Enhanced production of pectinase by Bacillus sp. DT7 using solid state fermentation docx

Enhanced production of pectinase by Bacillus sp. DT7 using solid state fermentation Des Raj Kashyap a , Sanjeev Kumar Soni a , Rupinder Tewari b, * a Department of Microbiology, Panjab University, Chandigarh 160014, India b Department of Biotechnology, Panjab University, Chandigarh 160014, India Received 22 May 2002; received in revised form 10 August 2002; accepted 14 August 2002 Abstract Bacillus sp. DT7 produced very high levels of alkaline and thermotolerant pectinase by solid state fermentation. Production of this enzyme was affected by nature of solid substrate, level of moisture content, presence or absence of carbon, nitrogen, mineral and vitamin supplements. Maximum enzyme production of 8050 U/g dry substrate was obtained in wheat bran supplemented with polygalacturonic acid (PGA; 1%, w/v) and neurobion (a multivitamin additive; 27 ll/g dry substrate) with distilled water at 75% moisture level, after 36 h of incubation at 37 °C. Ó 2002 Published by Elsevier Science Ltd. Keywords: Solid state fermentation; Bacillus; Pectinase 1. Introduction In recent years there has been a renewed interest in solid-state fermentation (SSF) processes for the pro- duction of bioactive compounds. While efforts continue largely to exploit filamentous fungi and yeasts for the production of various enzymes, attempts have also been made to explore possibilities of using bacterial strains in SSF systems (Pandey et al., 2000). Enzyme production by SSF using bacterial spp. has been reported for many enzymes such as xylanase (Gessesse and Mamo, 1999) and amylase (Babu and Satyanarayana, 1995) but re- ports on pectinase production by SSF using bacterial spp. are lacking in the literature. The use of SSF for the production of enzymes mainly from fungi may be due to the general belief that SSF technique is applicable only to filamentous fungi (Lonesane and Ghidyal, 1992). Studies on comparative production of pectinase by As- pergillus sp. using submerged fermentation (SmF) and SSF has shown the latter to be a better option (Solis- Pereyra et al., 1993; Minjares-Carranco et al., 1997). Moreover, the use of SSF has been reported to be more advantageous than SmF as it allows cheaper production of enzyme having better physiochemical properties than that produced by SmF (Solis-Pereyra et al., 1993). In this study, we report the production of very high levels of an alkali- and thermotolerant pectinase pro- duced by a mesophilic Bacillus sp. DT7 (Kashyap et al., 2000) using SSF conditions. 2. Methods 2.1. Solid state fermentation Solid substrates (5.0 g; wheat bran, rice bran, apple pomace) in 250 ml Erlenmeyer flasks were moistened with specified content of distilled water and autoclaved at 15 psi for 30 min. 2.0 ml of culture was used as an in- oculum in each flask of solid substrate, which were then incubated at 37 °C for specified time intervals. Visual observations regarding growth were made on each day and samples were drawn periodically to assess the en- zyme production. Enzyme was extracted with 25 ml, Tris–HCl buffer (0.01 M, pH 8.0) and filtered. The pooled filtrate of two extractions was centrifuged (10,000g for 15 min; 4 °C) and used as the source of enzyme. Pectinase activity in the extracted liquid was assayed by the calorimetric method described earlier (Kashyap et al., 2000). Pectinase activity in the supernatant was expressed as U/g of solid substrate used. * Corresponding author. Address: Department of Microbiology, Panjab University, Chandigarh 160014, India. Fax: +91-172-541409. E-mail address: rooptt1@glide.net.in (R. Tewari). 