3 Biotech DOI 10.

1007/s13205-013-0179-4

ORIGINAL ARTICLE

Bioconversion of lignocellulosic waste to bioethanol by Trichoderma and yeast fermentation

Received: 25 May 2013 / Accepted: 7 October 2013 The Author(s) 2013. This article is published with open access at Springerlink.com

Abstract The present work aimed at producing bioethanol using lignocellulosic waste sawdust and marine yeast fermentation. Lignocellulosic waste materials were converted into monosugars through acid hydrolysis and nally treated with cellulase enzyme derived from Trichoderma/Hypocrea. To enhance the conversion of the glucose from sawdust, the experimental conditions were statistically optimized. The efcient conversion of sawdust to glucose of 78.56 % was achieved under the conditions of pH 6.19, temperature 29 C, cellulase enzyme (8.16 IU ml-1) and sawdust (7.95 g l-1). The lignocellulosic waste-sawdust hydrolysis was used as the carbon source for the production of bioethanol. Bioethanol production of 85.6 % was achieved (55.2 g l-1) under the optimized conditions of temperature of 36.5 C, incubation time of 102 h and enzyme-treated sawdust of 45.14 ml l-1 and agitation of 330 rpm. This work achieved maximum bioethanol production using H. estonica and S. cerevisiae fermentation. Keywords Bioethanol Marine yeasts Trichoderma Lignocellulosic waste Mangroves

Introduction Fuel deciency is a global issue due to exhaustion of fossil fuel and growing climate change (EC 2003; Wingren et al.
K. Saravanakumar (&) K. Kathiresan Faculty of Marine Sciences, Centre of Advanced Study in Marine Biology, Annamalai University, Parangipettai 608502, Tamil Nadu, India e-mail: saravana732@gmail.com K. Kathiresan e-mail: kathirsum@rediffmail.com

2003; Li 2003; Qureshi et al. 2006; Sveinsdottir et al. 2009). In order to overcome this issue, different types of techniques have been invented for the possible conversion of cellulosic waste materials into glucose for the ethanol production, as an alternative way for fuel conservation (Sun and Cheng 2002). The bioconversion of lignocellulosic materials (i.e., agricultural residues, woods, and residues from pulp and paper industries, solid wastes) to bioethanol produces high yield of glucose after hydrolysis (McMillan 1994). The utilization of the lignocellulosic materials for the conversion of the biofuel involves only low cost (Li et al. 2007). Lignocellulose, the most abundant organic matter in the Earth, can be utilized to produce various renewable fuels and chemicals (Lau et al. 2010). For the digestion of these materials into glucose, many methods have been used such as thermal pretreatments, chemical pretreatments, biological pretreatments and enzymatic pretreatments (Meinita et al. 2012). Cellulolytic and hemicellulolytic enzymes are able to increase monosugars from the digestion of lignocellulose (Pakarinen et al. 2011). For this purpose, fungi in particular Trichoderma species that can produce cellulolytic enzymes are employed (Aro et al. 2005; Rodriguez Gomez et al. 2012). In general, yeasts are potential microorganisms for the production of bioethanol by sugar fermentation (Kathiresan et al. 2011; Senthilraja et al. 2011). However, most of the studies are restricted to microbes of terrestrial origin, but not of marine origin. Hence, the present work was undertaken for the conversion of sawdust (lignocellulosic wastes) to sugars for fuel fermentation using cellulolytic enzymes of the mangrove-derived Trichoderma and further conversion of sugars to bioethanol using the mangrove-derived yeast strain of Saccharomyces cerevisiae. Based on the data, the culture

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conditions were optimized statistically for the enzyme hydrolysis process and bioethanol fermentation.

