Bioresource Technology 98 (2007) 485490

Production of cellulase-free endoxylanase from novel alkalophilic thermotolerent Bacillus pumilus by solid-state fermentation and its application in wastepaper recycling
C. Asha Poorna, P. Prema

Biotechnology Division, Regional Research Laboratory (CSIR), Trivandrum 695 019, India Received 21 September 2005; received in revised form 17 February 2006; accepted 26 February 2006 Available online 17 July 2006

Abstract The present study aimed at optimization of culture condition for the enhanced production of extra cellular thermostable cellulase-free xylanase from Bacillus pumilus by solid-state fermentation. Batch studies were carried out to evaluate various agro-industrial residues such as rice bran, rice husk, rice straw, sawdust, coconut pith, sugarcane bagasse and wheat bran for enzyme production by the bacterial culture. The endoxylanase production was highest on wheat bran media (5582 U/gds), which was enhanced 3.8-fold (21 431 U/gds) by optimization of cultivation conditions. The enzymatic extracts was used in mixed wastepaper recycling, which resulted in a considerable improvement of the paper strength with high drainage and easy drying up. The results of enzyme application with recycled paper clearly indicated that the eVective use of enzymes in Wber separation could reduce the cost of carton paper production. 2006 Elsevier Ltd. All rights reserved.
Keywords: Bacillus pumilus; Cellulase-free xylanase; Solid-state fermentation; Lignocellulosic substrate; Recycling

1. Introduction Xylan is the most abundant non-cellulosic polysaccharide in hard wood (2035%) and soft wood (8%), which constitutes approximately one third of all renewable organic carbon sources on earth. Hydrolysis of xylan is an important step towards proper utilization of lignocellulosic material in nature. Chemical hydrolysis of lignicelluloses results in hazardous byproducts, forcing the use of microbial enzymes, which are speciWc in action for xylan hydrolysis and is an environment friendly option (Biely, 1985). Due to structural heterogeneity of xylan, complete degradation requires synergistic action of diVerent xylanolytic enzymes such as endo-xylanase, -xylosidase, -glucuronidase, -arabinofuranosidase and esterase. Among these, the most important one is endo-1-4- -xylanase,

Corresponding author. Fax: +91 249 1712. E-mail address: prema@csrrltrd.ren.nic.in (P. Prema).

which initiates the degradation of xylan into xylooligosaccharides and xylose (Collin et al., 2005). Xylanase have attracted considerable research interest because of their potential industrial applications in food and bread making, fruit juice clariWcation, beverage, animal feed, Wber separation (Beg et al., 2001), paper and pulp industries (Bajpai, 1997; Kenealy and JeVries, 2003). In order to make the enzyme applications more cost eVective at industrial level, its production using low cost substrates such as agrowastes has been recommended by many workers (Gupta et al., 2001). One alternative for this is use of solid-state fermentation (SSF), which is closer to natural system and has proved to be more eYcient in producing certain enzymes and metabolites (Pandey et al., 2000; Krishna, 2005). Filamentous fungi (Senthilkumar et al., 2005; Souza et al., 2001) and actinomycetes (Ninawe and Kuhad, 2005) prefer to grow well on moist substrates with less moisture content, whereas bacteria need high moisture contents and unable to grow in these conditions. There are only fewer reports related to successful utilization of bacteria for SSF

0960-8524/$ - see front matter 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2006.02.033

