Potential application of yeast cellulase-free xylanase in agrowaste material treatment to remove hemicellulose fractions

Bioresource Technology 63 (1998) 187-191 © 1998 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0960-8524/98 $19.00 ELSEVIER

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POTENTIAL APPLICATION OF YEAST CELLULASE-FREE XYLANASE IN AGROWASTE MATERIAL TREATMENT TO REMOVE HEMICELLULOSE FRACTIONS D. V. Gokhale, S. G. Patil & K. B. Bastawde* Division of Biochemical Sciences, NCIM, National Chemical Laboratory, Pune-411 008, (MS), India (Received 7 May 1996; revised version received 7 March 1997; accepted 14 April 1997)

be removed by cellulase-free xylanase pretreatment instead of hazardous chemical softening processes. Such a bioprocess will help in selective pretreatment processes (Hoq et al., 1992, 1994). There are very few reports on the production of microbial cellulase-free xylanase, especially from yeast strains (Bastawde et al., 1994). Earlier we reported that the yeast strain isolated from a natural decaying wood produces highest levels of extracellular cellulase-free xylanase (220 IU m1-1) under submerged conditions in shake flasks. In this manuscript, we describe the pretreatment effect on various agricultural materials and the time course effect on the hydrolysis of these materials, e.g. corn cob (40 mesh size), jute fibers, bleached as well as unbleached sugarcane bagasse pulp. The end product analysis of these hydrolyzates was monitored by HPLC.

Abstract Yeast cellulase-free xylanase was used for treatment of different agricultural waste residues including corn cob powder, raw jute fibers and sugarcane bagasse pulp. The high degree of hydrolysis (19.4%) was achieved by using 22 IU m l - 1 yeast xylanase in the case of treated bagasse pulp. Corn cob powder and raw jute fiber showed a slower rate of hydrolysis under the same conditions. The enzymatic hydrolysis was carried out using citrate buffer (50raM, p H 4.5) at 52-53°C for different time intervals. The hydrolysis products were analysed by HPLC, and xylose was found to be the major end product with traces of xylobiose and xylotriose at the beginning of hydrolysis. @ 1998 Elsevier Science Ltd. All rights reserved.

Key words: Agricultural residues, bagasse pulp, cellulase-free xylanase, corn cob, jute fiber, yeast strain. INTRODUCTION

METHODS

Increasing interest has been shown in using cellulase-free xylanase enzymes in the paper and pulp industry to modify pulp properties by removal of hemicelluloses and lignin residues without disturbing the required structural properties of cellulose microfibrils (Viikari et al., 1986). An advantage of this enzyme treatment process is to minimize the use of chlorine, thus reducing the concentrations of toxic chloro-organic wastes produced during chemical bleaching processes (Hoq et al., 1994; Nakamura et al., 1994). Industrial as well as academic scientists are looking for processes which are environmentally friendly and safe (Gosh and Dutta, 1983). Jute fiber is an important natural biodegradable agricultural product and could replace the chemically synthesized cellulose fibers in the near future (Hoq et al., 1994). Hemicellulose, mainly xylan, which acts as a hardening component in the cellulosic structure, can

Microorganism The yeast strain used in these studies was isolated from a decaying sandal wood, and it was maintained on both MGYP and PDA slopes as a stock culture (Bastawde et al., 1994). It is deposited with the National Collection of Industrial Microorganisms (NCIM), Division of Biochemical Sciences at National Chemical Laboratory, Pune-411 008, India, with an accession No. 3574 (Bastawde and Gokhale, 1991). Maintenance media (a) MGYP medium contained (g 1-1): malt extract, 3.0; glucose, 10.0; yeast extract, 3.0; Bacto-peptone, 5.0; and agar, 20.0. (b) PDA medium contained (g1-1): liquid extract of potatoes, 200.0; glucose, 20.0; yeast extract, 1.0; and agar 20.0. The pH for both media was adjusted to 6.5-7.0 before sterilization at 15 lb for 20 min.

