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Effect of gamma irradiation on the hydrolysis of lignocellulosic materials using cellulase derived from Aspergillus niger.

Introduction

Energy consumption has increased steadily over the last century as the world population has grown and more countries are becoming industrialized. Petroleum fuels have been the major resources to meet the increased energy demand. It has been predicted that the decline in worldwide crude oil production will begin before 2010 and that annual global oil production would decline from the current 25 billion barrels to approximately 5 billion barrels in 2050 (Campbell and Laherrere, 1998). Therefore, a potential solution to the ever-increasing energy demand is the use of materials from renewable sources.

Lignocellulosics are one of these materials and are in vast supply. Their hydrolysis yields fermentable sugars which can serve as chemical feedstocks and energy sources (Solomon et al., 1990, Kim et al., 2000; Ojumu et al., 2003a and b). Nigerian forests contain a lot of softwood and hardwood timbers that are of great commercial importance (Gbile, 1984), some examples are Khaya senegalensis (Mahogany), Tectona grandis (Teak) [hardwoods] and Triplochiton scleroxylon (Arere), Albizia zygia (Ayunre) [softwoods]. However, sawdust generated by mill processing of these woods is in large supply and currently constitutes a large portion of municipal waste. Unfortunately, because of the recalcitrant nature of lignocellulosic materials, their hydrolysis is not readily achieved. It has been reported that biodegradation of untreated natural lignocellulosic biomass is very slow, giving rise to the low extent of degradation, often under 20% (Fan et al., 1980). This low rate and extent of conversion inhibit the development of an economically feasible hydrolysis process. Hence, it is imperative to pretreat the biomass in order to obtain a suitable material for the bioconversion. Various pretreatment methods for enhancing bioconversion of lignocellulosics have been investigated by several workers to solve this recalcitrance problem (Solomon et al., 1990; Ojumu et al., 2003a, 2003b; Yang and Wyman, 2006). These include acid or alkali treatment, steam explosion, and extraction with aqueous organic solvents. Pretreatment is aimed at disrupting the lignocellulosic matrix to facilitate the separation of the constitutive polymers and subsequently, make the cellulose fraction more accessible to hydrolysis. Pretreatment of native lignocellulose causes reductions in crystallinity, decomposition of lignocellulosic biomass and removes secondary interactions between glucose chains (Fan et al., 1980).

Solomon et al. (1990) achieved hydrolysis of sawdust using cellulase with an activity of 0.056 IU/ml derived from Triplochiton scleroxylon. Ojumu et al. (2003b) produced cellulase enzyme of 0.0743IU/ml activity by Aspergillus flavus Linn isolate NSPR 101 using sawdust as substrate. In both cases cellulase activity was determined by Filter Paper Activity (FPA) and the authors used caustic swelling pretreatment method prior to the use of biological agents.

Although various methods of pretreatment have been reported (Jeoh and Agblevor, 2001; Bigelow and Wyman, 2002; Martin and Thomsen, 2007), few reports exist on the use of gamma irradiation (Martfnez et al., 1995; Lam et al., 2000; Betiku et al., 2009). Gamma radiation, if used in high dosage on lignocellulosics, causes a decrease in cell wall constituents or depolymerizes and delignifies the fiber (Al-Masri and Zarkawi, 1994). An increase in organic matter digestibility has been reported due to its cell wall degradation (Al-Masri and Guenther, 1995). However, its pasteurizing and sterilization capabilities for agricultural products have also been reported when used at low dosage (Kume et al., 1990; Kim et al., 2000). Kim et al. (2000) found that a gamma dose of 5-10 kGy was effective in reducing microbial contamination of medicinal herbs. However, research has shown that a higher cellulose degradation of agricultural by-products occurs for a combination of gamma radiation and chemical treatments as compared with chemical treatment or irradiation treatment alone (Banchorndhevakul, 2002). In a recent study carried out using Aspergillus flavus, Betiku et al. (2009) showed that sawdust of both soft- and hard-woods pretreated with physical, chemical and gamma irradiation methods yield far more cellulase than when pretreated with both physical and chemical methods only.

It has been reported that hydrolysis of lignocellulosics by cellulase is source dependent. Hence, cellulase derived from Aspergillus niger is employed in this work to study the extent of enzymatic hydrolysis of both softwood and hardwood samples, used to measure the effect of gamma irradiation as a pretreatment method, in addition, dosage for optimum recovery were also reported.