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(Gessesse and Mamo, 1999; Virupakshi et al., 2005). The aim of this work was to formulate an economic process for the production of endoxylanase utilizing cost-eVective agro-industrial wastes as substrate by Bacillus pumilus in SSF and to use the crude enzyme for recycling carton boxes and deinking of oYce paper. 2. Methods 2.1. Microorganisms A bacterial culture of B. pumilus (Subramaniyan et al., 1997) was used in this study. It was maintained on xylan agar slants containing (g/L) xylan 5.0 [Oat spelt xylan, Sigma Chemicals Co.], peptone 5.0, yeast extract 5.0, K2HPO4 2.0, MgSO4 7H2O 0.4 and agar 20.0 [pH 7.0] at 4 C. 2.2. Xylanase production by SSF and enzyme extraction Ten gram of the substrate was well mixed with mineral salts solution containing (g/L) KH2PO4, 2.0; MgSO4 7H2O, 0.4 in Erlenmeyer Xasks [250 ml] to get an initial moisture of 1:1 ratio, pH 7. It was autoclaved at 121 C for 20 min; cooled, inoculated with 18 h old seed culture (3.6 106 counts/ml) and incubated at 30 C. Flasks were removed at regular intervals of 24 h for 5 days and content was extracted in 0.1 M phosphate buVer, pH 7 (1:30 w/v) by mixing the contents using a shaker (at 150 rpm) for 30 min and Wltered through cheese cloth. The Wltrate was centrifuged at 10 000g for 20 min at 4 C and the supernatant was used as crude extract. All experiments were carried out in triplicate and the results were presented as the mean of three. 2.3. Analytical procedures Endoxylanase activity was determined as described earlier (Subramaniyan et al., 1997) at 50 C by using 0.5% oat spelt xylan dissolved in 50 mM phosphate buVer pH 7 as substrate. One unit of endoxylanase activity was deWned as the amount of enzyme that releases 1 mol of xylose under the experimental conditions. Endoxylanase production in SSF was expressed as U/g dry fermented substrate (gds). Biomass was estimated by direct method of dry mass (Pandey et al., 2001). Protein was estimated by Lowrys method (Lowry et al., 1951). 2.4. Enzyme production using agro-industrial residues and eVect of particle size of wheat bran The bacterial strain was cultivated as above with diVerent substrates (Soya meal untoasted, thur dhal husk, rice bran, rice husk, rice straw, sawdust, coconut pith, sugarcane bagasse and wheat bran). The eVect of particle size of wheat bran on enzyme production was evaluated by culturing the organism on wheat bran of diVerent particle sizes: 2 mm, 1 mm, 0.5 mm and 0.3 mm.

2.5. EVect of moisture level on enzyme production The inXuence of initial moisture level on the enzyme production was evaluated by varying the ratio of moisture of wheat bran to mineral salt solution (1:0.5, 1:1, 1:1.5, 1:2, 1:2.5 and 1:3, w/v). The level of mineral salts was maintained constant in all experiments whereas the water level was varied. 2.6. EVect of media pH, temperature and inoculum size on enzyme production EVect of media pH on enzyme production was estimated by culturing the strain in wheat bran of diVerent initial pH 7, 8, 9 and 10. EVect of temperature on enzyme production by B. pumilus was studied by incubating at diVerent temperatures 25, 30, 35, 40, 45 and 50 C for 120 h. EVect of inoculum size on enzyme production was estimated by inoculating Xasks with 2.5%, 5.0%, 7.5% and 10% (v/w) of 18-h inocula and incubated at 35 C. 2.7. Enzymatic treatment of carton and oYce paper For the preparation of paper pulp for this experiment, carton boxes and oYce papers were cut in to small pieces and soaked in distilled water for 10 min, then grinded in wet grinder for 15 min. The Wber freeing process included enzymatic pulping, i.e. treating the pulp with enzyme and separation stage by beating, i.e. grinding the pulp for separation. The pulp suspension of 400 g (consistency of 3.76%) was disintegrated in water at pH 6.0, 32 C for 10 min by agitating at 900 rpm. To this, enzyme was added at 2 U/g oven dry pulps and stirred for 1 h at 900 rpm at 50 C. The enzyme was diluted in distilled water before addition to get better dispersion in the pulp suspension (10% of the total reaction volume). The enzyme was inactivated by boiling the pulp for 5 min. Physical and mechanical properties of the pulp and paper were determined by preparing hand sheet, by passing the pulp through Buchner funnel which was layered with blotting paper, the water was removed by Wltering and sheets were pressed and dried in hot air oven at 50 C until the sheets have shown constant weight (Klungness and Ahmed, 2000). The Wber surface samples were examined and recorded using a JOEL JSM-6400 scanning electron microscope. 3. Results and discussion 3.1. Enzyme production using agro industrial residues and eVect of particle size Fig. 1 shows xylanase production from B. pumilus on various agro-industrial residues. Wheat bran (5582 U/gds) was found to be the best substrate followed by soya meal untoasted (4215 U/gds) and rice straw (1876 U/gds). The results also indicated that these substrates promoted high biomass, which would have been the reason for better pro-

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activity biomass

487

6000

0.7 0.6 0.5

7000

Xylanases activity (U/gds)

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Xylanases activity (U/gds)

6000 5000 4000 3000 2000 1000 0

activity protein

110 100 90 80 70 60 50 40

protein (mg/gds)

Biomass

3000 0.4 2000 1000 0 BG RS RH RB WB CP TDH SD SM -0.3 0.2

Agro industrial residues

Fig. 1. Production of endoxylanase and biomass at 96 h of incubation, by Bacillus pumilus using SSF on various agro industrial residues (BG bagasse, RS rice straw, RH rice husk, RB rice bran, WB wheat bran, CP coir pith, TDH thur dal husk, SD saw dust, SM soy meal Xakes).