was centrifuged (10,000 rpm) using a high speed centrifuge for 15 min and the supernatant was used for enzyme assay using the dinitrosalicylic acid (DNS) method (Miller 1959). Enzymatic hydrolysis Enzymatic hydrolysis was carried out in 125 ml screwcapped Erlenmeyer asks. Diluted acid-treated sawdust powder was suspended in distilled water in the asks. Water was added during the pretreatment in such a way so as to maintain the substrate concentration at 15 % (w/v) after the addition of the crude cellulase enzyme derived from H. estonica. Hydrolysis was performed at 50 C for 24 h at 120 rpm in an incubator shaker. 1 ml sample was periodically removed from each ask (0, 2, 8, 18, 24, 72, 120, and 168 h). Each sample was centrifuged for 10 min at 13,500g, and 500 ll supernatant was then removed and placed into a 1.5-ml Eppendorf tube containing the stop buffer (512 mM Na2CO3 and 288 mM NaHCO3; pH 10.0) (Sandhu et al. 2012). The buffered samples were stored at 4 C for subsequent glucose measurement; commercial glucose was used as the standard for the percentage calculation. Bioethanol production by S. cerevisiae Bioethanol production experiments were carried out according to the experimental setups derived from the center composite design. The enzyme-treated sawdust hydrolysis was used as the carbon source. The percentage of the bioethanol was calculated according to the method of Saravanakumar et al. (2013b).

Materials and methods Microorganisms and maintenance Trichoderma (Hypocrea) species (Trichoderma estonicum/ H. estonica SKS1 JQ611722) and yeasts species (Saccharomyces cerevisiae JN387604) were isolated from mangrove sediment, located in the south east coast of India using selective medium (Askew and Laing 1993) and yeast peptone agar medium (Fell 2005), respectively. The stock cultures were maintained at 4 C on slants of potato dextrose agar for H. estonica and yeast peptone agar for S. cerevisiae. Substrate Sawdust was obtained from local wood mills, passed through a 1.5-mm sieve to maintain uniform particle size, washed through distilled water to remove impurities present in it and nally dried at 60 C overnight. The sawdust was pre-hydrolyzed using 0.8 % of phosphoric acid by the method of Kathiresan et al. (2011). Cellulase enzyme production by Hypocrea estonica Spore suspension of Hypocrea estonica SKS1 (JQ611722) was used as microbial inoculum for the fermentation. It was prepared by culturing H. estonica in potato dextrose agar slant cultures at 30 C for 7 days and spore suspension was washed through the Tween-80 water (0.02 % v/v); the suspension was then assessed as nal spore count of 2.3 9 103 CFU ml-1. Then 10 ml of spore suspension was inoculated into a 250-ml Erlenmeyer ask containing 100 ml glucose pre-cultured medium (%, w/v) (glucose, 3; yeast extract, 0.8; K2HPO4, 0.4; MgSO47H2O, 0.2; pH, 7.0) and was cultured at 30 C, 200 rpm for 48 h in a rotary shaking incubator to prepare the mycelial suspension. At last, mycelial suspension was inoculated into a 250-ml Erlenmeyer ask containing the statistically optimized medium for cellulase production by H. estonica (Saravanakumar and Kathiresan 2013a) that consisted of 7.69 g l-1 of sawdust, 3.59 g l-1 of (NH4)2 SO4, under the temperature of 49 C at pH of 8.8 with inoculum size of 6.0 % (v/w) and was cultivated at 30 C for 120 h to produce cellulose.

Determination of bioethanol using gas chromatography Concentration of bioethanol in the distillate of culture ltrate was estimated using a Hewlett Packard 5890 Series II gas chromatography with chromosorb 105 column and nitrogen as a carrier gas. The temperature of the injection port, oven and detection port was 250, 120, and 250 C, respectively. For the analysis, 1 ll of liquid samples was injected into gas chromatography. The bioethanol concentration was determined using bioethanol standard plot and is expressed in percentage with help of instrumental default standard value of 65 g l-1 which is equivalent to 100 % (Saravanakumar and Kathiresan 2013a, 2013b).

Determination of cellulase enzyme activity 5 ml of the fermented residues was suspended in 150 ml distilled water and shaken at 120 rpm for 2 h. Then the ltrate

Optimization of the enzyme hydrolysis process Optimization of the environmental conditions for the maximum hydrolysis process of the sawdust to sugar using

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H. estonica-derived cellulase was studied using 30 runs of statistical model assessed from central composite design of response surface methodology. In this experiment, important factors such as pH (68), temperature (2050 C) and cellulase concentration (210 IU ml-1) on glucose conversion were studied. Y b0 Ri bi Xi Ri bii Xi2 Rij bij Xi Xj 1