*Author to whom correspondence should be addressed. 187

188

D. V. Gokhale, S. G. Patil, K. B. Bastawde

(c) Production medium: the production of cellulasefree xylanase was carried out in a Apergillus minimal medium (AMM) containing (g l-l): KH2PO4, 2.0; yeast extract, 1.0; peptone, 5.0; KCI, 0.5; NaNO3, 0.5; MgSO4.7H20, 0.5; xylan, or wheat bran, 10.0. The medium pH was adjusted to 5.5 before it was sterilized at 15 lb for 20 min. Xylanase production under submerged conditions Yeast cells were pregrown in 5.0 ml MGYP liquid medium in a boiling glass tube for two days at 28-30°C on a reciprocal shaker (200 rpm). This pregrown culture was used to inoculate 250ml Erlenmeyer flasks containing 50.0ml of AMM medium containing either 1% xylan or 1% wheat bran as an inducer carbon source for xylanase. These inoculated flasks were incubated at 28-30°C on a reciprocal shaker (200 rpm) for 18-20 h. Yeast cells were removed by high speed centrifugation at 10000 rpm for 20 min, and the clear supernatant liquid was used to determine the extracellular xylanase activity. Determination of xylanase activity Xylanase (EC 3.2.1.8) activity was determined as described earlier (Bastawde, 1992). A 0.5 ml sample of suitably diluted culture filtrate was mixed with 0.5 ml of 1% (w/v) oat spelts xylan (Sigma, St. Louis, Mo, USA) solution in citrate buffer (50 mm, pH 4.5) and incubated at 55°C for 30 min. The reaction was terminated by the addition of 1.0 ml of 3,5-dinitrosalicylic acid (DNS) solution. Reducing sugar released as xylose equivalent was measured by the Miller method (Miller, 1959). One unit of xylanase activity was defined as the amount of enzyme which produced one #mole of xylose per min per ml of the crude culture filtrate from soluble xylan substrate under standard assay conditions. Enzymatic hydrolysis of different substrates The enzymatic hydrolysis of different agricultural substrates was carried out as follows. Corn cobs were collected from a local market and were first milled to powder form (40 mesh size) in a Mini Willy mill. Raw jute fibers were obtained from Hindustan Lever Co., Bombay, while the bleached and unbleached bagasse pulps were received from Shrey-

ans Paper Mill Ltd, Ahmedgarh, Dist. Sangrur (Punjab), India. These cellulosic substrates were first treated with 0.1 N NaOH at 28-30°C for 16 h. The treated samples were washed with distilled water to remove the alkali. Twenty grams (wet wt) of alkali pretreated cellulosic subtrate was taken into 180 ml citrate buffer (pH4.5, 50mM) in 500ml conical flask, to which 20.0 ml crude xylanase was added to start the reaction. This hydrolysis reaction mixture was kept under constant shaking conditions (200rpm) at 53-55°C. The hydrolyzate samples were removed at intervals of every 4 h and checked for end product analysis as well as for reducing sugar yield. End product analysis by HPLC The end products of enzymatic hydrolysis of corn cob powder and bleached bagasse pulp were analyzed by using a high performance liquid chromatography (Hewlett-Packard 1082 B) pump equipped with an automatic injector, 50 #1 injection capacity loop, a 250 mm x 4.6 mm Shodex Ionpack (S-801/s) column, a chromatography computing integrator with RI detector (16 x ). The mobile phase used was deionized water, and the flow rate was adjusted to 1.0 ml min -~ at 50°C.

RESULTS Time course of enzymatic hydrolysis of bleached bagasse pulp The enzymatic hydrolysis of bleached bagasse pulp was carried out using different amounts of xylanase as well as with different concentrations of bleached bagasse pulp samples. Table 1 shows the enzymatic hydrolysis of 5.0 g (wet weight) bleached bagasse pulp with different enzyme concentrations (313, 783, 1566, 2060 and 3 1 3 0 I U g -1 of dried pulp). The hydrolysis was carried out for different time intervals, and samples of hydrolyzates were taken out for analysis of reducing sugars. The rapid initial increase in the rate of hydrolysis was followed by a decline in the hydrolysis rate. The further increase in the amount of enzyme did not affect the production of reducing sugars. The maximum hydrolysis of 5% bleached bagasse pulp was achieved with 2060 IU g - 1 of dried pulp within 24 h incubation.

Table 1. Release of reducing sugars from bleached bagasse pulp (5.0 g wet weight) treated with yeast xylanase at 55"C Reducing sugar (mg/g of pulp)

Total enzyme (IU) per gram of dry pulp

313 783 1566 2060 3130

2h

4h

8h

12h

24h

51 85 106 116 124

74 99 116 130 139

89 124 151 160 166

95 130 158 162 160

102 135 166 170 172

Potential appfication of yeast cellulase-freexylanase The second set of experiments was carried out by using different pulp concentrations using a fixed amount of enzyme (3760 IU). All other hydrolysis conditions were kept constant as described earlier. Table 2 indicates that bleached bagasse pulp at lower concentrations (5 and 10% wet weight) was hydrolyzed very rapidly (4 h), while the use of higher concentrations of BBP resulted in comparatively

200 ll 160 v

og

120

189

decreased yield of reducing sugars even after 24 h of incubation with the enzyme. The third set of experiments was performed with fixed substrate concentration of 10 g with varying xylanase units ranging from 156 to 6264IU (Table 3). The results shown in Table 3 also confirmed that 2 0 6 0 I U g -1 was enough to release maximum amounts of reducing sugars from BBP. The other untreated agricultural materials like jute fibers, corn cob powder and unbleached bagasse pulp showed lower extent of hydrolysis when treated with higher enzyme concentrations for longer periods of time (Table 4). The effect of alkali treatment on the same substrates prior to enzymatic hydrolysis increased the hydrolysis rates compared to those of untreated substrates (Fig. 1).