2mm

1mm

0.5mm

0.3mm

particle size
Fig. 2. EVect of wheat bran particle size (2.1, 0.5 and 0.3 mm size) on endoxylanase and protein production by Bacillus pumilus in SSF at 30 C at 96 h of incubation.

duction; however, in rice bran biomass was high but production was comparatively less. Production was very low with sawdust, rice husk and coir pith; the presence of polyphenol in high quantity in these substrates would have inhibited the growth and enzyme production. Hence, based on the result, wheat bran was selected and used as substrate for further optimization studies. The universal suitability of wheat bran as substrate is that it contains suYcient nutrients and is able to remain free even in high moist condition providing large surface area (Archana and Sathyanarayana, 1997). The biochemical composition of wheat bran (Lequart et al., 1999) indicated that wheat bran contained considerable amount of soluble sugar like glucose (42.5% dry wt), xylose (15.4% dry wt), arabinose (3.1% dry wt) and galactose (2.7% dry wt) required for the initiation of growth and replication of the microorganism. The degree of substitution of the main xylan chains by arabinose was higher in wheat bran (Lequart et al., 1999). It contained hemicellulose (45%), which may fulWll the role of inducers, and organic nitrogen sources (23%) that are essential for protein synthesis (Babu and Satyanarayana, 1996). Ninawe and Kuhad (2005) also reported wheat bran and corncob as an enhancer for xylanase production by Streptomyces cyaneus SN32.

InXuence of particle size of wheat bran on enzyme production is shown in Fig. 2. Maximum enzyme production was recorded when the particle size was at 0.5 mm (6532 U/ gds), followed by 1 mm (5081 U/gds). Enzyme production and biomass was too low and the protein content was too high at 0.3 mm, due to particle agglomeration, which would have inhibited oxygen transfer. The results clearly indicated that particle size of the substrate inXuenced xylanase production. Particle size of substrates is one of the most critical among several factors that inXuence fermentation process. The microorganism derives its energy by oxidation of organic carbon. The adherence and penetration of microorganism as well as enzyme action on the substrate depends upon the physical properties of the substrate, such as the crystalline and amorphous nature, the accessible area, surface area, porosity, particle size, etc. (Krishna, 2005). In SSF, particle size of the substrate determines the eYciency of the process and it is necessary to choose appropriate particle size for a particular process (Pandey et al., 2000). 3.2. EVect of moisture level on enzyme production EVect of initial moisture content on enzyme production is given in Table 1. The enzyme titer was relatively high when wheat bran was moistened with mineral salt in a 1:2.5

Table 1 EVect of initial moisture level, (i.e., substrate to mineral salt plus inoculum ratio 1:0.5, 1:1, 1:1.5, 1:2, 1:2.5 w/v), temperature (25, 30, 35, 40, 45, and 50 C) and inoculum sizes (2.5%, 5.0%, 7.5%, 10% v/w) on endoxylanase (activity U/gds) production by Bacillus pumilus grown on wheat bran for 4 days for initial moisture and 3 days for temperature and inoculum sizes at initial pH 9 and particle size of 0.5 mm Substrate:initial moisture 1:0.5 1:1.0 1:1.5 1:2.0 1:2.5 1:3.0 Enzyme (U/gds) 781 1568 3081 7071 9290 7473 Temperature (C) 25 30 35 40 45 50 Enzyme (U/gds) 158 9387 13 032 8545 7901 7214 Inoculum size (%, v/w) 2.5 5.0 7.5 10 Enzyme (U/gds) 543 8531 11 531 16 254