2 2 Y b0 b1 X1 b2 X2 b3 X3 b4 X4 b11 X1 b22 X2 2 2 b33 X3 b44 X4 b12 X1 X2 b13 X1 X3 b14 X1 X4

b23 X2 X3 b24 X2 X4 b34 X3 X4 2 Statistical optimization of bioethanol production In this experiment, S. cerevisiae (JN387604) was selected for optimization. The individual and interaction effects of temperature (C), incubation time (0120 h), and enzymetreated sawdust (1050 ml l-1) and agitation (100500) on bioethanol production were carried out. The maximum yield of bioethanol production was tested using 30 experimental setups derived from a statistical model-central composite design of response surface methodology. The

where Y is the predicted response (glucose), Xi, Xj the independent variables, b0 the offset term, bi the ith linear coefcient, bii the ith quadratic coefcient and bij is the ijth interaction coefcient. The experimental design in coded and uncoded value is presented in Table 1. In this study, the independent variables are coded as X1, X2, and X3, Thus, the second-order polynomial equation can be presented as follows:

Table 1 Center composite design of response surface methodology for the glucose production of experimental and predicted responses Std (A) pH (B) Temperature (C) 20 20 50 50 20 20 50 50 20 20 50 50 20 20 50 50 35 35 5 65 35 35 35 35 35 35 35 35 35 35 (C) Cellulase concentration (IU ml-1) 2 2 2 2 10 10 10 10 2 2 2 2 10 10 10 10 6 6 6 6 2 14 6 6 6 6 6 6 6 6 (D) Sawdust (mg l-1) 2 2 2 2 2 2 2 2 10 10 10 10 10 10 10 10 6 6 6 6 6 6 2 14 6 6 6 6 6 6 Glucose (%) Experimental 65.26 52.26 42.56 32.26 53.26 41.23 23.25 15.26 45.65 36.56 35.62 25.65 78.56 35.62 36.56 48.56 68.56 36.56 45.56 15.26 35.26 39.56 12.25 56.56 65.23 65.23 65.23 65.23 65.23 65.23 Predicted 63.13 44.33 34.68 31.08 52.91 31.96 19.11 13.36 52.32 31.85 36.04 30.77 70.89 48.27 49.26 41.84 67.71 41.49 49.89 15.01 39.03 39.87 27.61 45.28 65.23 65.23 65.23 65.23 65.23 65.23

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

6 8 6 8 6 8 6 8 6 8 6 8 6 8 6 8 5 9 7 7 7 7 7 7 7 7 7 7 7 7

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3 Biotech Table 2 Center composite design of response surface methodology for the bioethanol production of experimental and predicted response Std (A) Temperature (C) 0 50 0 50 0 50 0 50 0 50 0 50 0 50 0 50 25 75 25 25 25 25 25 25 25 25 25 25 25 25 (B) Incubation time 0 0 120 120 0 0 120 120 0 0 120 120 0 0 120 120 60 60 60 180 60 60 60 60 60 60 60 60 60 60 (C) Enzyme-treated sawdust (ml l-1) 10 10 10 10 50 50 50 50 10 10 10 10 50 50 50 50 30 30 30 30 10 70 30 30 30 30 30 30 30 30 (D) Agitation (rpm) 100 100 100 100 100 100 100 100 500 500 500 500 500 500 500 500 300 300 300 300 300 300 100 700 300 300 300 300 300 300 Bioethanol yield (%) Experimental 0.00 0.00 15.69 75.56 0.00 0.00 15.65 85.65 0.00 0.00 12.26 68.56 0.00 0.00 12.36 56.69 15.58 78.65 19.65 79.65 75.26 54.26 15.26 65.26 75.60 75.60 75.60 75.60 75.60 75.60 Predicted 4.71 9.51 19.99 82.40 4.24 8.58 19.09 81.04 21.21 18.69 25.81 80.92 15.28 12.30 19.45 74.10 -1.97 57.47 -8.25 68.82 49.04 41.75 16.12 25.67 75.60 75.60 75.60 75.60 75.60 75.60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

coded values of the fermentation factors were determined by the following equation: Y b0 Ri bi Xi Ri bii Xi2 Rij bij Xi Xj 3

where Y is the predicted response, Xi, Xj the independent variables, b0 the offset term, bi the ith linear coefcient, bii the ith quadratic coefcient and bij is the ijth interaction coefcient. The experimental design in coded and uncoded value is presented in Table 2. In this study, the independent variables are coded as X1, X2, X3 and X4. Thus, the secondorder polynomial equation can be presented as follows:
2 2 Y b0 b1 X1 b2 X2 b3 X3 b4 X4 b11 X1 b22 X2 2 2 b33 X3 b44 X4 b12 X1 X2 b13 X1 X3 b14 X1 X4 b23 X2 X3 b24 X2 X4 b34 X3 X4

A statistical program package Design Expert 8.0.6 was used for regression and model t analysis of the data was obtained, and then estimated the coefcient of the regression equation, and analyzed the variance of selected factors and model signicance.

