Evaluation of cellulase and xylanase production from Trichoderma harzianum using acid-treated rice straw as solid substrate

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ScienceDirect Materials Today: Proceedings 5 (2018) 22109–22117

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The 3rd International Conference on Green Chemical Engineering Technology (3rd GCET_2017): Materials Science

Evaluation of cellulase and xylanase production from Trichoderma harzianum using acid-treated rice straw as solid substrate Kok Yong Syuana, Lisa Ong Gaik Aia,b, Tong Kim Suana,b* a

Department of Biological Science, Faculty of Science, Universiti Tunku Abdul Rahman, Jalan Universiti, Bandar Barat, 31900, Kampar, Perak b Centre of Biodiversity Research, Universiti Tunku Abdul Rahman, Jalan Universiti, Bandar Barat, 31900, Kampar, Perak

Abstract Rice straw is an agricultural waste potentially producing cellulases and xylanases enzymes, which are important to industrials. The present study reports on the effect of the furan derivatives from the acid-treated rice straw on the production of cellulase and xylanase by Trichoderma harzianum. The content of furan derivatives in rice straw treated with hydrochloric acid, sulphuric acid, acetic acid and nitric acid was analyzed by HPLC. Hydrochloric acid-treated rice straw was found to contain the highest concentration of 5-hydroxymethylfurfural, 96.4 μg/g, but lower furfural content. The furan derivatives content of hydrochloric acid-treated rice straw with different number of times washing and enzymes production were studied by a 5-days solid state fermentation. The furan derivatives content did not show significant negative impact on the production of cellulase and xylanase. The highest cellulase and xylanase contents were found to be 12.7 U/g and 110.7 U/g respectively in 2washes of hydrochloric acid-treated rice straw. A 10-days of time growth profile was conducted to evaluate the cellulase and xylanase production of hydrochloric acid-treated (2-washes) and untreated rice straw. The maximum cellulase production was 16.1 U/g at day 2 using hydrochloric acid-treated rice straw. However, the maximum xylanase activity (271.5 U/g) was found to produce at day 3 in untreated rice straw. The morphology of untreated, hydrochloric acid-treated (2-washes) and fermented rice straw was characterized using SEM and FTIR. The compositions of cellulose and hemicellulose were increased after acid-pre-treatment, and reducing sugars was increased after solid state fermentation. The production of cellulase in hydrochloric acid-treated rice straw is higher as compared to untreated rice straw, yet, xylanase production is more significant with untreated rice straw. © 2018 Elsevier Ltd. All rights reserved. Selection and/or Peer-review under responsibility of The 3rd International Conference on Green Chemical Engineering and Technology (3rd GCET): Materials Science, 07-08 November 2017. Keywords: Trichoderma harzianum; HMF; furfural; cellulase; xylanase

* Tong Kim Suan. Tel: +60-5 4688 888 ext 4693; E-mail address: [email protected] 2214-7853 © 2018 Elsevier Ltd. All rights reserved. Selection and/or Peer-review under responsibility of The 3rd International Conference on Green Chemical Engineering and Technology (3rd GCET): Materials Science, 07-08 November 2017.