0960-8524/03/$ - see front matter Ó 2002 Published by Elsevier Science Ltd. doi:10.1016/S0960-8524(02)00206-7 Bioresource Technology 88 (2003) 251–254 2.2. Optimization studies for pectinase production The effect of environmental parameters in SSF was studied using wheat bran, unless otherwise stated, by altering physiochemical and culture conditions. Effect of different solid substrates: The effect of dif- ferent solid substrates on pectinase production was studied by using a variety of solid substrates (wheat bran, rice bran, apple pomace). The original moisture content of the apple pomace was 80%. Effect of moisture contents on pectinase production using wheat bran and rice bran was studied by adding different amount (50%, 60%, 66.7%, 75%, 77.8%, and 80%) of distilled water as the moistening agent to these solid substrates. Effect of salts: Different salts: CaCl 2 Á 2H 2 O, MgSO 4 Á 7H 2 O, CoCl 2 Á 2H 2 O, MnSO 4 Á 4H 2 O, H 3 BO 3 , ZnCl 2 , KCl and NaCl (1 mM, final concentration) were dissolved in the distilled water used to adjust moisture level in the solid substrate. Effect of carbon sources: Different carbon sources: glucose, mannitol, pectin, polygalacturonic acid (PGA), galactose, sucrose, lactose, maltose, sodium acetate (SA) and xylose were supplemented separately to a final concentration of 1% (w/v) in solid media. They were dissolved to required concentrations in 15 ml of distilled water, which was used as moistening agent per 5 g of solid substrate. Effect of nitrogen sources: Different organic and in- organic nitrogen sources: yeast extract, peptone, tryp- tone, glycine, urea, ammonium chloride, ammonium nitrate, ammonium sulphate and ammonium citrate were supplemented separately to a final concentration of 1% (w/v) in solid media after dissolving them in distilled water used for adjusting the moisture content. Effect of vitamin supplementation: Effect of neurobion (Nb) (a multivitamin solution of vitamin B 1 ,B 6 and B 12 ; E-Merck, India Ltd.) on pectinase production was studied by supplementing different concentrations (9–54 ll/g dry substrate) of Nb into the solid medium. For this 5.0 g wheat bran and 15 ml of distilled water were au- toclaved (15 psi, 30 min) separately. Nb was then added to the distilled water, which was subsequently used as moistening agent. Effect of optimized components: Effect of optimized components on pectinase production in optimized me- dium (wheat bran) was studied by supplementing these components alone or in combination in distilled water used as moistening agent. 3. Results and discussion Research on the selection of suitable substrates for pectinase production has mainly centered on tropical agro-industrial crops and residues. Of the various sub- strates reported in the literature, wheat bran has been the prime among all (Pandey et al., 2000). In the present study, out of three substrates (wheat bran, rice bran and apple pomace) used wheat bran yielded 4600 U of pec- tinase/g dry substrate at 75% moisture level after 36 h of incubation at 37 °C. When rice bran and apple pomace were used separately as prime substrates, maximum pectinase yields obtained were 3265.25 and 78.0 U/g dry substrate at 66.7 and 80% moisture contents respectively after 48 h of incubation at 37 °C. A decline in pectinase activity in wheat bran (26%) and rice bran (8.2%) was observed after 72 h of incubation. Moreover, the pro- duction of this enzyme using SSF process was much higher than the SmF process reported previously from the same bacteria (Kashyap et al., 2000). Higher pro- duction of pectinase in SSF process may be due to