Results Effect of the cellulase enzyme on sawdust hydrolysis process The interaction and individual effects of the experimental condition on hydrolysis process were studied by adopting the statistical design of the center composite model of 30

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experimental setups and are presented in Table 1 along with the predicted and experimental responses of glucose production. The acceptability of the statistical model was tested by the quadratic model along with the contour error plot for the predicted and experimental responses and further model tness tested by the normal standardized residual plot. The standard error value of 0.43 showed tness for the acceptable model and R2 value of the model was 0.87 which also proved the 87 % of model tness. The application of the response surface methodology based on the estimates of the parameters indicated an experimental relationship between the response and input variables with the glucose production expressed in the following quadratic mode: Glucose % 65:23 6:56X1 8:72X2 0:21X3 4:42X4 3:80X1 X2 0:54X1 X3 0:42X1 X4
2 1:34X2 X3 3:04X2 X4 7:20X3 X4 2:66X1 2 2 2 8:19X2 6:44X3 7:20X4

Table 3 Analysis of variance for the response of glucose production by H. estonica Source Model (A) Ph (B) Temperature (C) (C) Cellulase concentration (IU ml-1) (D) Sawdust (mg l-1) AB AC AD BC BD CD A2 B2 C2 D
2

Sum of squares

df Mean square

F value

p value prob [ F

8,011.377 14 572.2412 1,031.233 1,824.922 1.075267

7.290321 0.0002*** 0.0025** 0.0002***

1 1,031.233 13.13785 1 1,824.922 23.2494 1 1.075267

0.013699 0.9084NS

468.6968 231.04 4.6225 2.7889 28.6225 148.1089 828.8641 193.6786 1,841.955 1,139.255 1,420.334

1 468.6968 1 231.04 1 4.6225 1 2.7889 1 28.6225 1 148.1089 1 193.6786

5.971171 0.0274* 2.943437 0.1068NS 0.05889 0.03553 0.8115NS 0.8530NS

0.364649 0.5550NS 1.886899 0.1897NS 0.0054** 0.0002*** 0.0017** 0.0007*** 0.856NS 2.467455 0.1371NS

1 828.8641 10.55968 1 1,841.955 23.4664 1 1,139.255 14.51405 1 1,420.334 18.09498

5 where X1 (pH), X2 (temperature), X3 (cellulase) and X4 (agitation) are independent variables. Signicance of each coefcient is presented in Eq. (5), determined by the Students t test and p values (Montgomery 2001). The ANOVA results of the quadratic model for glucose production are given in Table 3. The value of R2 and adjusted R2 was close to 0.87 and it revealed a high correlation between the observed values and the predicted values. This means that regression model provides an excellent explanation of the relationship between the independent variables (factors) and the response (glucose production). The lack-of-t term was non-signicant as it was desired. The non-signicant value of lack-of-t observed (0.856) was more than probability of 0.05 and this revealed that the quadratic model was valid for the present study. In this case, the individual and interaction effects on the hydrolysis process were tested and the data are presented in 2 2 Table 3. The factors of the X1, X2, X4, X3X4, X2 2, X3, X4 were signicant but other factors and their interaction effects were not signicant on the glucose production (p \ 0.05). The optimization of the specic conditions for the glucose production was assessed. Statistically optimized conditions for the efcient sawdust hydrolysis process of glucose production were pH (6.19), temperature (29 C), cellulase enzyme (8.16 IU ml-1) and sawdust -1 (7.95 mg l ). This hydrolysed glucose was used further for the alcohol production as the carbon source. Optimization of bioethanol production by S. cerevisiae The interaction and individual effects of experimental conditions on bioethanol production process were studied