Materials and Methods

Sawdust samples

Two types of sawdust were used for this study; softwood (Triplochiton scleroxylon) and hardwood (Khaya senegalensis). The samples, carefully collected from Omolaye Sawmill, Ikirun, Osun State, Nigeria, were milled to yield fine particles. The fraction which passed 32-mesh but retained by 42-mesh were used in all the experiments. Samples were dried in a vacuum oven at 60[degrees]C for 24 h before pretreatment (Betiku et al., 2009). The cellulose component of typical softwood is reported to be 42%. The method proposed by Rivers et al. (1983) was used for the determination of cellulose content.

Pretreatment

The samples were exposed to [gamma]-ray ranging from 10kGy to 100kGy emitted from [sup.60]Co (cobalt-60 AECL), at a dose rate of 0.6 Gray per second. The irradiated samples was soaked in 1% (w/v) sodium hydroxide solution at a ratio of 1:10 (substrate:solution) for 2h at room temperature after which they were washed free of the chemical and autoclaved at 120[degrees]C (15 psig steam) for 1 h as prescribed by Ojumu et al. (2003b). The samples used for control studies were also subjected to the above pretreatment methods except for exposure to gamma radiation, this allows for the contribution of gamma radiation to be determined.

Enzymes production

A pure culture of Aspergillus niger provided by the Department of Microbiology, Obafemi Awolowo University, Ile-Ife, Nigeria, was used for cellulase production. The details of the enzyme production have been described elsewhere (Solomon et al., 1990; Ojumu et al., 2003b). The cellulase was harvested (and used immediately for the hydrolysis experiment) at the 12th hour of cultivation (when the activity is optimum) as observed in the previous study (Solomon et al., 1990; Ojumu et al., 2003b).

Enzymatic hydrolysis of sawdust

Hydrolysis of the sawdust samples with cellulase was conducted by suspending specified part of the dried sample in a 250 ml flask with 0.05 M citrate buffer (pH 5) for 1 h in an incubator at 45[degrees]C before adding the cellulase enzyme produced above in an amount corresponding to 10 ml enzyme solution per 1 g of dry sample. The enzyme solution was considered to be impure as no attempt was made to purity it. The hydrolysis was carried out at 45[degrees]C in an incubator shaker at 200 rpm for 12 h. Samples of the hydrolyzate were withdrawn every 1 h, centrifuged and the supernatant was analysed for reducing sugar.

Cellulase activity and reducing sugar concentration analysis

The cellulase activity was determined using Whatman No. 1 filter paper and was expressed as filter paper activity; this has been previously described in details by Ghose (1987). The total amount of reducing sugars which is expressed as equivalent glucose in 1.0 ml supernatant was determined by the modified dinitrosalicyclic acid (DNS) method of Miller (1959). The extent of hydrolysis was expressed as below (Mandels et al., 1976).

extent of hydrolysis = [weight of glucose formed x (162/180)] / dry weight of cellulose used x 100 (1)

Results and discussion

This work investigated the effectiveness of cellulase derived from Aspergillus niger in the hydrolysis of wood samples. In addition, the effect of gamma irradiation on the digestibility of the softwood and hardwood sawdust samples during hydrolysis based on the reducing sugar production was studied. The hydrolysis-time profile for the first eight hours (i.e. initial fast rate) showed that the extent of hydrolysis was improved when the wood samples were irradiated, irrespective of the species of wood used (data not shown). Although previously reported experiments also indicated that irradiation improved the digestibility of lignocellulosic materials at high dosage (Lam et al., 2000), the results indicated that significant hydrolysis was obtained at low dosage (10 kGy) for the wood (Table 1).