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1. Introduction Rice cultivation is one of the major agricultural activities in Malaysia in which it produced 2,252,000 tonnes of rice as reported in 2016 [1]. Rice straw is one of the nuisance causing trouble for the farmers after harvesting and around 63.8% of farmers responded practicing straw burning to eliminate this trouble fast and easy [2]. This practice, however, is a waste of valuable material where studies had found out various methods to utilize this biomass, including incorporation in the preparation of polymer composite [3,4], amalgamated with cement to produce fiber-cement bricks [5], assembled into fiber boards[6,7], and participated in the production of bio-based commodity such as bio-gas, bio-oil, bioethanol, bio-based chemicals and polymers [8,9,10]. It is also important to emphasize on the potential of rice straw as substrate in producing industrially important enzymes via fermentation, including cellulase and xylanase [11,12]. Despite of the various practical possibilities endowed by rice straw, the recalcitrant nature of the lignocellulose present in rice straw was known to arrest the usefulness and efficiency of this substrate, notably in enzymatic hydrolysis of its cell wall [13,14]. According to the study conducted by Rahnama, et al. [16], rice straws in Malaysia are made up of 39.74% of cellulose, 26.03% of hemicellulose, 9.22% of lignin and 12.48% of ash in average. The present of lignin was reported to be responsible for the recalcitrance nature of the cell wall and had exhibited a strong negative correlation with the efficiency of cell wall saccharification [14]. In order to permit the employed microorganisms the access of carbon source possessed by rice straw, the hemicellulose and cellulose must be released from the grip of lignin and broken down into monosaccharides [16]. To attain the goal of maximizing the efficiency of the fermentation process, it is advisable to subject rice straw for pre-treatment. Among the pretreatment methods available, acid treatment has gained much attention as it is one of the most cost-effective technique available [17]. With respect to the degradation of lignocellulosic components during acid treatment, hemicellulose, cellulose and lignin would decompose into their respective monomers. It is important to note that monomers produced will be further degrade into degradation compounds if the acid pre-treatment condition is harsh, for example furfural and 5-hydroxymethylfurfural (HMF) will be produced if further degradation of glucose and xylose monomers occurred [18,19]. Despite the cost-effectiveness of acid treatment, furfural and HMF, which is believed to have an inhibitory effect on the growth and enzyme production of microorganisms, will be produced as side products [20]. A lot of researches had been conducted to study the effect of furfural and HMF on microorganisms and the bioconversion of furfural and HMF [21, 22, 23,24]. In contrast, no research had quantified the content of furfural and HMF with respect to the effect on production of enzymes to date. Hence, the objectives of our research is to study the relationship between the content of furan derivatives and the effect of furan derivates on the enzyme production of locally isolated Trichoderma spp. 2. Materials and Methods 2.1. Fungal strain growth and identification The Trichoderma spp. strain was isolated by Baskaran [25] from cow dung and was maintained on potato dextrose agar (Himedia, India) at room temperature. The identification of the fungal strain was done via phenotypic and genotypic approach. The microscopic structure of the fungal strain was observed via slide culture technique described by Brown [26]. The slides prepared were studied using Olympus CX31 binocular microscope with the instruction given from Bissett [27, 28, 29, 30]. For the genotypic identification, DNA sample was isolated from the Trichoderma spp. following the procedure explained by Cenis [31]. The extracted DNA sample was subsequently added into 25-μL reaction volume containing 2μL of DNA samples, 2 mM of MgCl2, 0.14mM dNTPs, 0.75U Taq polymerases, 0.0048μM of each ITS1 and ITS4 primer, amplified via PCR assay with initial denaturation step at 94°C for 5 min, 35 cycles of 94°C for 30s, 55°C for 30s, and 72°C for 30s, ends with 5 min of 72°C for final extension step. The identification of the fungal isolate was confirmed with the phenotypic characteristic of both microscopic and its colony formation on potato dextrose agar.