Residual Lack-of-t Pure error Cor total NS not signicant

1,177.399 15 78.49328 1,177.399 10 117.7399 0 5 0 9,188.776 29

*** p \ 0.001; ** p \ 0.01; * p \ 0.05

by adopting the statistical design of the center composite for the bioethanol production. The acceptability of the statistical model was tested by the quadratic model along with the contour error plot for the predicted and experimental responses. The standard error value of the 0.43 showed tness for the acceptable model and R2 value of the model of 0.81 also proved 81 % of model tness. The application of the response surface methodology based on the estimates of the parameters indicated an experimental relationship between the response and input variables, which is expressed in the following quadratic model: Bioethanol yield % 75:60 14:86X1 19:27X2 1:82X3 2:39X4 14:41X1 X2 0:12X1 X3 0:83X1 X4 0:11X2 X3 2:67X2 X4 2:67X3 X4
2 2 2 2 11:96X1 11:33X2 7:55X3 13:68X4

6 where X1 (temperature), X2 (incubation time), X3 (enzymetreated sawdust) and X4 (agitation) are independent variables. Signicance of each coefcient is presented in Eq. (6), determined by the Students t test and p values

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(Montgomery 2001). The ANOVA results of the quadratic model for bioethanol production are given in Table 4. The value of R2 and adjusted R2 was close to 0.81 and it revealed a high correlation between the observed values and the predicted values. This means that regression model provides an excellent explanation of the relationship between the independent variables (factors) and the response (bioethanol production). The lack-of-t term was non-signicant. The non-signicant value of lack-of-t observed (0.52) was more than probability of 0.05 and this revealed that the quadratic model was valid for the present study. In this case, the individual and interaction effects on the hydrolysis process were tested and the data are presented in 2 2 2 Table 4. The factors of the X1, X2, X1X2, X2 1, X 2, X3, X4 were signicant but other factors and their interaction effects were not signicant on the bioethanol production (p \ 0.05). The optimization of the specic conditions for the bioethanol production was assessed. Statistically optimized conditions for the efcient bioethanol production process were temperature of 36.5 C, incubation time of 102 h and
Table 4 Analysis of variance for the response of bioethanol production by S. cerevisiae Source Model (A) Temperature (C) (B) Incubation hours Sum of squares df Mean square F value p value prob [ F

enzyme-treated sawdust of 45.14 ml l-1 and agitation of 330 rpm.

Discussion Bioethanol production from the lignocellulosic waste materials depends on the treatment of the sawdust and removal of the hemicelluloses and lignin from the sawdust for the production of monosugars such as glucose. This is a challenge to the current researchers on the renewable fuel production (Meinita et al. 2012). Marine-derived Trichoderma is a highly potential source for the bioprospecting research (Saravanakumar and Kathiresan 2012). These marine Trichoderma strains were used in this work. The present study successfully attempted the pretreatment of the sawdust with 0.8 % phosphoric acid followed by enzyme hydrolysis using H. estonica-derived cellulase for the maximum production of monosugars and then conversion of bioethanol by fermentation with yeast (S. cerevisiae). Statistically optimized conditions for the efcient sawdust hydrolysis process were pH (6.19), temperature (29 C), cellulase enzyme (8.16 IU ml-1) and 8 % phosphoric acid-treated sawdust (7.95 mg l-1). For suitability of each pre-hydrolysis treatment for maximum solubilization of hemicelluloses and lignin, the effect of pre-hydrolysis treatment of sawdust with the enzyme was investigated using H. estonica-derived cellulase enzyme. It resulted in maximum yield of glucose (78.56 %). The signicant conversion of the sawdust to the monosugars (glucose) was attained at temperature above 29 C. This is due to the fact that high temperature increases hemicellulose degradation and lignin transformation, thus increasing the potential of cellulose hydrolysis (Li et al. 2009), whereas low temperature increases the solubilization of the hemicellulose (Sun and Cheng 2002). Addition of dilute acid in steam treatment can effectively improve enzymatic hydrolysis, decrease the production of inhibitory compounds, and lead to more removal of hemicelluloses (Martin et al. 2002; Kathiresan et al. 2011; Matsushita et al. 2010; Wang et al. 2009). Saravanakumar and Kathiresan (2013a, b) have reported the potential of the marine strain over the terrestrial yeast strains and achieved the maximum yield of the bioethanol of 69.58 % under optimal conditions of temperature at 30 C, sawdust concentration of 6.84 mg l-1 under the agitation speed of 360 rpm in 89 h of incubation. Similarly, the present study attained maximum bioethanol production of 85.6 % under the optimized conditions of temperature of 36.5 C, incubation time of 102 h and enzyme-treated sawdust of 45.14 ml l-1 and agitation of 330 rpm; little variations of the optimized conditions were

