High sugar yields from sugarcane (Saccharum officinarum) bagasse using low-temperature aqueous ammonia pretreatment and laccase-mediator assisted enzymatic hydrolysis

Industrial Crops & Products 111 (2018) 673–683

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High sugar yields from sugarcane (Saccharum officinarum) bagasse using low-temperature aqueous ammonia pretreatment and laccase-mediator assisted enzymatic hydrolysis Kanak Raj, Chandraraj Krishnan

T

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Department of Biotechnology, Indian Institute of Technology Madras, Chennai 600036, India

A R T I C L E I N F O

A B S T R A C T

Keywords: Cellulose hydrolysis Aqueous ammonia soaking Low-temperature pretreatment Sugarcane bagasse Laccase-mediator system Lignocellulose

Sugarcane (Saccharum officinarum) bagasse (SCB) is a promising feedstock for cellulosic ethanol production. Combination of low energy pretreatments with efficient enzymatic saccharification is desirable to reduce process cost. SCB was converted into fermentable sugars with high yield by aqueous ammonia soaking (AAS) pretreatment and laccase-mediator assisted enzymatic hydrolysis. The optimal process conditions for AAS were determined as 20% aqueous ammonia, 50 °C, 48 h residence time and 1.2 mm particle size by employing response surface method. Hydrolysis of pretreated SCB by cellulases resulted in release of 57.30% of total sugars in the raw SCB. Addition of laccase to cellulase enzyme mixture enhanced the total sugar yield to 68.2%. When the mediator 1-hydroxybenzotriazole was added to the cellulase-laccase cocktail, the total sugar yield was improved to 78.4%. The mediator also reduced the amount of laccase required for maximum sugar yield. The glucan conversion of pretreated SCB with cellulase-laccase-mediator cocktail was 86%, which is higher than the reported levels of glucan conversion for SCB pretreated by AAS at high temperature. Therefore, the combination of low-temperature AAS and laccase-mediator assisted enzymatic hydrolysis would be useful to develop low-energy biomass conversion process.

1. Introduction Development of alternative renewable biofuels attracted attention due to the predictable exhaustion and adverse environmental impact of fossil fuels. Among biofuels, bioethanol produced from plant materials has been recommended as a potential alternative to gasoline (Goldemberg, 2007). Though commercial ethanol is produced from first generation feedstocks like corn, there are major social and environmental issues such as food-fuel conflict, overuse of chemical fertilizers and land-use. Whereas, the use of second-generation feedstock like lignocellulosic agricultural byproducts do not compete with food. Lignocelluloses are sources of low-cost sugars, renewable and sustainable for bioethanol production (Banka et al., 2015). Lignocelluloses consist of about 60–70% cellulose and hemicelluloses, which can be converted into ethanol. But, lignocelluloses also consist of lignin covalently bound to polysaccharides, forming a complex structure, which confers high resistance to structural disintegration and enzymatic hydrolysis of polysaccharides (Himmel et al., 2007). Hence, conversion of lignocelluloses to fermentable sugars requires a pretreatment to break the structural barrier to facilitate efficient enzymatic hydrolysis of cellulose

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and hemicellulose. The most extensively studied process for production of ethanol from lignocelluloses involves at least three major steps viz. (i) pretreatment of lignocellulose to disintegrate rigid structure (ii) enzymatic hydrolysis of cellulose and hemicelluloses to glucose and pentoses and (iii) co-fermentation of glucose and pentoses to ethanol. The estimated total production cost of ethanol involves major contribution from pretreatment and enzymatic hydrolysis. The ethanol production cost is higher than the realistic target price for commercialization (Goldemberg, 2007). The cost reduction is important since the successful process for conversion of lignocelluloses to bioethanol lies in making ethanol available to transportation sector at a reasonable price. The cost reduction of ethanol production can be reasonably achieved by combination of low-energy pretreatment and efficient enzyme cocktail for conversion of lignocelluloses to fermentable sugars. There are different pretreatment methods, which are broadly classified into chemical, mechanical and biological based on the type of reagents and physicochemical conditions (Mood et al., 2013). The pretreatments facilitate enzymatic hydrolysis of cellulose by modifying the structure of lignocellulose and partially solubilizing the lignin (Boonsombuti et al., 2013; Seguin et al., 2012). Acid and alkaline

Corresponding author. E-mail address: [email protected] (C. Krishnan).

https://doi.org/10.1016/j.indcrop.2017.11.047 Received 8 June 2017; Received in revised form 27 October 2017; Accepted 24 November 2017 0926-6690/ © 2017 Elsevier B.V. All rights reserved.

Industrial Crops & Products 111 (2018) 673–683

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assisted enzymatic hydrolysis. The present results showed an improvement in sugar yield from low-temperature AAS pretreated SCB, which would be useful for low-cost process development.

