Rubberwood fiber treatment by laccase enzyme and its application in medium density fiberboard.
Wood cell walls are composed of cellulose, hemicelluloses and lignin (Stefanidis et al, 2014). The mechanical property of wood fibers is mainly determined by cellulose and hemicelluloses, whereas lignin acts as an adhesive between the fibers (Gea et al., 2011; Winandy & Rowell, 2005). Cellulose is a long polymer chain of linear crystalline interrupted with regular amorphous region, primarily responsible for strength in the wood fiber (Muhammad Shahid Nazir & Mohd Azmuddin, 2013; Stefanidis et al., 2014). Cellulose crystallinity directly affects the physico-chemical behavior of the individual fiber such as modulus, hardness, stiffness, tensile, and swelling-shrinkage properties of individual fiber. Hemicelluloses are amorphous, highly branched polymer compared to cellulose (Zhou et al., 2009), acts as a linkage between cellulose and lignin. Lignin, on the other hand, is an amorphous phenolic compound that functions as a cementing material and stiffening agent for the cellulose molecules within the fiber cell wall (El Mansouri et al., 2007). Most of the lignin is found inside the cell wall as an intra-cellular lignin (70%), the rest are found in the middle lamella as extracellular lignin (Kunamneni et al., 2007; Tuor et al., 1995).
Laccase is a well-studied oxido-reductase enzyme, it take part in polymerization as well as depolymerization of lignin compound through free radical reaction (Nasir et al., 2013). Since Laccase enzymes are too large to penetrate into fibers (5580 kDa), it modify the amorphous extracellular component only (Gochev & Krastanov, 2007). Hence, due to a size exclusion reasons, laccase treatment is only a surface modification and it alters extracellular lignin without affecting the cellulose fiber. Although several researchers have studied the enzymatic hydrolysis of lingo-cellulosic fiber but the changes in physical and mechanical properties of fiber is still not fully understood (Kharazipour et al., 1997; Yaropolov et al., 1994). Thus this study deals the pretreatment reaction of laccase at two different times, on rubber wood fiber and the result was compared with untreated fiber. Such improved fibers can be used in many industrial applications like fiber board, pulp and paper industries etc (Nasir et al., 2013; Virk et al., 2012; Wu et al, 2011).
MATERIALS AND METHODS
A thermo-mechanically processed pulp of rubber wood fibers was supplied by Robin Resources (Malaysia) Sdn. Bhd. The pulp was then air dried to moisture content of 15-20% before applying any treatment. Laccase enzyme (Novo WA 20040) was supplied by Novozyme, Malaysia.
About 25 g of oven dried wood fibers of was suspended in 475 g of deionized water to make a solution of 5.0% consistency (mass pulp/mass suspension) in a 2 L Erlenmeyer flasks. A buffer solution of sodium acetate and acetic acid was used to maintain the pH. Two different reaction time (60 min and 120 min) were applied at constant temperature of 25[degrees]C, enzyme concentration of 7 U/g and 5 pH (Nasir et al., 2014). The two treatment were named as FT1 (Fiber treated) FT2 when treated at 60 min and 120 min respectively, whereas untreated was named as UT in later part of result and discussion. After each pulp treatment, fibers were sieved out immediately from the solution and transferred in to autoclave at 121[degrees]C for 15 min to stop further enzyme reaction. Treated fiber was dried in electric oven drier at 80-90[degrees]C for 24 hr to dye up the fiber until zero moisture content.
Preparation of medium density fiber boards
Air-dried treated fibre was used for preparation of MDF board. A total of 195 g fibre (185 + 5% allowance) was used for making the board with target panel density of 770 kg [m.sup.-3]. Soy-lignin adhesive was spread over fibres using air pressure gun. A locally designed rotating drum was used to mix the fibre and urea formaldehyde adhesive. A mat of dimension 220 mm x 220 mm was prepared after adding adhesive by weight of the fibre and pre-pressed to a thickness of 60 mm in cold press. The mat was then hot-pressed to a 6 mm thickness. Total pressing time was 240 s and applied pressure was 5 MPa and both these parameters were kept constant for all samples. After hot pressing, the boards were cooled in an incubator, conditioned at 25[degrees]C and 67% relative humidity. From each board, two samples were prepared for internal bonding, two for thickness swelling test and two for modulud of rupture (MOR) and modulud of elasticity (MOE) tests (Figure 1).