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2.2. Rice straw collection and acid pre-treatment Rice straw was collected from Padiberas National Berhad, Kompleks Bernas Sg. Manik, Perak, Malaysia. The collected rice straws were subjected to grinding process with the accompany of sieving process to standardize the size of the rice straws at 500 – 1500 μm. The processed rice straws were finally stored in a sealed container at room temperature prior to use. During acid pre-treatment, rice straw was treated in acid solution with the concentration of 3.75N, with of 1:20 ratio under the condition of 70°C for 1 hour. The acid pre-treatment was conducted with sulphuric acid (H2SO4), hydrochloric acid (HCl), nitric acid (HNO3), and acetic acid (CH3COOH). After the acid treatment, the mixture containing acid solution and rice straw was filtered through a layer of gauze to separate the rice straw and hydrolysate. The rice straw was subjected to Soxhlet-extraction approach for furan derivatives extraction using chloroform, while the hydrolysate was extracted via liquid-liquid extraction using chloroform as well. To evaluate the effect of the number of times washing on the furan derivatives content and enzyme production of Trichoderma strain, acid pre-treated rice straw was subjected to washing after acid pre-treatment and the number of washes were 2, 4, 6, 8, and 10 respectively. 2.3. Solid-state fermentation process and condition A substrate to moisture ratio of 1:1 was employed where 5g of rice straw was accompanied with the addition of 5 mL of 1% (w/v) peptone as a nitrogen source. The solid-state fermentation was initiated with the inoculation of five 50mm-diameter-plugs of Trichoderma spp. strain. The inoculated flasks were subsequently placed in an incubator and incubated at 30°C for the 5 days. For the time-profile study of the enzyme production of the Trichoderma spp. strain on acid-treated and untreated rice straw, the incubation period was up to 10 days. Enzyme was extracted with the addition of 0.05M sodium citrate buffer (pH 4.8), into the fermentation flask with the substrate to buffer ratio of 1:20 after fermentation. Then the solution was put onto a rotary orbital shaker at 200 rpm for 1 hour in room temperature. The filtrate was later being collected via filtration using gauze to remove the residue of biomass and substrate and subjected to centrifugation at 7000rpm for 30 min at 4°C. The resulting supernatants were used in determination of enzyme activity. 2.4. Determination of enzyme activity For xylanase activity measurement, xylose released from the cleavage of 1.8 mL of 1% (w/v) xylan in 0.05M sodium citrate buffer (pH 4.8) by 0.2mL of enzyme extract was measured. The mixture of xylan, enzyme extract and buffer was incubated at 50°C for 5 min, before the addition of DNS reagent to stop the reaction. The determination of xylose released were measured via DNS method described by Miller [32] and was presented as 1 U of enzyme per gram of rice straw can produce 1μmol xylose per minute. For the cellulase enzyme activity assay, 0.5mL of enzyme extract was added into 0.5mL 2% of CMC in 0.05M sodium citrate buffer (pH4.8) and the solution was subjected to incubation at 50°C for 30min. After the incubation, the determination of reducing sugar was carried out using DNS method as described above with a glucose standard curve as reference. The enzyme activity of the measured cellulase was presented as as 1 U of enzyme will produce 1 μmol glucose per minute. The product of Soxhlet-extraction and liquid-liquid extraction was dissolved in methanol and subjected to HPLC (High Performance Liquid Chromatography) analysis. The concentration of furan derivatives in rice straw was presented in mg of furan derivatives per gram of rice straw as calculated according to (eq1). =

×

×

×

(1)

is the weight of rice From equation (1), indicates the mass of furan derivatives (furfural or HMF) in mg, straw used in the extraction process in g, is the concentration of furan derivatives measured during HPLC analysis in mg/L, is the volume of methanol used in dissolving the product in L, and is the dilution factor. A standard curve of furfural and HMF was plotted as reference. For the concentration of furan derivatives in hydrolysate, the in equation (1) was replaced with , which represent the mass of hydrolysate involved in the extraction process in g.

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2.5. Instrumental analysis The content of furan derivatives was determined via HPLC system (LC-20AD, Shimadzu Scientific Instruments, Inc, USA) employing a Gemini 5u C18 110A column (150mm x 4.6mm, Phenomenex Inc., USA). The samples were dissolved in methanol and carried in mobile phase containing 0.2% H2SO4 and methanol (80/20, v/v) running through the column at the flow rate of 1mL/min for 10 min. Furfural and HMF was detected by SPD-20A, prominence HPLC UV-Vis detector at 285nm with the retention time of 4.2 min and 3.1 min respectively. The changes of functional groups in rice straw was studied using FTIR system (Spectrum RX-1, PerkinElmer, Inc, UK) using KBr pellet method. The micro-structure of untreated, acid-treated rice straw and acid-treated fermented rice straw were studied via SEM-EDX system (JSM-6701F FESEM, JEOL, Ltd., Japan) after coated with platinum. 3. Results and Discussions 3.1. Identification of the Trichoderma spp. isolated The Trichoderma spp. isolated grows rapidly on PDA agar whereby its mycelium forming white tuft covering the surface of the agar plate completely within 3 – 4 days. Flat pustules-like structures are observed with yellowish green shade powdery surface which later turn into dark greyish-green colour during spore formation after 4th day of growth. It is also important to note that the fungal isolate was found to be able to change the colour of the PDA agar from yellow into yellowish orange in colour. For microscopic structure of the Trichoderma spp., as shown in Figure 1, the conidia of the fungal isolates were sub-globous in shape, attaching on conidiophore which is in verticillate branching appearance. The primary branching of the conidiophore is slightly bend towards the apex and eventually growing longer towards the base. Furthermore, the lack of sterile elongation of the conidiophore is also one of the unique to take into consideration.