pretreatments preferentially remove hemicellulose and lignin respectively. Most pretreatment methods are associated with one or combination of high-energy consumption, high capital cost and generation of toxic compounds. Mechanical methods are energy intensive and require high capital costs. Thermochemical pretreatments generate toxic compounds inhibiting biocatalysts and require specialized equipment to withstand high temperature, high pressure and corrosion. A desirable economic pretreatment process would involve minimum energy, low-capital expenditure, generation of minimum or no toxic compounds and facilitate efficient saccharification and fermentation. One approach to achieve cost-effective pretreatment of lignocellulose is using low-temperature methods. The alkaline pretreatments are more efficient than acids for delignification of lignocellulose at low temperature. A wide range of temperature has been reported for delignification of lignocellulose by alkaline pretreatments. Delignification of lignocellulose by alkaline reagents is mainly due to cleavage of aryl ether bonds in lignin and hydrolysis of ester bonds in carbohydrate complex. Like alkaline pulping process, extensive lignin removal from lignocellulose requires high temperature (> 170 °C). Since partial delignification is adequate for enzymatic saccharification of carbohydrate polymers, low temperature alkaline extraction of lignin is sufficient. Aqueous ammonia soaking (AAS) is a promising low-energy pretreatment, which can partially remove lignin at temperatures ranging from 30 to 70 °C (Chandel et al., 2013; Kim and Day, 2013). The reported AAS-pretreatments show preservation of high level of cellulose and hemicellulose and generate very low or no toxic compounds, which can lead to high sugar yields during enzymatic hydrolysis and efficient microbial fermentation. Because of the high volatile nature of ammonia, it can be easily recovered after the pretreatment for recycling. Biological delignification of lignocellulose using microbial and enzyme biocatalysts has been reported at low temperature (Wan and Li, 2012). The enzyme laccase (phenol oxidase, EC 1.10.3.2), a copper containing phenol oxidase produced by white rot fungi catalyzes lignin degradation (Heap et al., 2014). Though the laccase producing fungi can be used for biological pretreatment of lignocellulose, the method requires very long residence time and results in loss of sugars due to the growth of fungus. Instead, the use of laccase for delignification minimizes both pretreatment time and sugar loss. Laccases act on electron rich phenolic substrates by using molecular oxygen as the final electron acceptor (Bento et al., 2010; Kunamneni et al., 2008) However, the utility of laccase for delignification of lignocellulosic biomass is limited by the stearic and redox limitations in the bulky lignocellulosic structure (Heap et al., 2014). This limitation has been overcome by using mediators, which are low molecular weight chemical oxidants that act as electron carriers between laccases and the lignin substrates. The laccase-mediator system has been employed in pulp processing industries to enhance the enzymatic bleaching of kraft pulp (Bourbonnais et al., 1997). Recently, the laccase-mediator system has attracted attention for improving the enzymatic hydrolysis of cellulose in the chemical pretreated lignocellulosic feedstocks (Rico et al., 2014). Among various lignocellulosic materials, sugarcane (Saccharum officinarum) bagasse (SCB) is one of the potential feedstocks available worldwide in abundance and it has great strategic and economic advantages for bioethanol production (Goldemberg, 2007). Next to Brazil, India is the largest producer of sugarcane. SCB is composed of 60–65% cellulose and hemicelluloses, which can be converted into fermentable sugars (Krishnan et al., 2010). SCB has been subjected to different pretreatments including AAS prior to enzymatic hydrolysis. The efficiency of enzymatic hydrolysis and sugar yields from SCB with AAS pretreatment is lower than that with thermochemical pretreatments (Chandel et al., 2013). Though high-temperature can enhance the efficiency of hydrolysis, there is loss of hemicellulose during pretreatment. The lower carbohydrate content in the pretreated SCB would lead to low sugar yield during enzymatic hydrolysis. Therefore, to improve hydrolysis efficiency and sugar yields, we employed a combination of optimized low-temperature AAS pretreatment and laccase-mediator

2. Materials and methods 2.1. Chemicals Cellulase (Cellic CTec2) was obtained from Novozyme, Denmark as gift sample. Purified laccase, 1-hydroxybenzotriazole (HBT), 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) and HPLC grade sugars were purchased from Sigma–Aldrich, Inc., St. Luis, USA. Commercial laccase was obtained from Synkromax Biotech, Chennai, India as a gift sample. All other chemicals were of analytical grade. 2.2. SCB and composition analysis Sugarcane was obtained from fields in Tamil Nadu, the southern part of India. The cane was crushed in a manual cane-crusher and the fibrous bagasse was obtained. The soluble sugars in the bagasse were removed by washing with distilled water. The washed bagasse was air dried at room temperature (30 ± 2 °C) in a fume hood and ground using a lab blender and stored in airtight zip lock bags for further use. The composition of untreated and AAS pretreated SCB was determined according to Laboratory Analytical Procedure (LAP) of the National Renewable Energy Laboratory (Sluiter et al., 2012). The concentrations of glucose, xylose and arabinose in the biomass hydrolyzate were determined using HPLC. The levels of glucan, xylan and arabinan were determined according to NREL calculation. Moisture content was measured using moisture analyzer (KERN MLS_N Version 2.0). 2.3. AAS pretreatment The pretreatment of SCB was done in 100 ml bottle (Scott Duran) without agitation. The solid to liquid ratio was 1:10. Appropriate amounts of SCB and aqueous ammonia were mixed in the bottles and closed with airtight caps. The bottles were incubated in a temperaturecontrolled incubator at different temperatures for various residence times as per the experimental design. The pretreated SCB was washed with distilled water until the pH was 7, air-dried in fume hood overnight at room temperature (30 ± 2 °C) and stored in zip lock bags for further use. 2.4. Optimization of AAS pretreatment The pretreatment conditions were optimized for production of maximum sugar yields during enzymatic hydrolysis. The optimization was carried out by employing response surface methodology (RSM). RSM is a collection of statistical and mathematical techniques employed for improvement and optimization of processes (Gilmour, 2006). The method uses empirical modeling of relationship between the process variables and the responses obtained based on a design of experiment. The actual variables are transformed into dimensionless coded variables as in Eq. (1)

xi =

Xi − Xo , i = 1,2,3,.... ΔXi

(1)

where, xi, Xi and Xo are coded value, actual value and center point value of the variables respectively and Δxi is the step change of the actual value. The coded value is dimensionless and the actual values are expressed in actual units. Residence time (h), pretreatment temperature (°C), aqueous ammonia (%) and particle size of SCB (mm) were selected as process variables for optimization. SCB particles of different size distribution (< 0.3 mm, 0.3–1.2 mm, 1.2–2.1 mm, 2.1–3.0 mm, and 3.0V3.8 mm) were obtained using screens with different sizes, ASTM mesh nos. 7, 10, 18, and 50 (Test Sieves, Mumbai, India). 674

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‘Design Expert’ software. The predicted optimal conditions were validated by performing additional experiments under these conditions. Sugar yield in enzymatic hydrolysis of pretreated SCB was used as the response (Y). Sugar yield was defined as the percent recovery of sum of glucose and xylose from raw SCB after pretreatment and hydrolysis. It was calculated using the equation as given below

Table 1 Coded symbols and actual values of the process variables. Variable

Unit

Ammonia Concentration Particle size Residence time Temperature

% mm h °C

Coded symbol

Coded Level

A B C D

-α

−1

0

1

+α

5 0.3 12 23

10 1.2 24 32

15 2.1 36 41

20 3 48 50

25 3.9 60 59

Sugaryield (%) =

3

∑ i=1

3

βi Xi +

∑ i=1

2

βii Xi2 +

∑ i=1

2.5. Enzymatic hydrolysis of pretreated SCB Enzymatic hydrolysis of pretreated SCB was carried out in 15 ml scintillation vials. Each vial contained 0.2 g (dwt) of pretreated SCB, Cellic CTec2 cellulase (30 FPU/g dwt of substrate) and sodium citrate buffer (50 mM, pH 4.8) in a total volume of 10 ml. To avoid microbial growth, chloramphenicol (1 mg/ml) and cycloheximide (1 mg/ml) were added to the hydrolysis mixture. The reaction mixture was incubated at 50 °C with agitation at 150 rpm for 72 h. The amount of glucose and xylose released during hydrolysis was determined by using HPLC.