&

Identification of soluble products of pretreated subtrate xylanolysis

8O ef

~- 40

I

I

500

t000

I

,

1500

I

2000

I

I

2500

3000

3500

X y l a n a ~ A c t i v i t y (IU)

Fig. 1. Enzymatic hydrolysis of different agrowaste materials: e, jute fiber; A, corn cob powder; I, bleached bagasse pulp.

HPLC, as well as paper chromatography analysis, of corn cob powder and bleached bagasse pulp hydrolysates revealed mainly xylose with traces of xylobiose and xylotriose as the end products during initial hydrolysis period. As the time progressed xylotriose and xylobiose disappeared after 12h hydrolysis showing predominantly xylose as the only end product of hydrolysis (Fig. 2).

Table 2. Effect of yeast cellulase-free xylanase (3760 IU) on different concentrations of pretreated bleached bagasse pul

Total pulp used (g, dry weight)

1.2 2.4 3.6 4.8

Reducing sugar (mg/g of dry pulp) 4h

8h

12h

24h

151 121 119 106

151 145 132 110

151 149 137 106

162 150 137 121

Table 3. Effect of different enzyme concentrations on pretreated bleached bagasse pulp (2.4 g dry weight)

Total enzyme used (IU per gram of pulp)

156 390 783 1566 2060 3130 6364

Reducing sugar (mg/g dry pulp) 4h

8h

12h

24h

57 73 91 12l 132 136 143

61 84 96 145 148 152 165

68 87 102 149 158 162 165

74 84 98 149 162 164 169

Table 4. Effect of yeast cellulase-free xylanase on the hydrolysis of different agrowaste materials

Agrowaste materials

Bagasse pulp Corn cob powder Jute fibers

Reducing sugars released (mg) per gram of the substrate in 24 h Treated

Untreated

172 96 18

4.6 3.4 2.8

190

D. V. Gokhale, S. G. Patil, K. B. Bastawde

(a)

(b)

(d)

(c)

(e)

l

I 0

I 5

I 10

I 0

I 5

I 10

I 5

I 0

I 10

I 0

I 5

I 10

I 0

| 5

I 10

Min

Fig. 2. Enzymatic hydrolysis pattern of corn cob powder at different time intervals, (a) 1 h, (b) 2 h, (c) 8 h, (d) 12 h and (e) 16 h. Hydrolysis products formed are: AC -- acetal group; AR -- arabinose; X1 -- xylose; X2 -- xylobiose; X3 xylotriose; X, -- xylan. Xyiose production from pretreated corn cob powder We attempted to produce xylose from alkali-treated corn cob, a cheap agricultural waste product. Twenty grams of powdered alkali-treated corn cob was incubated with yeast xylanase (76 IU g - i of corn cob) which yielded 1.5 g of xylose within 20 h amounting to 8% conversion. The xylose produced in this hydrolysis was partially purified on an activated charcoal column.

DISCUSSION Cellulase-free xylanase produced by the newly isolated yeast strain has shown the capacity of producing appreciable amounts of reducing sugar in the form of xylose from different agricultural waste residues. Okeke and Obi (1995) reported on the saccharification of agro-waste materials using Sporotrichum pruinosum and Arthrogruphis sp. which produced both cellulase and xylanase activities. Such enzyme complexes have limited applications in industries, like paper and pulp, fruit juice clarification and textile. Currently, technologists and research scientists are looking towards cleaner process technology for pulp and paper manufacturing which are eco-friendly. The crude xylanase preparation from Aureobasidium pullulans was used to remove hemicellulose from unbleached sulphite pulp in which the pulp was treated with 0.03 g NaOH per gram of pulp at 80°C

