuence of husk of pulses The inXuence of husk of pulses (bengal, green and red gram) on the production of pectinase from sunXower head in both submerged and solid-state conditions is as shown in Figs. 6–8. Husk of green gram seemed to be relatively a better supplement for the production of endo- and exo-pectinase in both submerged and solid-state conditions when compared to other husks. Both, green gram and red gram husks showed a linear increase in the production of endoand exo-pectinase up to 20% concentration in both submerged and solid-state conditions. However, bengal gram husk reduced the production of endo- and exo-pectinase after the concentration of 10%, in both submerged and solid state conditions. Addition of any natural substance as 25
DMF27 endo
Pectinase activity (U/ml and U/g)
DMF45 endo DMF27 exo
20
DMF45 exo 15
10
5
Pectinase activity (U/ml and U/g)
DMF27 endo DMF45 endo
25
DMF27 exo 20
DMF45 exo
15 10 5 0 0
5
10 15 20 Red gram husk (%)
25
Fig. 8. InXuence of red gram husk as supplement on the production of pectinase from sunXower head by A. niger in submerged and solid-state conditions.
supplement to the main substrate for the production of desired end product is not uncommon in fermentation practices, especially in solid-state conditions. Husk of pulses, chieXy available in the region, as other agro wastes in addition to sunXower head, are known to be rich in not only nitrogen but also mineral contents. Therefore, an attempt was made in the present study to use husk of pulses as supplement to the substrate sunXower head with an aim to enhance the yield of pectinase. Among all the husk of pulses, the inXuence of green gram husk was considerably more to give better yield of endo-pectinase and exo-pectinase, particularly in solid-state condition. 3.5. Standardized bioprocess
0 0
5 10 15 20 Bengal gram husk (%)
25
Fig. 6. InXuence of bengal gram husk as supplement on the production of pectinase from sunXower head by A. niger in submerged and solid-state conditions.
The development strategies that are employed in Wnding the substrate and microorganisms for economic value and their potentials in the fermentation process are most critical and important. The successful approach to achieve this
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objective is to understand the multifacet interactions of the substrate and organism in the prevailing conditions. Several researchers have reported the production of a wide range of fungal pectinases from variety of substrates under optimum conditions. Hours et al. (1988) recorded 1062–1300 U/g pectinase production from apple pomace with an adequate addition of organic nitrogen using Aspergillus foetidus. Blandino et al. (2002) recorded 20–25 U/g of pectinase from wheat employing Aspergillus awamori. Galiotou-Panayotou and Kapantai (1993) observed the production of 14.5 U/ml pectinase production by A. niger on supplemented citrus pectin. However, no literature as per our survey, indicated the production of fungal pectinase from sunXower head except, the pectinase production by yeast, Cryptococcus albidus from olive vegetation waters enriched with sunXower calathide meal (Federico et al., 1988). An attempt was made here for the production of pectinases from the substrate sunXower head in both submerged and solid-state conditions employing DMF 27 and DMF 45 strains of A. niger respectively. Standardized bioprocess with all optimized conditions resulted maximum production of both endo-pectinase (19.8 U/g) and exo-pectinase (45.9 U/g) by A. niger DMF 45 in SSF compared to endo-pectinase (18.9 U/ml and exopectinase (30.6 U/ml) by A. niger DMF 27 in SmF (data not shown). Similar to these observations, Solis-Pereyra et al. (1993) while investigating eVects of diVerent carbon sources on the synthesis of pectinase by A. niger in submerged and solid-state fermentations, recorded 18.8 and 4.9 times higher endo- and exo-pectinase productivities, respectively. 4. Conclusions Results of this study indicated that deseeded sunXower head could be an attractive and promising substrate especially in solid-state fermentation for the production of pectinases by A. niger DMF 45. It would be worth to look into the complex nutritive value of both sunXower head and green gram husk for their further exploration to produce fungal pectinase. Acknowledgements We thank the Department of Microbiology, Gulbarga University for providing necessary facilities for the present work. References Aguilar, G., Huitron, C., 1990. Constitutive exo pectinase produced by Aspergillus sp. CH-Y-1043 on diVerent carbon source. Biotechnol. Lett. 12 (9), 655–660.