3

×

∑

βij Xi Xj + ε

j=i+1

(3)

Where, (G + X)P is the amount of glucose and xylose released during hydrolysis of pretreated SCB (in mg/g dwt pretreated SCB), (SR)P is the solids recovered after pretreatment of raw SCB (in mg/g dwt), and (G + X)R is the amount of glucose and xylose present in the raw SCB (in mg/g dwt raw SCB)

The experiments were designed according to central composite design (CCD) at five coded levels −α, −1, 0, +1, +α, where α=2 (Box and Hunter, 1957). The actual values of variables corresponding to the coded levels are given in Table 1. The experimental design consisted of 2k factorial points, 2k axial points and 6 center points for replications, where k is the no of processing variables. The software Design Expert 8.0.7.1 (State Ease Inc., Minneapolis, MN) was used to obtain the experimental conditions as shown in Table 2. All the experiments were performed in triplicates and the average values of the responses were used for statistical analysis. The results were fitted to a second order polynomial equation as given in Eq. (2).

Y = β0 +

(G + X ) P × (SR) P × 100 (G + X ) R

(2)

where, Y is the response Xi and Xj are process variables, β0 is the intercept constant, βi is the linear coefficient, βii is the quadratic coefficient and βij is the two-factor’s interaction coefficient and εi is the random error. The coefficients were estimated by least square method. The optimal variables for maximum sugar yields were determined by numerical method as described by Myers et al. (2009). All the statistical analysis and generation of response surface were performed using the

2.6. Enzyme assays FPase was assayed using Whatman No.1 filter paper strips. The reaction medium containing 30 mg Whatman No.1 filter paper and appropriately diluted enzyme in 50 mM sodium citrate buffer of pH 4.8

Table 2 Experimental design matrix of central composite design and sugar yields. Run number

A: Ammonia (%)

B: Particle size (mm)

C: Residence time (h)

D: Temperature (°C)

Sugar yieldsa (%) Observed

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 a

10 10 10 20 10 20 15 20 15 20 20 20 10 10 20 15 10 20 10 15 15 25 15 15 5 15 15 15 15 15

3.0 3.0 1.2 1.2 1.2 3.0 2.1 1.2 2.1 3.0 3.0 3.0 3.0 1.2 1.2 2.1 3.0 1.2 1.2 2.1 2.1 2.1 3.9 2.1 2.1 2.1 2.1 0.3 2.1 2.1

24 48 24 48 48 24 36 24 36 48 48 24 24 48 48 36 48 24 24 36 12 36 36 36 36 36 36 36 60 36

32 50 50 50 32 50 41 32 41 32 50 32 50 50 32 41 32 50 32 41 41 41 41 23 41 59 41 41 41 41

Sugar yields refer to the percent recovery of sum of glucose and xylose from raw SCB.

675

28.83 38.24 51.96 57.30 55.48 42.58 43.85 40.36 43.54 37.70 39.21 40.20 43.37 55.98 47.16 42.75 39.64 51.80 36.39 43.51 29.83 43.83 50.79 30.62 27.56 47.45 43.09 72.21 46.09 41.87

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

Predicted 1.2 1.3 1.8 2.2 1.8 1.4 1.3 1.5 0.9 1.1 2.2 0.9 1.9 2.6 2.1 2.5 0.7 2.1 2.3 1.7 1.8 0.8 1.2 0.9 1.1 1.4 0.7 1.2 1.7 2.1

31.42 39.98 49.27 58.47 52.01 46.18 39.67 41.01 39.67 40.01 43.23 34.67 42.93 55.22 55.25 39.67 36.77 52.52 37.77 39.67 34.06 42.91 49.38 32.40 36.42 47.03 39.67 70.97 45.31 39.67

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Table 3 Analysis of variance (ANOVA) for reduced quadratic model. Source

Sum of square

Degree of freedom

Mean square

F value

P-value Prob > F

Model Residual (error) Lack of fit Pure error Corrected total

2183.27 347.27

7 20

311.90 17.36

17.96

< 0.0001

346.19 1.08 2533.42

17 3 29

20.36 0.36

56.54

0.0033

Std. dev. = 4.17; Mean = 43.77; C.V.% = 9.52; PRESS = 800.35; R2 = 0.8628; Adj R2 = 0.8147; Pred R2 = 0.6837; Adeq Precision = 16.169.

was incubated at 60 °C for 60 min. The reducing sugars released in the reaction was estimated by 3,5-dinitrosalicylic acid method. One unit of FPase is defined as the amount of enzyme required to release 1 μmol of reducing sugars as glucose equivalents per min under the assay conditions. Laccase activity was measured as the oxidation of ABTS. The reaction mixture containing 0.5 mM ABTS, 0.1 ml of appropriately diluted enzyme and 100 mM sodium acetate buffer (pH 5) in a total volume of 1 ml was incubated at 24 °C for 10 min. The absorbance of the reduced product in reaction mixture was measured at 420 nm. One unit of enzyme activity was defined as the amount of enzyme oxidizing 1 μmol of ABTS per min under the assay conditions.

Fig. 1. Correlation of predicted and actual sugar yields from different runs of the experimental design.

Table 4 Significance of the coefficients of regression.