for 1 h with 2.5% pulp consistancy. In the present studies we used lower concentrations of alkali (0.02 g NaOH per gram of pulp) at 28-30°C for 16 h with 2.0% pulp consistancy. This alkali-treated bagasse pulp was incubated with yeast cellulase-free xylanase resulting in production of 150 mg of reducing sugar as xylose per gram of pulp. Christov and Prior (1994) used Aureobasidium pullulans xylanase and were able to produce 12.8 mg of reducing sugar per gram of pulp after 24 h. The higher yields of reducing sugar obtained in our studies could be due to the use of bleached pulp for enzyme treatment. In the future the chlorine used for pulp bleaching may be replaced by such cellulase-free xylanase enzymes to get better quality paper (Kantelinen, 1992; Sinner and Presselmayr, 1992; Turner et al., 1992). There are many drawbacks of the chlorine pulp bleaching process, such as the release of chloride compounds into the atmosphere and their discharge into waste water which further increases the toxicity of whole ecosystem. Viikari et al. (1994) have rightly emphasized the need for the industrial use of xylanase in Kraft pulp bleaching process. The Indian paper and pulp industry, as of today, is not as large as the USA, Europe or Scandinavian countries'. As reported by Subrahmanyam (1990), the total capacity of paper production by Indian paper and pulp companies amounts to only 3.0 million tonnes per annum. The pollution discharge load produced by these companies is quite large. Bajpai and Bajpai (1994) have recently given some

Potential application of yeast cellulase-free xylanase thoughts on the above matter and emphasized biological wastewater treatments. The other alternative way of treating the paper pulp is with cellulase-free xylanase treatment in combination with mild chemical treatments (Viikari et al., 1991; Koponen, 1991; Tolan, 1992; Turner et al., 1992 and Viikari et al., 1994). We are reporting here the possible applications of our yeast cellulase-free xylanase mainly in three different areas: (1) production of xylose from agro-waste materials like corn cob; (2) removal of hemicellulose fractions from bleached or unbleached pulp, as well as from the jute fibers used in textile industries without harming the cellulosic micro-fibril structures; (3) minimizing the use of chlorine for the bleaching process by partially replacing it with enzyme, making the overall process economically and environmentally friendly.

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Hoq, M. M., Alam, M. M., Mohiuddin, G. & Gomes, I. (1992) Enzyme biotechnology in developing countries: production and application of thermostable xylanase for jute fibre upgradation. In Harnasing Biotechnology for the 21th Century, Proceeding of the ninth International Biotechnology Symposium and exposition, ed. Michael and Bose, pp. 568-573. ACS. Hoq, M. M., Hempel, C. & Dectwer, W. D. (1994). Cellulase-free xylanase by Thermomyces lanuginosus RT 9: Effect of agitation, areation, and medium composition production. J. Biotechnol., 37, 49-58. Kantelinen, A. (1992) Enzyme in bleaching of kraft pulp, VI'T Publication, 114, Technical Research centre of Finland, Espoo., pp. 86. Koponen, R. (1991). Enzyme system prove their potential. Pulp Paper Inst., 33, 20-25. Miller, L. G. (1959). Use of dinitrosalicylic acid reagent for determination of reducing sugars` Anal. Chem., 31, 426-428. Nakamura, S., Nakai, R., Wakabayashi, K., Ishiguro, Y., Aono, R. & Horikoshi, K. (1994). Thermophilic alkaline xylanase from newly isolated alkaliphillic and thermophillic Bacillus sp. strain TAR-1. Biosci. Biotech. Biochem., 58, 78-81. Okeke, B. C. & Obi, S. K. C. (1995). Saccharification of agro-waste materials by fungal cellulases and hemicellulases` Bioresource Technology, 51, 23-27. Sinner, M. & Presselmayr, W. (1992). Chlorine is out, bring in the enzymes, Pulp Paper Int., 9, 87-89. Subrahmanyam, P. V. R. (1990). Waste management in pulp and paper industry. J Indian Assoc. Environ. Manag., 17, 79-94. Tolan, J. S. (1992) The use of enzyme to enhance pulp bleaching. Tappi Proceedings, 1992 Pulping Conference, Boston, MA, November 1-5, pp. 13-17. Tappi Press. Turner, J. C., Skerker, P. S., Burns, B. J., Howard, J. C., Alonso, M. A. & Andres, J. L. (1992). Bleaching with enzymes instead of chlorine mill trails. Tappi J., 75, 83-89. Viikari, L., Ranua, M., Kantelinen, A., Linko, M. & Sundquist, J. (1986) Bleaching with enzymes, Biotechnology in the Pulp and Paper Industry, 3rd Int. Conf., Stockholm, pp. 67-69. Viikari, L., Sundquist, J. & Kettunen, J. (1991). Xylanase enzyme promote pulp bleaching. Paper Timber, 73, 384-389. Viikari, L., Kantelinen, A., Sundquist, J. & Linko, M. (1994). Xylanases in bleaching: From an idea to the industry. FEMS Microbiol. Rev., 13, 335-350.