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ratio (9290 U/gds). The importance of water in any SSF system is attributed to the fact that majority of the viable cells require the moisture content of 7080% for the synthesis of new cells. The signiWcance of moisture implies that while preparing a substrate, it is necessary to consider the exact quantity of water addition to the substrate to satisfy the requirement of the system (Pandey et al., 2000). Babu and Satyanarayana (1996) correlated microbial growth and product synthesis with the level of moisture content selected. Low moisture may reduce the solubility of lignin and swelling capacity of substrate, and so higher water tension minimizes microbial growth. Higher moisture content results in swelling of substrate, thereby facilitating better utilization of the substrate by the microorganism (Pandey et al., 2000). A reduction in enzyme yield at very high moisture level more than 75%, may be due to the steric hindrance of growth of the organism through reduction in inter particle space, decreased porosity, gummy texture, alteration in wheat bran particle structure and impaired oxygen transfer (Feniksova et al., 1960). 3.3. EVect of medium pH, temperature and inoculum size on enzyme production The result of inXuence of initial media pH on enzyme production is shown in Fig. 3. The optimum pH for the production of xylanase by B. pumilus was in the range of pH 8 (8853 U/gds) to 9 (10 125 U/gds). At pH 7 (6931 U/ gds) and 10 (6521 U/gds) the production of xylanase were slightly decreased. There is a report related production of xylanase at alkaline pH 9 by Aspergillus Wscheri Fxn 1 (1024 U/g) in wheat bran (Senthilkumar et al., 2005) and S. cyaneus SN32 using wheat bran (632 IU/ml) and corn cob (616 IU/ml) at 42 C (Ninawe and Kuhad, 2005). There was a considerable increase in the pH value after the growth was established. Each microorganism holds a range pH for its growth and activity with optimum value between this
50
activity protein

range. The initial pH inXuences many enzymatic systems and the transport of several species of enzymes across the cell membrane. Most bacterial species are unable to grow at reduced moisture level and alkaline pH. The optimum pH for the production of endoxylanase by B. pumilus ranged from 8.0 to 9.0. From the analysis of result, it was evident that the optimum temperature (Table 1) for the production of extracellular xylanase was 35 C at 72 h of incubation, followed by 40 C, and was productive even at 50 C. At 25 C, a very low titer of enzyme was recorded. Results indicated that temperature was an inXuencing factor for enhanced production. The maximum cell growth and enzyme yield of B. pumilus coincided at 35 C. Highest levels of xylanase production in fungal system have already been reported to occur generally at temperatures optimum for growth of culture in SSF (Sudgen and Bhat, 1994). Archana and Sathyanarayana (1997) have reported xylanase activity (19.8 U/DBB) at 50 C. There are reports related to synthesis of endoxylanase, pectinase and cellulase when supplemented with other carbon sources (Olsson et al., 2003). Similar reports were there for xylanase production by A. sulphureus (Lu et al., 2003) and S. cyaneus SN32 (Ninawe and Kuhad, 2005). The inoculum level at 10% (v/w) with a cell count of 3.6 106 counts/ml was found to be optimum for xylanase production at 72 h of incubation by SSF using wheat bran as substrate by B. pumilus. An inoculum size below 5% was inadequate for good growth and enzyme production in SSF (Table 1). Inoculum size 7.5% has also found to be very productive by B. pumilus in SSF. Similar reports on -amylase by B. coagulans B49 have been described with maximum production at 10% (Babu and Satyanarayana, 1996) and B. licheniformis A99 (Archana and Sathyanarayana, 1997) at 15% inoculum size. An incubation period of 72 h (21 431 U/gds) was best suited for maximum enzyme titer and the total soluble pro-

9000

48 46

Endoxylanase activity (U/gds)

10500

160 20000 140 16000

Xylanases activity (U/gds)

Protein (mg/gds)

7500 6000 4500 3000 1500

120 12000 100 8000 80 4000 0

Xylanase activity U/gds protein mg/gds

protein (mg/gds)

44 42 40 38

60

24

48

72

96

120

Time course
6 7 8

pH

10

11

Fig. 3. EVect of initial media pH on endoxylanase and level of protein production by Bacillus pumilus on wheat bran as substrate in SSF at a particle size of 0.5 mm.

Fig. 4. Time course of endoxylanase production and level of protein produced by Bacillus pumilus using fresh wheat bran of particle size 0.5 mm, initial pH and moisture level of mineral salt added as 9% and 70%, respectively, at a temperature of 35 C and 18 h grown inoculum of 10% (v/w) by SSF.