X-Ray measurements were conducted on a Rigaku MiniFlex II, bench top X-ray diffractometer (XRD) analyzers. The fiber specimens were pressed at 10 tone hydraulic pressure into a circular shape of a tablet with 14 mm diameter and 0.5 mm of thickness. The X-ray diffractometer was operated at a voltage of 30 kV with a current density of 15 mA. The scanning range was from 2q = 10[degrees] to 50[degrees] at a scan speed of 0.015[degrees]/s. The data was collected using a fixed time mode with angular intervals of 0.015[degrees]. The method adopted was based on Kim and Holtzapple (2005). Crystallinity Index (Crl) was calculated from Equation 1.
Crystallinity Index (Crl) = [([I.sub.002] - [I.sub.am]/[I.sub.002]] x 100 ... (1)
Where, [I.sub.002] is the intensity of the diffraction from the 002 plane 2e = 22.0-23.0, and [I.sub.am] is the intensity of the background scatter measured at 2e = 16.0-18.0.
Medium density fiber boards (MDF) performance Test
Modulus of rupture, internal bonding and maximum load bearing capacity were used to determine the performance of the board. Mechanical testing of samples was done using a universal testing machine and results were analysed using TRAPEZIUM X-software. All testing was performed according to ASTM D10370-6a (ASTM 2006) and statistical analysis was performed on SYSTAT 9.0 software.
RESULTS AND DISCUSSION
Physical Properties of Treated Fiber
An initial experiment was cried out to understand the real effect of laccase treatment on rubber wood fiber. Figure 2 exhibited, the untreated fibers having the least crystallinity index of 65.5% whereas it was improved in treated fibers to 74.4% and 72.87% in FT1 and FT2, respectively. The X-ray-based measurement of crystallinity index (CrI) indicates that laccase treatment increased the crystallinity of rubber wood and maximum was observed at 1h of treatment time. However, the CrI started to reduce when the reaction was prolonged for 2h. The change in crystallinity index was due to the removal of extracellular lignin from the fiber surface that ultimately improves the crystallinity (Lionetto et al., 2012; Wu et al., 2011), But in the next two hour fiber showed a decreasing trend. It was expected that a prolong treatment, lignin started to precipitate back on the fiber surface that results in the fall of crystallinity which is supported by the FE-SEM result (Kumar et al., 2009).
Figure 3 depicted the FE-SEM picture of treated and untreated fiber at 5000x magnification. From the close view of micrograph it was obvious that an enzyme treatment brought a distinguish change in the fiber surface. An untreated fiber (UT) had uneven furrowed surface whereas a treated fibers have a very smooth surface. The uneven surface of wood fiber could be due to extra-cellular lignin found naturally on fiber surface which was either removed in lignification process or become even surface due to deposition. Furthermore FT2 fiber exhibits slightly thicker layer of deposition as compared to the FT1 treated fiber. Although laccase act specifically on lignin, the deposition composition should be studied thoroughly. Thus it was concluded that the smoothness of the fiber surface could be due to the removal of loosely bonded lignin and then precipitation of the hydrolyzed lignin, as a smooth layer on the fiber surface which is similar to the finding of Kumar et al. (2009).
Thermal Properties of Treated Fiber
Figure 4 shows the Thermo-gravimetric curves were all wood fibers (either treated or untreated fibers) exhibited two exothermic peaks overlapping with regions of weight loss (Jawaid & Abdul Khalil, 2011). Thermal stability of treated and untreated fibers was analyzed on TGA. Thermal degradation of wood fibers occurs in two stages, it initiates from the amorphous cellulose at around 300[degrees]C followed by the crystalline cellulose degradation at a higher temperature around 350[degrees]C (Quintana et al., 2015). It was observed that initial degradation of untreated fiber (UT) was started at temperature 331[degrees]C whereas this temperature was found higher for treated fibers (FT1 and FT2). Final degradation temperature of treated fiber was also increased from 449[degrees]C of untreated fiber to 491[degrees]C of treated fiber. Among the treated fibers FT1 exhibits higher degradation temperature as compared to FT2 due to its higher crystallinity index (CrI). It was observed that Tg value of treated fiber was higher and increases with the increase in crystallinity of the fiber. Therefore it was concluded that a treated fiber were having a higher decomposition temperature as compared to untreated fiber (Li & Pickering, 2008).