Fig. 1. Microscopic morphology of the fungal strain isolated examined under 1000x magnification.

Based on the colony morphology and microscopic structure of the fungal isolate, the fungal isolate is classified into the genus Trichoderma section Pachybasium according to the description provided by (Bissett, 1991) as Trichoderma harzianum. Rapid growth and flat conidiogenous pustules-structure formation of the Trichoderma spp. isolate is equivalent with the characteristics reported by Castle [33] on Trichoderma harzianum. Study of Sun [34] agreed with the phenotypic characterisation of the Trichoderma spp. isolate as Trichoderma harzianum as well, denoting the characteristic of Trichoderma harzianum conidia as non-catenulate and produced at the apex of the phialide and the branch of conidiophores. On the other hand, secretion of diffusible yellow pigments into PDA is reported to be a trait of Trichoderma harzianum by Tan [35]. The identity of the Trichoderma spp. isolates is later confirmed with molecular identification using ITS 1- and ITS 4-primer pair as Trichoderma harzianum with 100% query cover and 99% identity.

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3.2. Evaluation of different acid-types and the concentration of furan derivatives produced In order to evaluate the relationship between furan derivatives content and their effect on enzyme production of Trichoderma harzianum, the furan derivatives content produced during the acid pre-treatment needed to be determined first. In accordance to the data presented in Table 1, HCl pre-treatment had produced the highest concentration of total furan derivatives readily extracted from rice straw, which is 100.08 μg/g, followed by H2SO4 pre-treatment which halved the yield from HCl pre-treatment (49.58 μg/g). Among the total furan derivatives content yield, HCl pre-treatment produced mostly HMF which consist of 96.33% of the total furan derivatives content it produced, whereas H2SO4 pre-treatment produced both HMF and furfural equally, which is 23.82 μg/g and 25.77 μg/g respectively. Because only the furan derivatives present in rice straw bares the responsibility of causing any effect on the enzyme production during solid-state fermentation, hence HCl pre-treatment which yield the highest concentration of furan derivatives content retain in rice straw is selected for the downstream experiment. Table 1. Furan derivatives content in rice straw and hydrolysate during different acid pre-treatments. Sample

μg of HMF per gram of rice straw

μg of furfural per gram of rice straw

Total furan derivatives content in rice straw

μg of HMF per gram of hydrolysate

μg of furfural per gram of hydrolysate

Total furan derivatives content in hydrolysate

CH3COOH

0.21

2.69

2.90

0.34

0.04

0.39

H2SO4

23.82

25.77

49.58

5.95

0.08

6.02

HCl

96.41

3.67

100.08

2.78

0.10

2.87

HNO3

4.96

4.20

9.16

0.37

0.40

0.76

In accordance to the evaluation of furan derivatives content produced after different acid pre-treatments, the HCl pre-treatment was shown to have the best efficiency in producing total furan derivatives, which surprisingly surpass the efficiency of diprotic acid, H2SO4. This phenomenon was supported by the findings of Shi, et al. [36], which reported on the degradation of monosaccharides occurs much faster in HCl solution compared to H2SO4 solution. This may be due to the dissociation of H2SO4 in H2O required it to first dissociate into H3O+ and HSO4- ions before the latter was dissociated into H3O+ and SO42- ions. With the same normality of acid concentration, the concentration of the released H+ ions should be equal between all acid pre-treatment studied, however, the dissociation of HSO4ions to H3O+ and SO42- ions is not favourable due to the reason that it is a weak acid which does not readily dissociate into the H3O+ and SO42- ions. Nonetheless, the role played by Cl- ions and HSO4- ions on the degradation of monosaccharides into furfural and HMF may be one of the factors affecting the concentration of furfural and HMF produced during acid pre-treatment [37]. The yield of HMF was found to be higher than furfural in HCl pretreatment, in which the possible explanation for this occurrence may be the Cl- ions dissociated from HCl solution may have the tendency on acting on glucose molecules hydrolysed from cellulose. In addition, with the higher tendency of hydrolysing cellulose instead of hemicellulose of HCl pre-treatment, the products of degradation, especially HMF tends to be trapped inside of the rice straw as described above. 3.3 Evaluation on the effect of number of washes on the content of furan derivatives and enzyme production According to the illustration of Figure 2 and 3, the furan derivatives content and the enzyme production of Trichoderma harzianum has positive correlation, which is contrary with the previous findings reported on the inhibitory effect of furan derivatives of furan derivatives on enzyme production [38,39]. One of the reasonable explanation on this occurrence is that the increase in washing not only neutralize the pH in rice straw by washing away the acid, but also the hydrolysed cellulose and hemicellulose polymers. In Figure 3, the xylanase activity dropped drastically from 110.65 U/g to 24.78U/g in 2-wash and 4-wash rice straw respectively. The xylanase activity does not varied much in 4-wash, 6-wash and 8-wash which consists of 24.78 U/g, 23.57 U/g and 28.31 U/g respectively. Interestingly, the CMCase activity had a drastic drop from 2-wash (12.67 U/g) to 4-wash (4.76 U/g) similar with the trend exist in xylanase activity with response to number of washes.