the reason that solid substrate not only supplies the nutrient to the microbial cultures growing in it, but also serves as anchorage for the cells (Pandey et al., 2000) allowing them to utilize the substrate effectively. However, some of the nutrients in the solid substrate may be available in suboptimal concentrations, or even not present in the substrates. In such cases, it would be necessary to sup- plement them externally. Therefore, wheat bran was supplemented with different salts (final concentration, 1 mM), carbon (final concentration, 1% w/v) and nitrogen (1%, w/v) sources and Nb (27 ll/g dry substrate) to evaluate their effect on pectinase production at 75% moisture level. Addition of CaCl 2 Á 2H 2 O and MgSO 4 Á 7H 2 O en- hanced pectinase production by 28.0% and 11.6% re- spectively, whereas CoCl 2 Á 2H 2 O and MnSO 4 Á 4H 2 O did not have any effect on pectinase production (Table 1). Salts such as H 3 BO 3 , ZnCl 2 , KCl and NaCl inhibited the pectinase production up to 12%. Carbon sources such as PGA, SA, pectin and lactose were found to enhance pectinase production from 11% to 44% (Table 2). When various nitrogen sources were supplemented in wheat bran medium, yeast extract (YE), peptone and ammonium chloride were found to enhance pectinase production up to 24% (Table 3). Addition of glycine, Table 1 Production of pectinase from Bacillus sp. DT7 in wheat bran using various salts Salt (1 mM) Relative activity (%) Control 100.0 CaCl 2 Á 2H 2 O 128.0 MgSO 4 Á 2H 2 O 11.5 CoCl 2 Á 2H 2 O 102.0 MnSO 4 Á 4H 2 O 104.8 H 3 BO 3 89.0 ZnCl 2 87.5 KCl 93.4 NaCl 93.0 Note: Incubated at 37 °C for 36 h incubation. 100% activity is equiv- alent to 4600 U/g dry substrate. 252 D. Raj Kashyap et al. / Bioresource Technology 88 (2003) 251–254 urea and ammonium nitrate inhibited pectinase pro- duction, which may be due to poor growth of Bacillus in the medium containing these nitrogen sources whereas, tryptone had no effect on pectinase production in solid medium. Effect of Nb on pectinase production in solid medium was studied by supplementing different concentrations (9–54 ll/g dry substrate) of this multivitamin solution in wheat bran. Pectinase production was enhanced by 65.8% when 27 ll/g dry substrate of multivitamin solu- tion was added to wheat bran medium. Increasing the concentration of Nb above 27 ll/g dry substrate had no effect on pectinase production. The effect of optimized components alone or in combinations on pectinase production by Bacillus sp. DT7, was studied in wheat bran medium at 37 °C for 36 h. Of the different components supplemented combina- tion of Nb (27 ll/g dry weight) and PGA (3%, w/w), enhanced pectinase production by 75% (Table 4), whereas combination of Nb with other components such as YE (3%, w/w) þ PGA; PGA þ SA (3%, w/w), YE þ PGA þ SA and PGA þ SA þ YE þ CaCl 2 Á 2H 2 O increased pectinase production from 43% to 70% de- pending on the combinations used. From the present study, it is apparent that SSF process for pectinase production from a bacterial source is a better option than SmF. Moreover, as wheat bran is a cheap and readily available byproduct, the production of pectinase using SSF may be a cost-effective affair. As this enzyme has been successfully used for degumming of fibre crops in our previous study (Kashyap et al., 2001), the low cost of its production may further broaden the scopes for its use in industries involved in treatment of fibre crops. References Babu, K.R., Satyanarayana, T., 1995. a-amylase production by the thermophilic Bacillus coagulans in solid state fermentation. Process Biochem. 