2.7. Laccase assisted enzymatic hydrolysis of pretreated SCB The laccase assisted enzymatic hydrolysis of SCB was carried out in two modes viz. sequential treatment and simultaneous treatment (Heap et al., 2014; Rico et al., 2014). In sequential treatment, the AAS pretreated SCB was first treated with laccase at 50–400 U/g dwt substrate in 50 mM sodium citrate buffer of pH 4.8 for 72 h. Then the laccasetreated SCB was washed with distilled water and subsequently hydrolyzed with cellulase. In the simultaneous treatment, the AAS pretreated SCB was treated with a mixture of cellulase and laccase. The reaction mixture containing AAS pretreated SCB and cellulase-laccase mixture in 50 mM sodium citrate buffer of pH 4.8 was incubated at 50 °C for 72 h. After the reaction, the amount of simple sugars released was determined by HPLC method. The simultaneous treatment was also carried out by the addition of mediator (HBT) to the cellulase-laccase mixture.

Sum of square

Degree of freedom

Mean square

F value

P-value Prob > F

A-Ammonia B-Particle Size C-Residence Time D-Temperature BC CD B2

63.24 698.77 191.23 325.03 79.05 68.78 757.15

1 1 1 1 1 1 1

63.24 698.77 191.23 325.03 79.05 68.78 757.15

3.64 40.24 11.01 18.72 4.55 3.96 43.61

0.0708 < 0.0001 0.0034 0.0003 00454 0.0604 < 0.0001

in the SEM for imaging analysis. 2.10. Thermogravimetry Thermogravimetric analysis (TG Analysis) was performed using NETZSCHSTA449F3 Jupiter. TG scans were performed in the range of 25 °C–700 °C at a heating range of 10 °C/min. The experiments were carried out under inert argon atmosphere using 10–40 mg of sample. The gas flow of 20 ml/min was maintained in order to prevent any thermo oxidative degradation. 2.11. Fermentation of SCB hydrolyzate

2.8. X-ray diffraction (XRD)

SCB was pretreated by SAA under optimized conditions and hydrolyzed using cellulase supplemented with laccase and HBT. The solid residue in the hydrolysate was removed by centrifugation under aseptic condition and the supernatant was filtered through sterile 0.2 μm membrane. Pre-sterilized solutions of yeast extract and beef extract were added to the hydrolyzate under aseptic conditions to a final concentration of each at 2 g/L Candida tropicalis Y-27290 obtained from ARS Culture Collection (NRRL) was used for fermentation. The culture was maintained in yeast agar medium for routine use by sub-culturing. Seed culture was prepared by growing the strain in shake flasks overnight at 30 °C with agitation at 200 rpm. After inoculation of the hydrolysate with the yeast cells, the flasks were incubated at 30 °C with agitation (200 rpm). Fermentation samples were collected at different time intervals and the concentrations of sugars, ethanol and xylitol were determined using HPLC. The cell growth was monitored by measuring absorbance of the culture samples at 600 nm.

The untreated and AAS pretreated SCB samples were analyzed by XRD using D8 ADVANCE diffractometer equipped with a sealed tube of CuKα source. The samples were cast on microscopic slides prior to observation. Scans were collected from 2θ = 10° to 60° in steps of 0.04° at a time interval of 0.2 s. The recitation (CuKα) level used was 1.5406 A°. The crystallinity index (CrI) of SCB was determined according to Segal et al. (1959) as given below.

CrI = (I002 − Iam) × 100/ I002

Source

(4)

where, I002 is intensity of the diffraction from the 002 plane at 2θ = 22.4° and Iam is intensity of amorphous region at 2θ = 18.4°. 2.9. Scanning electron microscopy (SEM) The morphological structure of untreated and pretreated SCB was studied by SEM using Quanta 200 FEG HRSEM. SCB particles were placed on a carbon conductive tape on pin-mount specimen holder made up of 60% gold and 40% palladium. SCB particles were pressed to ensure that no space was left between the particles and the loose particles were blown by a spray duster. The sample holder was then placed

2.12. HPLC quantification of sugars and fermentation products The sugars and alcohol in hydrolysate and fermentation samples were quantified using a HPLC (Shimazu Corporation, Japan) system connected to a refractive index (RI) detector and Aminex HPX-87H 676

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Fig. 2. Response surface plots for the effect of (A) residence time and temperature and (B) residence time and particle size on the sugar yields during enzymatic hydrolysis of pretreated SCB.

ANOVA and Tukeys test were performed using Microsoft Excel. 3. Results and discussion The composition of SCB used in this study was 37.96% cellulose, 20.54% xylan, 1.51% arabinan and 23.44% lignin. The observed composition is in the range reported for SCB (Krishnan et al., 2010). The lignin content is high compared with other agro-residues. The removal of lignin is important to achieve high sugar yields during enzymatic hydrolysis. There are several reports on partial removal of lignin facilitating high sugar yields. Alkaline pretreatments using alkali or ammonia are known to solubilize lignin and the level of delignification is related directly with the temperature. High temperature alkaline pretreatment removes 55% lignin (Aita et al., 2011), whereas low temperature pretreatment like AAS has been reported to remove 41–45% lignin in SCB (Chandel et al., 2013). Low temperature pretreatment is desirable since it does not generate toxic sugar derivatives and recovers high levels of polysaccharides (Kim and Day, 2013). Hence in the present study the conditions of low-temperature AAS pretreatment for efficient hydrolysis of SCB were optimized by employing statistical method.

Fig. 3. XRD spectra of SCB before and after pretreatment. UNSCB – Untreated SCB, PTSCB – Pretreated SCB.

column (Bio-Rad). A 5 mM H2SO4 was used as a mobile phase, at a flow rate of 0.6 mL/min. The column oven and RI detector were maintained at 60 °C. HPLC grade sugars and alcohol were used as standards for identification and quantification.