Medium Density Fiber Board Performance Analysis
Thickness swelling results of the MDF observed at 2 hr and 24 hr is depicted in Figure 5. A treated fiber exhibits greater dimensional stability as compared to untreated fiber. A lower thickness swelling in treated fiber could be due to the change in surface morphology of the fiber. Since FT1 fiber had least free hydroxyl group due to interfacial bonding between fibers, it exhibits the lowest thickness swelling. Whereas in case of FT2, lignin formed a thin layer over the fiber, it reduces the accessibility of water to the fiber, which ultimately improves the dimensional stability of the fiber. However, the standard deviation error bar shows an overlapping trend that explains that the difference was least significant.
Figure 6 shows the mechanical test (MOE, MOR and IB) results of MDF prepared from different fibers. A very significant and unexpected change in properties of the board is visible when fiber is treated at different reaction condition. The figure demonstrates an increase in the tensile and bending propertied when the fiber FT1 is treated at optimum condition. The increment in bonding strength of the fiber is probably because of the exposed hydroxyl groups on the fiber surface due to removal of lignin from the fiber that helps in interfacial bonding between fibers (Li & Pickering, 2008). Moreover, the strength of MDF reduces drastically and exhibited lower than the untreated fiber when the fiber FT2 was treated for prolonged time. It was assumed that in a prolong enzymatic reaction, lignin molecules starts precipitating and form a uniform layer on wood fiber surface that mask the free hydroxyl groups on wood fiber surface (Figure 3, FT2). That ultimately hindered the interfacial bonding between the fibers. However the strength of FT2 boards below the untreated fiber (UT) boards is still need to be investigated.
In the preliminary study of laccase effect natural fibers were carried on for two different times. The crystallinity index was observed maximum up to 74.4 %, at 1 hr enzyme reaction condition then after it decreased considerably and remained constant for a wide range of around 71-72%. Fiber treated with enzyme exhibited superiority in fiber surface structure and thermal degradation over other treated and untreated fibers. A treated fiber with improved crystallinity exhibited improved individual performance and collectively improved the performance of MDF. However in this work only one parameter was considered for study but to obtain a highly crystalline and mechanically strong fiber, a thorough study of various reaction parameters such as time, concentration and temperature of the reaction is recommended.
Furthermore, the fiber with improve mechanical strength and crystalline structure can be utilized for various other purposes also in order to produce high quality product.
The authors acknowledged the Universiti Sains Malaysia for awarding Post-doctoral fellowship to Dr. Mohammad Nasir.
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Mohammed Nasir , Othman Sulaiman  *, Rokiah Hashim , Kaizar Hossain , Arun Gupta  and Mohd Asim 
 Division of Bioresource, Paper and Coatings Technology, School of Industrial Technology, Universiti Sains Malaysia, 11800 Penang, Malaysia.
 Division of environment technology, School of Industrial Technology, Universiti Sains Malaysia, 11800 Penang, Malaysia.
 Faculty of Chemical and Natural Resource Engineering Universiti Malaysia Pahang, 23600 Pahang, Malaysia.
 Institute of Tropical Forestry and Forest Products (INTROP), Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia.
(Received: 11 May 2015; accepted: 10 June 2015)
* To whom all correspondence should be addressed. E-mail: email@example.com
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|Author:||Nasir, Mohammed; Sulaiman, Othman; Hashim, Rokiah; Hossain, Kaizar; Gupta, Arun; Asim, Mohd|
|Publication:||Journal of Pure and Applied Microbiology|
|Date:||Sep 1, 2015|
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