28,097.22 14 2,006.944 5,299.67 8,909.677 1 5,299.67

4.824282 0.0023** 12.73932 0.0028** 0.0003***

1 8,909.677 21.41704 1 79.64327

79.64327 (C) Enzymetreated sawdust (ml l-1) (D) Agitation (rpm) AB AC AD BC BD CD A

0.191446 0.6680NS

136.8993 3,320.641 0.2116 53.4361 0.1849 113.8489 29.75703 3,924.794 3,519.94 1,563.842 5,129.922

1 136.8993 1 3,320.641 1 0.2116 1 53.4361 1 0.1849 1 113.8489 1 29.75703 1 3,924.794 1 3,519.94 1 1,563.842

0.329078 0.5747NS 7.982139 0.0128* 0.000509 0.9823NS 0.128449 0.7250NS 0.000444 0.9835NS 0.273669 0.6085NS 0.07153 9.4344 0.7928NS 0.0078**

B2 C2 D
2

8.461214 0.0108* 3.759156 0.0716NS 0.0031** 0.52NS

1 5,129.922 12.33128

Residual Lack-of-t Pure error Cor total NS not signicant

6,240.133 15 416.0088 6,240.133 10 624.0133 0 5 0 34,337.35 29

*** p \ 0.001; ** p \ 0.01; * p \ 0.05

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attributed due to the higher availability of monosugars in the Trichoderma-derived enzyme-treated sawdust hydrolysis. Thus, the temperature has a signicant role in the bioethanol production by increasing the enzyme hydrolysis of the sawdust concentration and also the bioethanol yield due to increased production of fermentable monosugars (Fromanger et al. 2010; Zuroff and Curtis 2012). The present work suggested that the maximum production of the bioethanol from the sawdust could be achieved by proper method of acid and enzyme pretreatment and yeast fermentation employing H. estonica and S. cerevisiae.
Acknowledgments The authors are thankful to the authorities of Annamalai University, India and to Ministry of Human Resource Development, Govt. of India, New Delhi. Conict of interest of interest. The authors declare that they have no conict

Open Access This article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited.

References
Aro N, Pakula T, Penttiillae M (2005) Transcriptional regulation of plant cell wall degradation by lamentous fungi. FEMS Microbiol Rev 29:719739 Askew DJ, Laing MD (1993) An adapted selective medium for the quantitative isolation of Trichoderma sp. Plant Pathol 42: 686690 Fell JW (2005) The role of nucleotide analysis in the systematics of the yeast genera Cryptococcus sp. and Rhodotorula sp. Stud Mycol 38:129146 Fromanger R, Guillouet SE, Uribelarrea JL, Molina-Jouve C, Cameleyre X (2010) Effect of controlled oxygen limitation on Candida shehatae physiology for ethanol production from xylose and glucose. J Ind Microbiol Biotechnol 37:437445 Kathiresan K, Saravanakumar K, Senthilraja P (2011) Bio-ethanol production by marine yeasts isolated from coastal mangrove sediment. Int Multidiscip Res J 1(1):1924 Lau MJ, Lau MW, Gunawan C, Dale BE (2010) Ammonia ber expansion (AFEX) pretreatment, enzymatic hydrolysis and fermentation on empty palm fruit bunch ber (EPFBF) for cellulosic ethanol production. Appl Biochem Biotechnol 162: 18471857 Li K (2003) The role of enzymes and mediators in lignocellulose degradation. In: Goodell B, Nicholas D, Schultz T (eds) American Chemical Society Symposium Series 845. Wood deterioration and preservation, advances in our changing world. American Chemical Society, Washington, DC, pp 196209 Li A, Antizar-Ladislao B, Khraisheh M (2007) Bioconversion of municipal solid waste to glucose for bioethanol production. Bioprocess Biosyst Eng 30(3):189196 Li XH, Yang HJ, Roy B, Wang D, Yue WF, Jiang LJ, Park EY, Miao YG (2009) The most stirring technology in future: cellulose enzyme and biomass utilization. Afr J Biotechnol 8:24182422 n C, Galbe M, Wahlbom CF, Hagerdal BH, Jonsson LJ (2002) Mart Ethanol production from enzymatic hydrolysates of sugarcane