Blandino, A., Iqbalsyah, T., Pandiella, S.S., Cantero, D., Webb, C., 2002. Polygalacturonase production by Aspergillus awamori on wheat in solid-state fermentation. Appl. Microbiol. Biotechnol. 58, 164–169. Boccas, F., Roussos, S., Gutierrez, M., Serrano, L., Viniegra, G.G., 1994. Production of pectinase from coVee pulp in solid-state fermentation system: selection of wild fungal isolate of high potency by a simple three-step screening technique. J. Food Sci. Technol. 31 (1), 22–26. Budiatman, S., Lonsane, B.K., 1987. Cassava Wbrous waste residue: a substitute to wheat bran in solid-state fermentation. Biotechnol. Lett. 9 (8), 597–600. Federico, F., Gianfranco, M., Maurizio, S., Maurizio, P., 1988. Pectic enzyme production by Cryptococcus albidus var. albidus on olive vegetation waters enriched with sunXower calathide meal. Biol. Wastes 25, 291–301. Galiotou-Panayotou, M.P.R., Kapantai, M., 1993. Enhanced polygalacturonase production by Aspergillus niger NRRL-364 grown on supplemented citrus pectin. Lett. Appl. Microbiol. 17, 145–148. Ghildyal, N.P., Ramakrishna, S.V., Devi, P.N., Lonsane, B.K., Asthane, H.N., 1981. Large-scale production of pectolytic enzyme by solid-state fermentation. J. Food Sci. Technol. 18, 248–251. Hours, R.A., Voget, C.E., Ertola, R.J., 1988. Some factors aVecting pectinase production from apple pomace in solid-state-cultures. Biol. Wastes 24, 147–157. Larios, G., Garcia, J.M., Huitron, C., 1989. Endo-polygalacturonase production from untreated lemon peel by Aspergillus sp. CH-Y-1043. Biotechnol. Lett. 11, 729–734. Miller, G.L., 1959. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal. Chem. 31, 426–428. Pandey, A., 1991. EVect of particle size of substrate on enzyme production in solid-state fermentation. Bioresour. Technol. 37, 169–172. Pandey, A., Selvakumar, P., Carlos, R.S., Poonam, N., 1999. Solid-state fermentation for the production of industrial enzymes. Current Sci. 77 (1), 149–162. Pandey, A., Soccol, C.R., Mitchell, D., 2000. New developments in solidstate fermentation—Part I: bioprocesses and applications. Process Biochem. 35, 1153–1169. Pandey, A., 2003. Sold-state fermentation. Biochem. Eng. J. 13, 81–84. Ranganna, S., 1979. Manual of Analysis of Fruit and Vegetables Products. Tata McGraw-Hill Publ., Co. Ltd., New Delhi, p. 634. Sebastian, F.C., Jorge, A.A., Roque, A.H., 1996. Pectinase production proWle of Aspergillus foetidus in solid-state cultures at diVerent acidities. Biotechnol. Lett. 18 (3), 251–256. Shivakumar, P.D., Krishnanand, 1995. Anaerobic degradation of pectin by mixed consortia and optimization of fermentation parameters for higher pectinase activity. Lett. Appl. Microbiol. 20, 117– 119. Solis-Pereyra, S., Favela-Torres, E., Viniegra-Gonzalez, G., GutierrezRojas, M., 1993. EVect of diVerent carbon sources on the synthesis of pectinase by Aspergillus niger in submerged and solid state fermentation. Appl. Microbiol. Biotechnol. 39, 36–41. Solis-Pereyra, S., Favela-Torres, E., Gutierrez, Rojas, M., Roussos, S., Saucedo Castaneda, G., Gunasekaran, P., Viniegra-Gonzalez, G., 1996. Production of pectinases by Aspergillus niger in solid-state fermentation at high initial glucose concentrations. World J. Microbiol. Biotechnol. 12, 257–260. Taragano, V., Sanchez, V.E., Pilosof, A.M.R., 1997. Combined eVect of water activity depression and glucose addition on pectinase and protease production by Aspergillus niger. Biotechnol. Lett. 19 (3), 233–236.