3.1. Optimization of low-temperature SAA pretreatment by RSM RSM was employed to select suitable low-temperature ammonia pretreatment conditions for maximum saccharification of SCB. Four process variables viz. aqueous ammonia concentration, residence time, particle size and temperature were used for optimization. The solid to liquid ratio was maintained at 1:10. The yields of sugar obtained from enzymatic hydrolysis of pretreated SCB were used as the responses. The

2.13. Statistical analysis All experiments were carried out in triplicates and the average values with standard deviations were presented. Statistical calculations, 677

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the response surface plots for effect of residence time in combination with temperature or particle size on sugar yields. As seen in Fig. 2a, increase in temperature and residence time had a positive effect on sugar yields. The sugar yields increased slowly with increase in residence time at high temperature. Similarly, when residence time was high, a slow increase in sugar yields was observed with increase in temperature. Whereas, when one of these two variables are low, the sugar yields increased rapidly with increase in the level of the other variable. These trends show that the optimal region lies at the higher levels of these variables. The plot of residence time vs particle size showed that the increase in residence time significantly improved sugar yield at low particle size (Fig. 2b). Whereas at high particle size, the residence time had negligible effect on sugar yields. As seen in Table 2 the observed maximum sugar yield was 72.21% for SCB pretreated with ammonia concentration of 15% at 41 °C for 36 h. The yield is in the range reported for SCB pretreated by AAS (Chandel et al., 2013; Kim and Day, 2013). The observed high glucan conversion at low temperature is due to small particles size (0.3 mm), which increases the surface area of biomass for enzymatic hydrolysis. Similarly, decrease in particle size of biomass has been reported to improve enzymatic hydrolysis (Khullar et al., 2013). Since larger particle size is favorable for energy efficient industrial process, the optimal conditions for larger particle size were determined from the model. The predicted optimal conditions were 20% aqueous ammonia, 48 h residence time, 50 °C and 1.2 mm particle size. These results suggest that higher severity is required for larger particle size of the biomass. However, the predicted sugar yield of 58.47% for the larger particle size (1.2 mm) was less than the sugar yields corresponding to small particle size (0.3 mm). The decrease in sugar yields is attributed to decrease in surface area due to larger particle size. The validation of predicted conditions and sugar yields was performed by carrying out three replicates of experiments under the optimal conditions, which showed sugar yield of 58.51%, similar to the predicted value.

Fig. 4. TG analysis of SCB before and after pretreatment. (A) Weight loss of SCB with variation in temperature (B) DTG curve for SCB before and after pretreatment. UNSCB – Untreated SCB; PTSCB – Pretreated SCB.

plan of experiments and the observed sugar yields are given in Table 2. The CCD design showed significant changes in sugar yields from the enzymatic hydrolysis of SCB pretreated at different conditions. The highest sugar yield obtained was 72.21% (Run No. 28) and the lowest sugar yield was 28.83% (Run No. 1). The significant difference in sugar yields indicates the variable effect of the chosen parameters on sugar yields. Further, the experimental data were fitted into a reduced quadratic model as shown in Eq. (5).

3.2. Physicochemical characterization of untreated and AAS pretreated SCB The changes in composition and crystallinity of SCB after SAA pretreatment under the optimized conditions were determined. The SAA pretreatment significantly altered the composition of SCB. The SCB pretreated at optimized conditions contained 43.35% cellulose, 26.94% xylan, 2.40% arabinan and 18.75% lignin. There was no loss in cellulose and hemicelluloses and the observed recovery of these polysaccharides was higher than that reported for pretreatment at higher temperature. The AAS pretreatment of SCB with dilute ammonia at 70 °C for 96 h has been reported to retain 94% of glucan and 78% of hemicellulose (Kim and Day, 2013). Chandel et al. (2013) reported a loss of 68% xylan during AAS with 20% ammonia at 70 °C for 24 h. AAS pretreatment of SCB with dilute ammonia at low temperature of 30 °C for very long residence time of 40 days completely retained glucan and solubilized 27% of hemicellulose (Kim et al., 2010). Retention of hemicellulose along with cellulose would significantly improve the total sugar yield during enzymatic hydrolysis. The reported results indicate that the loss of hemicellulose during aqueous ammonia pretreatment depends on temperature and residence time. Accordingly, the present results showed that at low temperature of 50 °C and short residence time of 48 h, the loss of hemicellulose was low at 6%. Therefore, the optimized low-severe pretreatment conditions are suitable for recovery of cellulose and hemicellulose at high levels. Similar to present results, low temperature AAS pretreatment of wheat straw with aqueous ammonia at 50 °C for 60 h has been reported to retain high levels of cellulose and hemicellulose (Gao et al., 2012). The increase in the concentration of cellulosic polysaccharides in the pretreated bagasse is primarily due to the removal of lignin during the pretreatment. Alkaline reagents like ammonia are well known to remove lignin (Chiaramonti et al., 2012; Mood et al., 2013). However,

Y = 6.17863 + 0.32465 × A − 25.17257 × B + 1.45457 × C + 1.10004 × D − 0.20581 × BC − 0.019198 × CD + 6.33011 × B2

(5)

The results of ANOVA of the model are given in Table 3. The low pvalue of < 0.0001 for the model with a F-value of 17.96 suggest that the model is highly significant. The coefficient of determination (R2) of 0.8628 implies that there is a strong correlation between the actual and predicted values as shown in Fig. 1. This model is further confirmed by the reasonable agreement between predicted and adjusted determination coefficients. The low coefficient of variation (CV = 9.52%) signifies the precision and reliability of the experiments. The signal level is adequate since the observed signal to noise ratio was high at 16.169. A desirable ratio of signal to noise is greater than 4. These statistical tests indicate the suitability of the model for prediction of sugar yields under different combinations of process variables within the range of design space. The significance of coefficients of linear and quadratic terms was assessed based on the p-values. The linear effects of particle size and temperature were found to be greater than that of residence time (Table 4). Two interaction terms BC (particle size and residence time) and CD (residence time and temperature) had low p-values indicating their significant interaction effects. Among the quadratic terms, only B2 (particle size) was significant with a p-value of < 0.0001. Fig. 2 shows 678

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Fig. 5. SEM pictures for SCB after pretreatment. A, B – Untreated SCB at 800 x and 1200 x magnifications; C, D – Ammonia pretreated SCB at 800 x and 1200 x magnifications.