bagasse using recombinant xylose-utilising Saccharomyces cerevisiae. Enz Microb Technol 31:274282 Matsushita Y, Yamauchi K, Takabe K, Awano T, Yoshinaga A, Kato M, Kobayashi T, Asada T, Furujyo A, Fukushima K (2010) Enzymatic saccharication of Eucalyptus bark using hydrothermal pretreatment with carbon dioxide. Biores Technol 101: 49364939 Mcmillan JD (1994) Fuels production In: Himmel ME, Baker JO, Overend RP (eds) ACS symposium series 566. American Chemical Society, Washington, DC, pp 292324 Meinita MD, Hong YK, Jeong GT (2012) Comparison of sulfuric and hydrochloric acids as catalysts in hydrolysis of Kappaphycus alvarezii (cottonii). Bioprocess Biosyst Eng Jan 35(12):123 Miller GL (1959) Use of DNS reagent for the measurement of reducing sugar. Anal Chem 31:426428 Montgomery DC (2001) Design and analysis of experiments, 5th edn. John Wiley and Sons, New York Pakarinen A, Zhang J, Brock T, Maijala P, Viikari L (2011) Enzymatic accessibility of ber hemp is from GH families 10 and 11 with thermostable cellulases in lignocellulose hydrolysis. 108(12):28232834 Qureshi TA, Mirbahar KB, Samo MU, Soomro NM, Solangi AA, Memon A (2006) Clinical study of experimentally induced anaphylactic shock in goats. Int J Pharmacol 2:357361 Rodriguez Gomez D, Lehmann L, Olkjar et al (2012) Examining the potential of plasma-assisted pretreated wheat straw for enzyme production by Trichoderma reesei. Appl Biochem Biotechnol 166(8):20512063 (Data provider: Technical University of Denmark Document type) Sandhu H, Nidumolu U, Sandhu S (2012) Assessing risks and opportunities arising from ecosystem change in primary industries using ecosystem-based business risk analysis tool. Hum Ecol Risk Assess 18(1):4768 Saravanakumar K, Kathiresan K (2012) Bioprospecting potential of marine-derived Trichoderma. Asian Pac J Trop Biomed 18 Saravanakumar K, Kathiresan K (2013a) Thermostable cellulase produced by trichoderma (Hypocrea estonica). J Biotechnol Sci 1(1):2241 Saravanakumar K, Senthilraja P, Kathiresan K (2013b) Bioethanol production by mangrove derived marine yeast, Sacchromyces cerevisiae King Saud University Journal of King Saud UniversityScience http://dx.doi.org/10.1016/j.jksus.2012.12.005 Senthilraja P, Kathiresan K, Saravanakumar K (2011) Comparative analysis of bioethanol production by different strains of immobilized marine yeast. J Yeast Fungal Res 2(8):113116 Sherrard EC, Harris EE (1932) Factors inuencing properties of isolated wood lignin. Ind Eng Chem 24:103 Sun Y, Cheng J (2002) Hydrolysis of lignocellulosic material from ethanol production: a review. Bioresour Technol 83:111 Sveinsdottir M, Baldursson SRB, Orlygsson J (2009) Ethanol production from monosugars and lignocellulosic biomass by thermophilic bacteria isolated from Icelandic hot springs. Icelandic Agric Sci 22:4558 Wang Y, Sakamoto Y, Kamiya Y (2009) Remediation of actual groundwater polluted with nitrate by the catalytic reduction over copper-palladium supported on active carbon. Appl Catal A 361:123129 Wingren A, Galbe M, Zacchi G (2003) Techno economic evaluation of producing ethanol from softwood: comparison of SSr and SHF and identication of bottlenecks. Biotechnol Prog 19:11091117 Zuroff TR, Curtis WR (2012) Development of symbiotic consortia for lignocellulosic biofuel production. Appl Microbiol Biotechnol 93:14231435

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