1

120

0.8

100 80

0.6

60 0.4

40

0.2

20

Xylanase Activity (U/g)

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Concentration of furan derivatives (μg/g)

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0

0 2

4

6

8

10

Number of washes

1

14 12

0.8

10 0.6

8

0.4

6 4

0.2

2

0

CMCase Activity(U/g)

Concentration of furan derivatives (μg/g)

Fig. 2. Furan derivatives and xylanase activity on acid-treated rice straw with series of number of times washing ( indicates HMF content present in HCl-treated rice straw; indicates furfural content present in HCl-treated rice straw; indicates xylanase activity in U/g).

0 2

4

6

8

10

Number of washes Fig. 3. Furan derivatives and CMCase activity on acid-treated rice straw with series of number of times washing ( content present in HCl-treated rice straw; indicates furfural content present in HCl-treated rice straw; activity in U/g)

indicates HMF indicates CMCase

3.4. Time profile study on the enzyme production on acid-treated and untreated rice straw In order to evaluate the effect of furan derivatives content in rice straw on xylanase and CMCase production of Trichoderma harzianum, a time profile study was conducted. From the illustration of Figure 4, xylanase production on untreated rice straw was slightly higher than the HCl pre-treated rice straw in overall. There are two peaks can be observed across the 15-days study which is at day-3 (271.5 U/g) and day-6 (142.5 U/g) on untreated rice straw. For HCl pre-treated rice straw, the trend of the peaks is similar to the untreated rice straw, but with lesser xylanase activity (183.7 U/g on day 3; 130.6 U/g on day 6). On the other hand, CMCase production on HCl pre-treated rice straw is higher than the yield on untreated rice straw in which its peak is on day-2 turning out 16.13 U/g and 2.5 U/g respectively as illustrated in Figure 5.

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Xylanase activity (U/g)

300 250 200 150 100 50 0 1

2

3

4

5 6 Number of days

7

Concentration of CMCase (U/g)

Fig. 4. Xylanase production of Trichoderma harzianum on untreated rice straw (

8

9

10

) and HCl-treated rice straw (

).

18 16 14 12 10 8 6 4 2 0 1

2

3

4

5 6 Number of days

Fig. 5. CMCase production of Trichoderma harzianum on untreated rice straw (

7

8

9

10

) and HCl-treated rice straw (

)

From the study of Norazlina, Meenalosani and Ku Halim [40], the highest yield of xylanase produced by Trichoderma sp. on untreated sugarcane bagasses was reported to be occurred on day 6 with the yield of 316.25 U/g. Furthermore, a study conducted by Rezenda, et al. [41], the highest yield of xylanase of Trichoderma harzianum rifai on sugarcane bagasses is reported at day 7 with 288 U/mL of yield. It is interesting that the Trichoderma harzianum used in this study had its peak at day 3 on untreated rice straw. The possible explanation is that the structural composition of rice straw that differ from sugarcane bagasses might play a role in which rice straw might contains more easily accessible carbon source, especially xylan, for the consumption of the fungal isolate to turn out xylanase on earlier days. The decreased in yield on HCl-treated rice straw was supported by the study of several studies reporting on the inhibition of furan derivatives present on the enzyme yield [38, 39]. On the other hand, the CMCase production does not received any huge negative impact from furan derivatives present in HCl pre-treated rice straw, but higher yield in CMCase might suggest that the acid pre-treatment might as well turned out CMC during the process of hydrolysis of cellulose.