reported increase is higher than that of the present level due to higher delignification and loss of hemicellulose. Though crystallinity increases after pretreatment, the removal of lignin facilitates higher accessibility of cellulose to cellulases, which results in enhancement of saccharification (Zhang and Wu, 2014). The degradation of polysaccharides and lignin in SCB after AAS pretreatment was further studied by thermogravimetry. The TGA curves of the percent weight loss of compounds during thermal degradation of SCB samples with gradual increase in temperature is shown in Fig. 4A. There are significant differences between the curves of untreated and pretreated SCB indicating differences in composition. There was prominent weight loss around 100 °C and the maximum weight loss was observed in the region of 190–400 °C. Similar profile of weight loss has been reported for sugarcane bagasse and the weight losses at 100 °C and 190–400 °C have been attributed to loss of moisture and degradation of polysaccharides and lignin. While hemicellulose and cellulose are degraded in a narrow range of temperature around 250 °C and 350 °C respectively, lignin degrades in broad range of temperature ranging from 190 to 400 °C (El-Sayed and Mostafa, 2014). The gradual weight loss at higher temperature indicates the decomposition of lignin and it attained a final left-over weight of 17.35% for untreated SCB. The onset temperature of decomposition increased to around 260 °C for AAS pretreated SCB due to removal of extractives and partial delignification. Similar to present observation, SCB pretreated by other methods has been reported. The first derivative thermogravimetric (DTG) curve of rate of weight loss corresponding to thermal degradation is shown in Fig. 4B. The peaks of the DTG curves reveal the degree of thermal

the level of delignification by ammonia depends on the pretreatment conditions. High-severe pretreatment conditions result in higher removal of lignin (Chandel et al., 2013; Zhang and Wu, 2014). The selective removal of lignin is important for obtaining high sugar concentration during enzymatic saccharification. The present pretreatment conditions resulted in 37% removal of lignin. A slightly higher level of removal of lignin 41–45% after aqueous ammonia pretreatment of SCB at 70 °C has been reported (Chandel et al., 2013). High temperature aqueous ammonia pretreatment of SCB 160–170 °C has been reported to remove 55% of lignin (Zhang and Wu, 2014). Though lignin is a major barrier for enzymatic hydrolysis of cellulose in lignocellulose, for efficient enzymatic hydrolysis of cellulose, complete removal of lignin is not necessary. A partial removal of lignin has been reported to facilitate efficient enzymatic hydrolysis (Gao et al., 2012). Similarly, partial delignification of SCB by AAS pretreatment enhanced enzymatic hydrolysis by 2.5 folds compared with untreated SCB. In addition to lignin content, crystallinity index affects the enzymatic hydrolysis of glucan in SCB. To determine the change in CrI of SCB after pretreatment, XRD analysis was performed. Fig. 3 shows the XRD spectra of untreated and AAS pretreated SCB. The CrI of untreated SCB was 38.24%. The CrI of AAS pretreated SCB increased by 7.2% to 45.47%. Similar increase in CrI of SCB after alkaline pretreatment has been reported (Chandel et al., 2013). The increase in CrI of SCB is attributed to the removal of amorphous components (lignin and hemicelluloses), which results in higher concentration of crystalline cellulose compared with the untreated SCB. Pretreatment of SCB by AAS at 70 °C has been reported to increase the CrI by 15% (Chandel et al., 2013). The 679

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degradation corresponding to the relative change in composition of samples. The main weight loss peaks for the untreated SCB were observed around 280 °C and 350 °C corresponding to the degradation of hemicelluloses and lignin (Zhang and Wu, 2014). These results showed higher thermal stability of AAS pretreated SCB because of increase in the level of crystalline cellulose, as observed by XRD. The enzymatic hydrolysis of lignocelluloses depends on both chemical and morphological structures. The change in morphology like breakages and pore formation caused by pretreatments has great effect on enzymatic hydrolysis, since these changes improve accessibility of cellulose to enzymes. Hence, SEM analysis of AAS pretreated SCB was carried out to study the morphological changes. The SEM pictures of SCB samples are shown in Fig. 5. There were significant changes in structure after AAS pretreatment of SCB. The native SCB showed smooth morphology due to the presence of amorphous compounds like lignin and hemicellulose on the surface (Fig. 5A). Similar morphological structure has been reported for native SCB (Chandel et al., 2013; Zhang and Wu, 2014). Compared to the SEM images of native SCB, the AAS pretreated SCB showed rough surface with distortions (Fig. 5B). This is because of the ability of ammonia to remove lignin with swelling of the biomass (Swain and Krishnan, 2015). The SEM results indicate that in addition to lignin removal the distortion in structure and exposure of cellulose fibers contributed to the enhancement of enzymatic hydrolysis of AAS pretreated SCB. Fig. 6. Effect of addition of laccase to cellulase on sugar yields during enzymatic hydrolysis of AAS pretreated SCB. (A) SCB was treated with cellulase and laccase sequentially (B) SCB was treated with a cellulase and laccase simultaneously. Laccase units refer to units per gram of the pretreated SCB. Significant difference in sugar yields was observed between groups as determined by one-way ANOVA (F(3,4) = 8.23, p < 0.034) for Fig. A and, (F(3,4) = 44.33, p < 0.0015) for Fig. B. The post hoc Tukey test showed significantly higher sugar yield after the addition of 200 U/g of laccase. Bars with same letter indicates same group.