Extended hydrolysis experiment (up to 96 h) showed that reducing sugar yield was more than double when the samples were treated with gamma irradiation, irrespective of the level employed (Figures 1 and 2). This observation is corroborated by previous workers that digestibility of cellulose and lignocellulosic materials are enhanced by high-energy irradiation (Banchorndhevakul, 2002; Betiku et al., 2009). The statistical analysis of the results obtained for irradiated softwood sample at different dosage levels (10 to 100 kGy) revealed that by exposing the sample to a 70 kGy dose, the highest hydrolysis rate and the maximum conversion of cellulose were obtained. Further increase in the irradiation dosage contributed insignificantly to the conversion while for hardwood sample, the optimal result was obtained at 90kGy level of irradiation (Table 1, Figures 1 and 2). The t-test analysis of the data revealed that the irradiated hardwood sawdust hydrolysed better than the irradiated softwood sawdust at both 70 and 90kGy irradiation dosage levels, as shown by the relatively higher yield of reducing sugar obtained from the irradiated hardwood (Figures 3 and 4). This observation could be attributed to percent composition of the cellulose in the wood samples, available specific surface area for the reaction, lignin content of the substrate, availability of the active site for enzymes, all of which favoured the hardwood sawdust (Cowling, 1975; Fan et al., 1980). Dunlap and Chiang (1980) observed this occurrence and stated that irradiation appears to be strongly species selective; for example, the digestion of aspen carbohydrate is essentially complete after a dosage of 108 rad, while spruce is only 14% digestible at this dosage. Betiku et al. (2009) also reported same trend in their study of hydrolysis of the same species of woods using cellulase derived from Aspergillus flavus. Optimal gamma irradiation observed for the hydrolysis of softwood and hardwood sawdust samples were 40kGy and 90 kGy, respectively (Betiku et al., 2009). The variation observed in the gamma irradiation for pretreatment of softwood sawdust sample may be attributed to the source of the wood samples.

The kinetic data obtained were found to deviate from the kinetic model of Ghose and Das (1971). This means that the kinetic pattern suggested by the empirical model for the initial phase of reaction may not be applicable to all the stages of hydrolysis beyond a certain period of hydrolysis. The possible cause of the deviation could be the existence of factors like the build-up of resistant cellulose during the course of hydrolysis, the onset of the effect of product inhibition, the heterogeneity of the lignocellulosic materials, interrelation between [C.sub.1] and [C.sub.x] of the cellulase enzyme. Such a deviation was observed by Ghose (1969).

In this work, A. niger have been shown to be a good source of cellulase and in addition, irradiated woods have been demonstrated to be good candidates for cellulose biomass conversion into useful products; however, hardwood (Khaya senegalenesis) is more suitable for this process compared with softwood (Triplochiton scleroxylon) since it yielded more reducing sugars.

[FIGURE 1 OMITTED]

[FIGURE 2 OMITTED]

[FIGURE 3 OMITTED]

[FIGURE 4 OMITTED]

References

[1] Al-Masri, M.R., and Guenther, K.D., 1995, "The effect of gamma irradiation on in vitro digestible energy of some agricultural residues," Das Wirtschaftseigene Futter, 41, pp. 61-68.

[2] Al-Masri, .M.R., and Zarkawi, M., 1994, "Effect of gamma irradiation on cell-wall constituents of some agricultural residues," Radiat. Phys. Chem., 44, pp. 661-663.

[3] Banchorndhevakul, S. (2002), "Effect of urea and urea-gamma treatments on cellulose degradation of Thai rice straw and corn stalk," Radiat. Phys. Chem., 64, pp. 417-422.

[4] Betiku, E. Adetunji, O.A., Ojumu, T.V., and Solomon, B.O., 2009, "A Comparative Study of the Hydrolysis of Gamma Irradiated Lignocelluloses," BJChE, 26(2), pp. 251-255.

[5] Bigelow, M., and Wyman, C.E., 2002, "Cellulase production on bagasse pretreated with hot water," Appl. Biochem. Biotechnol., 100, pp. 921-934.

[6] Campbell, C.J., and Laherrere, J.H., 1998, "The end of cheap oil" Sci Ambas., 3, pp. 78-83.

[7] Cowling, E.B., 1975, Physical and chemical constraints in the hydrolysis of cellulose and lignocellulosic materials," Biotechnol. Bioeng. Symp., 5, pp. 163-181.

[8] Dunlap, C.E., and Chiang, L.C., 1980, "Cellulose degradation--a common link". In: M.L. Schuler, ed., Utilizing and Recycle of Agricultural Wastes and Residues, CRC Press, New York, pp. 19-65.

[9] Fan L.T., Lee, Y-H., and Beardmore, D.H., 1980, "Major chemical and physical features of cellulosic materials as substrates for enzymatic hydrolysis," Adv. Biochem. Eng., 14, pp. 101-117.

[10] Gbile, K.O., 1984, "Botanical name of common trees in Nigeria", Iwalaye Press, Ibadan, Oyo State, Nigeria.