30, 305–309. Gessesse, A., Mamo, G., 1999. High level xylanase production by an alkalophilic Bacillus sp. by using solid state fermentation. Enzyme Microb. Technol. 25, 68–72. Kashyap, D.R., Chandra, S., Kaul, A., Tewari, R., 2000. Production, purification and characterization of pectinase from a Bacillus sp. DT7. World J. Micobiol. Biotechnol. 16, 277–282. Kashyap, D.R., Vohra, P.K., Soni, S.K., Tewari, R., 2001. Degum- ming of buel (Grewia optiva) bast fibres by pectinolytic enzyme from Bacillus sp. DT7. Biotechnol. Lett. 23, 1297–1301. Table 4 Production of pectinase from Bacillus sp. DT7 in wheat bran supple- mented with optimized components Component Relative activity (%) Control 100 PGA 144.0 SA 140.0 YE 125.0 Nb 166.0 CaCl 2 Á 2H 2 O 129.0 PGA þ YE 143.5 PGA þ Nb 175.0 PGA þ SA 148.4 PGA þ CaCl 2 Á 2H 2 O 146.5 YE þ SA 148.4 SA þ Nb 163.0 SA þ CaCl 2 Á 2H 2 O 144.0 YE þ Nb 170.5 YE þ CaCl 2 Á 2H 2 O 130.8 PGA þ SA þ YE 149.0 PGA þ SA þ Nb 169.8 PGA þ CaCl 2 Á 2H 2 O 149.0 SA þ YE þ Nb 169.8 PGA þ YE þ CaCl 2 Á 2H 2 O 145.2 SA þ YE þ Nb 164.6 SA þ YE þ CaCl 2 Á 2H 2 O 163.0 YE þ Nb þ CaCl 2 Á 2H 2 O 167.5 PGA þ SA þ YE þ Nb 169.0 PGA þ SA þ YE þ Nb þ CaCl 2 Á 2H 2 O 170.0 Note: Incubated at 37 °C for 36 h. 100% pectinase activity is equivalent to 4600 U/g dry substrate. Table 2 Production of pectinase from Bacillus sp. DT7 in wheat bran using various carbon sources Carbon source (final concentration, 1% w/v) Relative activity (%) Control 100 Pectin 117.8 Glucose 77.2 Galactose 75.5 Mannitol 104.0 Sucrose 82.4 SA 141.1 Lactose 111.3 PGA 143.8 Maltose 39.0 Xylose 64.5 Note: Incubated at 37 °C for 36 h. 100% activity is equivalent to 4600 U/g dry substrate. Table 3 Production of pectinase from Bacillus sp. DT7 in wheat bran using various nitrogen sources Nitrogen source (1%, w/v, final concentration) Relative activity (%) Control 100 YE 124.8 Peptone 121.4 Tryptone 102.0 Glycine 0 Urea 0 Ammonium chloride 110.7 Ammonium nitrate 0 Ammonium sulphate 76.42 Note: Incubated at 37 °C for 36 h. 100% pectinase activity is equivalent to 4600 U/g dry substrate. D. Raj Kashyap et al. / Bioresource Technology 88 (2003) 251–254 253 Lonesane, B.K., Ghidyal, N.P., 1992. Solid substrate cultivation. In: Doelle, H.W., Mitchell, D.A., Rolz, C.E. (Eds.), Exoenzyme. Elsvier Science Publishers Ltd., London, pp. 191–209. Minjares-Carranco, A., Trejo-Aguilar, B.A., Aguilar, G., Vniegra- Gonzalez, G., 1997. Physiochemical comparison between pectinase producing mutants of Aspergillus niger adapted either to solid-state fermentation or submerged fermentation. Enzyme Microb. Tech- nol. 21, 25–31. Pandey, A., Soccol, C.R., Mitchell, D., 2000. New developments in solid state fermentation. I. Bioprocesses and products. Process Biochem. 35, 1153–1169. Solis-Pereyra, S., Favela-Torre, E., Viniegra-Gonzalez, G., Gutierraez- Rojas, M., 1993. Effect of different carbon sources on the synthesis of pectinase by Aspergillus niger in submerged and solid state fermentations. Appl. Microbiol. Biotechnol. 39, 36– 41. 254 D. Raj Kashyap et al. / Bioresource Technology 88 (2003) 251–254 . Enhanced production of pectinase by Bacillus sp. DT7 using solid state fermentation Des Raj Kashyap a , Sanjeev Kumar Soni a , Rupinder Tewari b, * a Department of Microbiology,. levels of alkaline and thermotolerant pectinase by solid state fermentation. Production of this enzyme was affected by nature of solid substrate, level of moisture content, presence or absence of. the thermophilic Bacillus coagulans in solid state fermentation. Process Biochem. 30, 305–309. Gessesse, A., Mamo, G., 1999. High level xylanase production by an alkalophilic Bacillus sp. by using solid state