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3.5. Instrumental analysis a

b

c

Fig. 6. FTIR analysis of (a)untreated rice straw; (b) fermented HCl pre-treated rice straw; (c) and HCl-treated rice straw.

From Figure 6, there is a broad trough at around 3400cm-1 indicates OH-group stretching, in which the HCl pretreated and fermented rice straw have a deeper trough suggesting hydrolysis may have occurred on them and the concentration of OH-group increased. The trough at around 2900 cm-1 indicates the presence of CH3 group, where the three spectra have no difference in this region. Furthermore, the trough at around 1630 cm-1 indicating the presence of C=O and N-H stretching, which does not have much difference between the three spectra. Finally, the trough at around 1060 cm-1 indicating the presence of C-O-C linkage from glycosylic bond, in which the untreated rice straw shows the most distinct trough, followed HCl-treated rice straw, and fermented rice straw. It is important to note that for fermented rice straw spectra the region at around 1060 cm-1 does not only decrease in volume but there is a formation of two distinct trough can be observed. This suggest that the fermentation process might cleave the C-O-C bond and form new compounds. a

b

c

Fig. 7. SEM analysis of (a) untreated rice straw; (b) HCl pre-treated rice straw; (c) and fermented HCl pre-treated rice straw.

The surface morphology of the untreated rice straw under SEM analysis was as shown in Figure 7(a), in which the surface is wrapped smoothly by lignin. However, the lignocellulosic structure of HCl pre-treated rice straw in Figure 7(b) was ruptured exposing the cellulose and hemicellulose. The pipeline-structure on HCl pre-treated rice straw was where the cellulose should be before acid pre-treatment. The structure of HCl treated rice straw which subjected to fermentation, as shown in Figure 7(c), further degrade by the enzymes secreted by Trichoderma harzianum in which the cleavage was deeper and the lignocellulosic structure was further hydrolysed.