3.3. Improvement of enzymatic hydrolysis of pretreated SCB by laccasemediator system The AAS pretreated SCB under the optimized conditions for large particle size was further treated with laccase to improve the sugar yield during enzymatic hydrolysis. The ammonia pretreated SCB was treated with laccase and cellulase in sequential mode, where the ammonia pretreated SCB was first treated with laccase and then hydrolyzed with cellulase. The sugar yield increased with the increasing levels of laccase resulting in a maximum of 20% increase in sugar yield with laccase at 200 U/g solids compared with the control (Fig. 6A). Further increasing laccase to 400 U/g showed lower hydrolysis yield compared with the maximum yield obtained with laccase at 200 U/g. There was a significant difference in sugar yields between groups as estimated by oneway ANOVA (F(3,4) = 8.23, p < 0.034). When the pretreated SCB was treated with laccase and cellulase simultaneously, the profile of change in sugar yields with laccase levels was similar to that of sequential treatment with laccase and cellulase. A maximum increase in sugar yield of 19% was observed with laccase at 200 U/g (Fig. 6B). Oneway ANOVA showed significant difference in sugar yields between the groups (F(3,4) = 44.33, p < 0.0015). A significant enhancement of sugar yield by the addition of 200 U/g of laccase compared to cellulase without laccase was revealed by post hoc Tukey’s test. Addition of higher laccase (400 U/g) to cellulase decreased the sugar yield. Similarly, decrease in sugar yields during enzymatic hydrolysis of steam pretreated biomass in the presence of laccase has been reported (OlivaTaravilla et al., 2015). It was hypothesised that the decrease in glucose formation in enzymatic hydrolysis with laccase was caused by grafting of phenolic radicals onto lignin. The presence of laccase in enzymatic hydrolysis of Sigmacell with lignin extract reduced glucose yield by 37% compared with assays without laccase (Oliva-Taravilla et al., 2016). This was attributed to formation of phenolic oligomers by laccase oxidation, and their role in inhibition of endoglucanases, cellobiohydrolases and xylanases. Similar to present results, increase in the sugar yields during enzymatic hydrolysis of pretreated lignocelluloses by both sequential and simultaneous treatment using laccase and cellulase has been reported (Palonen and Viikari, 2004). A 13–15% increase in sugar yields has been reported for enzymatic hydrolysis of steam-pretreated softwood by simultaneous and sequential treatments with laccase. Moilanen et al. (2011) have reported 12% increase in the hydrolysis yield of steam-

Fig. 7. Enzymatic hydrolysis of AAS pretreated SCB with addition of laccase-mediator to cellulase. Laccase loading was varied at a constant concentration of mediator (HBT) and cellulase (A) Effect of laccase loading on sugar yield. Significant difference in sugar yields was observed between groups as determined by one-way ANOVA (F(3,4) = 116.95, p = 0.0002). The post hoc Tukey test showed that sugar yield was significantly higher after the addition of 50 U/g of laccase. Bars with same letter indicates same group. (B) Comparison of glucan conversion by cellulase-laccase enzyme mixture with and without mediator.

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Table 5 Comparison of effect of aqueous ammonia pretreatment conditions on compositional change and glucan conversion. Ammonia (g/g)

Temperature (°C)

Time (h)

Lignin removal (%)

Sugar loss (%)

Glucan conversion (%)

Cellulase loadingb (IU/g dwt biomass)

References

0.14 2.00 2.00 0.30

70 70 50 30

96 24 48 960

45.0 41.5 37.0 46.0

22.0 68.0 6.0 27

78.0 70.0 86.0 NRa

30 FPU Spezyme + 15 CBU Novo188 15 FPU Celluclast + 17.5 CBU Novo188 30 FPU CTec2 NR

Kim and Day (2013) Chandel et al. (2013) Present study Kim et al. (2010)

a b

NR- Not reported. FPU-FPase Unit; CBU − Cellobiase Unit.

enhanced by using low molecular weight chemical oxidants as mediators, which act as electron carriers between laccases and the lignin substrates (Bourbonnais and Paice, 1997; Rico et al., 2014). In lignocellulose, the complex network of lignin and carbohydrate polymers further hinders the accessibility of lignin to laccase. Therefore, small mediator molecules are highly useful for enzymatic depolymerization of lignin in lignocellulose. The use of 1-HBT as a mediator for enzymatic pulping of wood with laccase has been reported (Camarero et al., 2007). There are a few reports on enzymatic hydrolysis of thermochemical pretreated lignocellulosic biomass using laccase mediator system. Laccase-mediator treatment of steam pre-treated spruce wood and alkaline peroxide pretreated Eukalyptus wood improved sugar yields during enzymatic hydrolysis (Moilanen et al., 2011; Rico et al., 2014). The enhancement of sugar yields during enzymatic hydrolysis of lignocelluloses treated with laccase-mediator system is caused by enhanced modification and removal of lignin, which in turn reduces nonspecific binding of cellulases (Moilanen et al., 2014). Since the composition of lignin in pretreated lignocelluloses varies with the method of pretreatment, the laccase-mediator would modify lignin differently. The effect of laccase-mediator system on ammonia-pretreated lignocellulose has not been investigated. Hence the effect of laccasemediator system on enzymatic hydrolysis of aqueous ammonia pretreated SCB was studied. Fig. 7A shows the change in sugar yields of enzymatic hydrolysis of pretreated SCB using cellulase supplemented with laccase and HBT. Compared to cellulase alone, the cocktail of cellulase with laccase at 50 U/g and 2.5% (w/v) HBT enhanced the sugar yield by 35.5%. Further increase in the level of laccase did not increase the level of enhancement of sugar yields. While laccase at 200 U/g with HBT showed 24.3% enhancement of sugar yield, laccase at 400 U/g with HBT showed 22.2% lower sugar yield compared with the control. One-way ANOVA (F(3,4) = 116.95, p = 0.0002) showed significant difference in sugar yields between groups. The post hoc Tukey’s test showed significantly higher sugar yield after the addition of 50 U/g of laccase. Though the trend of change in sugar yields during hydrolysis by cellulase-laccase-mediator mixture is similar to cellulaselaccase mixture, the amount of laccase required for maximum

Fig. 8. Fermentation of SCB hydrolyzate obtained from laccase-HBT assisted enzymatic hydrolysis of AAS pretreated SCB by C. tropicalis.

pretreated spruce by sequential treatment of laccase and cellulase. Therefore, the level of enhancement of sugar yields observed for ammonia pretreated SCB is higher than that reported for steam pretreated softwood. The increase of sugar yield by laccase is primarily attributed to lignin degradation by the oxidative action of laccase on lignin, which enhances the accessibility of cellulose to enzymatic hydrolysis (Rico et al., 2014). As lignin causes unproductive binding of cellulases, the degradation of lignin increases the availability of cellulases to act on cellulose fibers. Laccase supplemented cellulase can be further used for simultaneous saccharification and fermentation (SSF), since laccase can retain considerable activity in the presence of ethanol that is simulataneousl produced with enzymatic hydrolysis. Purified laccases have been shown to lose about 10–20% activity in 4–5% ethanol (Rodakiewicz-Nowak et al., 1999). Since, cellulosic ethanol production results in about 5% ethanol, laccase can be used in SSF to enhance enzymatic hydrolysis. During the enzymatic degradation of lignin polymer, the reactivity of laccase decreases with the size of substrate due to steric hindrance (Christopher et al., 2014; Heap et al., 2014). The laccase action is

Fig. 9. Mass balance for conversion of sugars in SCB to ethanol using AAS pretreatment and laccase-HBT assisted enzymatic hydrolysis.