[11] Jeoh, T., and Agblevor, F.A. (2001), "Characterization and fermentation of steam exploded cotton gin waste," Biomass Bioenergy, 21, pp. 109-120.

[12] Kim, M.J., Yook, H.S., and Byun, M.W., 2000, "Effects of gamma irradiation on microbial contamination and extraction yields of Korean medicinal herbs," Radiat. Phys. Chem., 57, pp. 55-58.

[13] Kume, T., Ito, H., Ishigaki, I., LebaiJuri, M., Othman, Z., Ali, F., Mutaat, H.H., Awang, M.R., and Hashim A.S., 1990, "Effect of gamma irradiation on microorganisms and components in empty fruit bunch and palm press fiber of oil palm wastes," J. Sci. Food Agric., 52, pp. 147-157.

[14] Lam, N.D., Nagasawa, N., and Kume, T., 2000, "Effect of radiation and fungal treatment on lignocelluloses and their biological activity,". Radiat. Phys. Chem., 59, pp. 393-398.

[15] Mandels, M., Andreotti, R., and Roche, C., 1976, "Measurement of saccharifying cellulase," Biotechnol. Bioeng. Symp., 6, pp. 21-33.

[16] Martfnez, J.M., Granado, J., Montanr, D., Salvad, J., and Farriol, X., 1995, "Fractionation of residual lignocellulosics by dilute-acid prehydrolysis and alkaline extraction: Application to almond shells," Bioresour Technol., 52, pp. 59-67.

[17] Martin, C., and Thomsen, A.B., 2007, "Wet oxidation pretreatment of lignocellulosic residues of sugarcane, rice, cassava and peanuts for ethanol production," J. Chem. Technol. Biotechnol., 82, pp. 174-181.

[18] Miller, G.L., 1959, "Use of dinitrosalicyclic acid reagent for determination of reducing Sugars," Biotechnol. Bioeng. Symp., 5, pp. 193-219.

[19] Ojumu, T.V., Attah-Daniel, B.E., Betiku, E., and Solomon, B.O., 2003a, "Auto-hydrolysis of lignocellulosics using extremely low acid under high temperature in a batch process," Biotechnol. Bioprocess Eng., 8, pp. 291-293.

[20] Ojumu, T.V., Solomon, B.O., Betiku, E., Layokun, S.K., and Amigun, B. (2003b), Cellulase production by Aspergillus flavus Linn isolate NSPR 101 fermented in sawdust, bagasse and corncob," Afr. J. Biotechnol., 2, pp. 150-152.

[21] Solomon, B.O., Layokun, S.K., Mwesigye, P.K., and Olutiola, P.O., 1990, "Hydrolysis of sawdust by cellulase derived from Aspergillus flavus Linn Isolate NSPR 101: Beyond the initial fast rate period," JNSChE, 9, pp. 46-50.

[22] Yang, B., and Wyman, C.E., 2006, "BSA treatment to enhance enzymatic hydrolysis of cellulose in lignin containing substrates," Biotechnol. Bioeng., 94, pp. 611-617.

E. Betiku * (1), K.O. Oluoti (2) and B.O. Solomon (3)

(1), (2) Biochemical Engineering Laboratory, Dept. of Chemical Engineering, Obafemi Awolowo University, Ile-Ife, 220005, Osun State, Nigeria

* Corresponding Author E-mail: ebetiku@oauife. edu.ng

(3) Present Address: National Biotechnology Development Agency, P.M.B. 5118, Wuse-Abuja, Nigeria
Table 1: Mean for extent of conversion of irradiated softwood and
hardwood sawdust after 8 hours at 40[degrees]C.

Dosage Levels Mean Extent of Mean Extent of Hydrolysis After
 (kGy) Hydrolysis after 8hrs (hardwood)
 8hrs (softwood)

 0 0.276 0.314
 10 0.625 0.681
 20 0.616 0.643
 30 0.622 0.664
 40 0.618 0.678
 50 0.624 0.628
 60 0.614 0.623
 70 0.628 0.652
 80 0.632 0.651
 90 0.634 0.645
 100 0.635 0.637
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Author:Betiku, E.; Oluoti, K.O.; Solomon, B.O.
Publication:International Journal of Biotechnology & Biochemistry
Date:Dec 1, 2010
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