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4. Conclusion In conclusion, the HCl pre-treatment shows its superiority in producing furan derivatives, especially furfural, among the other acid types tested in this study. However, the furan derivatives content can be reduced along with the increase in number of washes. The xylanase and CMCase activity were surprisingly decrease with the decrease of furan derivatives during washing, in which this phenomenon maybe caused by loss of monosaccharides during washing process. Time profile study suggests that the furan derivatives content present in rice straw does not have significant impact on the production of xylanase and CMCase from Trichoderma harzianum. References [1] Department of Statistic Malaysia, Statistical Handbook, Department of Statistic Malaysia, Putrajaya, Malaysia, 2017. [2] Rosmiza, M.Z., Amriah, B., and Rosniza Aznie, C.R., IPEDR, 54 (2012) 245 – 249. [3] Yao, F., Wu, Q., Lei, Y., and Xu,Y., Ind. Crops Prod., 28 (2008) 63 – 72. [4] Ismail, M.R., Yassen, A.A.M., and Afify, M.S.A., Fiber Polym, 12, 5 (2011) 648 – 656. [5] Basta, A.F., Sefain, M.Z., and El-Rewainy, I., Bioresources, 6,2 (2011) 1359 – 1375. [6] Yang, H.S., Kim, D.J., Lee, Y.K., Kim, H.J., Jeon, J.Y., and Kang, C.W., Bioresour. Technol., 95 (2004) 61 – 65. [7] El-Kassa, A.M., and Mourad, A-H.I., Mater. Des., 50 (2013) 757 – 765. [8] Zhang, R., and Zhang, Z., Bioresour. Technol., 68 (1999) 235 – 245. [9] Binod, P., Sindhu, R., Singhania, R.R., Vikram, S., Devi, L., Nagalakshmi, S., Kurien, N., Sukumaran, R.K., and Pandey, A., Bioresour. Technol., 101 (2010) 4767 – 4774. [10] Abraham, A., Mathew, A.K., Sindhu, R., Pandey, A., and Binod, P., Bioresour. Technol., 215 (2016) 29 – 36. [11] Colina, A., Sulbaran-De-Ferrer, B., Aiello, C., and Ferrer, A., Appl. Biochem. Biotechnol., 105 – 108 (2003) 715 - 724. [12] Narra, M., Dixit, G.,Divecha, J., Madamwar, D., and Shah, A.R., Bioresour. Technol., 121 (2012) 355 – 361. [13] Chang, V.S., and Holtzapple, M.T., Appl. Biochem. Biotechnol., 84 – 86 (2000) 5 – 37. [14] Chen, F., and Dixon, R.A., Nat. Biotechnol. 25, 7 (2007) 759 – 761. [15] Rahnama, N., Mamat, S., Shah, U.K.M., Ling, F.H., Rahman, N.A.A., and Ariff, A.B., Bioresources, 8, 2 (2013) 2881 – 2896. [16] Iranmahboob, J., Nadim, F., and Monemi, S., Biomass Bioenergy., 22 (2002) 401 – 404. [17] Jonsson, L.J., and Martin, C., Bioresour. Technol., 199 (2016) 103 – 112. [18] Mosier, N., Wyman, C., Dale, B., Elander, R., Lee, Y.Y., Holtzapple, M., and Ladisch, M., Bioresour. Technol., 96 (2005) 673 – 686. [19] Behera, S., Arora, R., Nandhagopal, N., and Kumar, S., Renew. Sust. Energ. Rev., 36 (2014) 91 – 106. [20] Saeman, J.F., Ind. Eng. Chem., 37 (1945) 45 – 53. [21] Taherzadeh, M.J., Gustafsson, L., Niklasson, C., and Lidén, G., Appl. Microbiol. Biotechnol., 53 (2000) 701 – 708. [22] Almeida, J.R.M., Bertilsson, M., Gorwa-Grauslund, M.F., Gorsich, S., and Lidén, G., Appl. Microbiol. Biotechnol., 82 (2009) 625 – 638. [23] Ran, H., Zhang, J., Gao, Q., Lin, Z., and Bao, J., Biotechnol. Biofuels., 7, 51 (2014) 1 – 12. [24] Ask, M., Bettiga, M., Mapelli, V., and Olsson, L., Biotechnol. Biofuels, 6, 22 (2013) 1 -13. [25] Baskaran, I., Bachelor of Science Final Year Project, UTAR, Perak, Malaysia, 2011. [26] Brown, A., and Smith, H., Benson’s microbiological applications: laboratory manual in general microbiology (13th eds), McGraw-Hill Education, New York, 2015, pp. 151 - 153. [27] Bissett, J., Can. J. Bot., 62 (1984) 924 – 931. [28] Bissett, J., Can. J. Bot., 69 (1991) 2357 – 2372. [29] Bissett, J., Can. J. Bot., 69 (1991) 2373 – 2471. [30] Bissett, J., Can. J. Bot., 69 (1991) 2418 – 2420. [31] Cenis, J.L., Nucleic Acids Res. 20, 9 (1992) 2380. [32] Miller, G.L., Anal. Chem., 31, 3 (1959) 426 – 428. [33] Castle, A., Speranzini, D., Rghei, N., Alm, G., Rinker, D., and Bissett, J., Appl. Environ. Microbiol., 64, 1 (1998) 133 – 137. [34] Sun, S.H., Huppert, M., and Cameron, R.E., Antarctic Research Series: Terresterial Biology III, 30 (1978) 1 – 26. [35] Tan, S.H. Bachelor of Science Final Year Project, UtTAR, Perak, Malaysia, (2013). [36] Shi, Y., Yokoyama, T., Akiyama, T., Yashiro, M., and Matsumoto, Y., Bioresource, 7, 3 (2012) 4085 – 4097. [37] Marcotullio, G., and De Jong, W., Green Chem., 12 (2010) 1739 – 1746. [38] Klinke, H.B., and Ahring, B.K., Appl. Microbiol.Biotechnol., 66 (2004) 10 – 26. [39] Mills, T.Y., Sandoval, N.R., and Gill, R.T., 2, 26, Biotechnol. Biofuels. (2009) 1 - 11. [40] Norazlina, I., Meenalosani, N., and Ku Halim, K.H., Int. J. Energy Res., 3, 2 (2013) 99 – 105. [41] Rezende, M.I., Barbosa, A.D.M., Vasconcelos, A.F.D., and Endo, A.S., Braz. J. Microbiol., 33 (2002) 67 – 72.