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tein was very less (Fig. 4). There was an increase of 3.8-fold by optimization from initial value of 5582 U/gds under cultivation conditions and a shift in maximum production from fourth day to third day. The bactobran retained 93% and 86% of activity at 96 and 120 h, respectively. 3.4. Enzyme application in recycling of waste paper The eVect of crude enzyme on waste paper utilization was tried. The SEM (scanning electron micrograph) micrographs of pulp are given in Figs. 5 (untreated) and 6 (treated). The SEM micrograph of enzyme treated pulp showed a signiWcant change on the surfaces, the surface of untreated pulp appeared smoother than that of xylanase treated Wbers. Fibers of treated pulp underwent a peeling process giving rise to Xakes and Wlaments of materials detached from Wber surfaces, due to xylan hydrolysis. Enzyme treatment increases the Wber swelling, which facili-

tates reWning, which in turn results in better physical properties (Kenealy and JeVries, 2003). Enzyme addition prior to reWning can improve strength properties at a Wxed reWning level. 4. Conclusions SigniWcance of this optimization studies related to maximization of endoxylanase production utilizing economical carbon sources- wheat bran as a substrate as well as increase of 3.8-fold and shift in production from fourth to third day of fermentation. The use of low-cost raw materials leads to reduction in the culture medium cost that generally range from 25% to 50% of total production cost. This increase in yield and decrease in production cost is a promising methodology for hyper production of alkaline thermostable xylanase. The results of enzyme application with recycled paper clearly indicate that the eVective use of enzymes in Wber separation can reduce the cost of carton paper production and is a promising alternative in the present scenario of biobleaching of Kraft pulp. Acknowledgements The authors are grateful to the Director, Regional Research Laboratory, Trivandrum for providing the facilities to carry out the above work. Asha Poorna, C. thankfully acknowledges the CSIR, Government of India for providing a SRF to carry out this work. References
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Fig. 5. SEM micrograph of the untreated pulp, after grinding, the Wber surface observed to be smooth.

Fig. 6. SEM micrograph of enzyme treated pulp, the Wber surface observed to be rough with wrinkles and peeling of micro Wbrils.

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C. Asha Poorna, P. Prema / Bioresource Technology 98 (2007) 485490 Pandey, A., Soccol, C.R., Rodriguez-Leon, J.A., Nigam, P., 2001. SolidState Fermentation in Biotechnology. Asiatech Publishers, Inc., New Delhi. p. 221. Senthilkumar, S.R., Ashokkumar, B., Chandra Raj, K., Gunasekaran, P., 2005. Optimization of medium composition for alkali-stable xylanase production by Aspergillus scheri Fxn 1 in solid-state fermentation using central composite rotary design. Bioresour. Technol. 96, 1380 1386. Souza, D.F., Souza, C.G.M., Peralta, R.M., 2001. EVect of easily metabolizable sugars in the production of xylanase by Aspergillus tamarii in solid-state fermentation. Process Biochem. 36, 835838. Subramaniyan, S., Prema, P., Ramakrishna, S.V., 1997. Isolation and screening for alkaline thermostable xylanase. J. Basic Microbiol. 37, 431437. Sudgen, C., Bhat, M.K., 1994. Cereal straw and pure cellulose as carbon sources for the growth and production of plant cellwall degrading enzyme by Sporotrichum thermophilie. World J. Microbiol. Biotechnol. 10, 444451. Virupakshi, S., Gireesh Babu, K., Satish, R.G., Naik, G.R., 2005. Production of a xylanolytic enzyme by a thermoalkaliphilic Bacillus sp. JB-99 in solid-state fermentation. Process Biochem. 40, 431435.

Krishna, C., 2005. Solid-State Fermentation SystemsAn Overview. Crit. Rev. Biotechnol. 25, 130. Lequart, C., Nuzillard, J.M., Kurek, B., Debeire, P., 1999. Hydrolysis of wheat bran and straw by an endoxylanase: production and structural characterization of cinnamoyl-oligosaccharides. Carbohydr. Res. 319, 102111. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.L., 1951. Protein measurement with the folin phenol reagent. J. Biol. Chem. 193, 265275. Lu, W., Li, D., Wu, Y., 2003. InXuence of water activity and temperature on xylanase biosynthesis in pilot-scale solid-state fermentation by Aspergillus sulphureus. Enzyme Microb. Technol. 32, 305311. Ninawe, S., Kuhad, R.C., 2005. Use of xylan-rich cost eVective agro-residues in the production of xylanase by Streptomyces cyaneus SN32. J. Appl. Micrbiol. 99, 11411148. Olsson, L., Christensen, T.M.I.E., Hansen, K.P., Palmqvist, E.A., 2003. InXuence of the carbon sources on the production of cellulase, hemicellulases and pectinases by Tricoderma reesi Rut C-30. Enzyme Microb. Technol. 33, 612619. Pandey, A., Soccol, C.R., Mitchell, D., 2000. New development in solid state fermentation: I bioprocesses and products. Process Biochem. 35, 11531169.