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temperature AAS pretreated SCB using cellulase-laccase-mediator system is higher than the reported sugar yields for AAS pretreated SCB. Besides, laccase-HBT addition to cellulase did not show any toxic effect on microbial utilization of glucose and xylose in fermentation producing ethanol. Therefore, the combination of low temperature AAS pretreatment and laccase-mediator system assisted hydrolysis would be useful to develop low-energy process for conversion of plant biomass to fermentable sugars and subsequent fermentation of sugars to ethanol and co-products.

enhancement of hydrolysis is decreased by the presence of mediator. The glucan conversion of pretreated SCB by the addition of laccase or laccase-mediator system to cellulase is shown in Fig. 7B. While addition of only laccase to cellulase enhanced glucan conversion to a maximum of 75.69%, the laccase-mediator system increased the glucan conversion to 86%. The level of laccase corresponding to maximum glucan conversion was 200 U/g when mediator was not present. Interestingly, the presence of mediator reduced the amount of laccase to 50 U/g to achieve maximum glucan conversion. Hence, the mediator not only enhanced the sugar yield, but it reduced the amount of laccase required for maximum sugar yield. This result clearly showed that the mediator enhanced the efficiency of laccase, hence less amount of laccase was required to obtain the maximum sugar yield. The enhancement in glucan conversion was 18% higher for addition of laccase-mediator to cellulase compared with addition of laccase to cellulase. The sugar yield obtained using cellulase-laccase-HBT mixture from low temperature AAS pretreated SCB is higher than the reported sugar yields for SCB pretreated by AAS (Table 5). Therefore, the results demonstrate that the sugar yield during enzymatic hydrolysis of lowtemperature AAS pretreated lignocellulose is enhanced by the addition of laccase-mediator systems to cellulases. However, the cost of mediator like HBT is high that again adds to process cost. This can be offset by minimizing cellulase loading instead of simply adding laccase-mediator.

Acknowledgements The authors greatly acknowledge IIT Madras for awarding half time teaching assistantship to Kanak Raj. References Aita, G.A., Salvi, D.A., Walker, M.S., 2011. Enzyme hydrolysis and ethanol fermentation of dilute ammonia pretreated energy cane. Bioresour. Technol. 102 (6), 4444–4448. Banka, A., Komolwanich, T., Wongkasemjit, S., 2015. Potential Thai grasses for bioethanol production. Cellulose 22 (1), 9–29. Bento, I., Silva, C.S., Chen, Z., Martins, L.O., Lindley, P.F., Soares, C.M., 2010. Mechanisms underlying dioxygen reduction in laccases. Structural and modelling studies focusing on proton transfer. BMC Struct. Biol. 10 (1), 28. Boonsombuti, A., Luengnaruemitchai, A., Wongkasemjit, S., 2013. Enhancement of enzymatic hydrolysis of corncob by microwave-assisted alkali pretreatment and its effect in morphology. Cellulose 20 (4), 1957–1966. Bourbonnais, R., Paice, M.G., Freiermuth, B., Bodie, E., 1997. Borneman S. Reactivities of various mediators and laccases with kraft pulp and lignin model compounds Env. Appl. Environ. Microbiol. 63 (12), 4627–4632. Box, G.E., Hunter, J.S., 1957. Multi-factor experimental designs for exploring response surfaces. Ann. Math. Stat. 1, 195–241. Camarero, S., Ibarra, D., Martinez, A.T., Romero, J., Gutiérrez, A., José, C., 2007. Paper pulp delignification using laccase and natural mediators. Enzyme Microb. Technol. 40 (5), 1264–1271. Chandel, A.K., Antunes, F.A., Silva, M.B., da Silva, S.S., 2013. Unraveling the structure of sugarcane bagasse after soaking in concentrated aqueous ammonia (SCAA) and ethanol production by Scheffersomyces (Pichia) stipitis. Biotechnol. Biofuels 6 (1), 102. Chiaramonti, D., Prussi, M., Ferrero, S., Oriani, L., Ottonello, P., Torre, P., Cherchi, F., 2012. Review of pretreatment processes for lignocellulosic ethanol production, and development of an innovative method. Biomass Bioenergy 46, 25–35. Christopher, L.P., Yao, B., Ji, Y., 2014. Lignin biodegradation with laccase-mediator systems. Front. Energy Res. 2, 1–13. El-Sayed, S.A., Mostafa, M.E., 2014. Pyrolysis characteristics and kinetic parameters determination of biomass fuel powders by differential thermal gravimetric analysis (TGA/DTG). Energy Convers. Manag. 85, 165–172. Gao, A.H., Bule, M.V., Laskar, D.D., Chen, S., 2012. Structural and thermal characterization of wheat straw pretreated with aqueous ammonia soaking. J. Agric. Food Chem. 60 (35), 8632–8639. Gilmour, S.G., 2006. Response surface designs for experiments in bioprocessing. Biometrics 62, 323–331. Goldemberg, J., 2007. Ethanol for a sustainable energy future. Science 315 (5813), 808–810. Heap, L., Green, A., Brown, D., van Dongen, B., Turner, N., 2014. Role of laccase as an enzymatic pretreatment method to improve lignocellulosic saccharification. Cat. Sci. Technol. 4 (8), 2251–2259. Himmel, M.E., Ding, S.Y., Johnson, D.K., Adney, W.S., Nimlos, M.R., Brady, J.W., Foust, T.D., 2007. Biomass recalcitrance: engineering plants and enzymes for biofuels production. Scince 315 (5813), 804–807. Khullar, E., Dien, B.S., Rausch, K.D., Tumbleson, M.E., Singh, V., 2013. Effect of particle size on enzymatic hydrolysis of pretreated Miscanthus. Ind. Crops Prod. 44, 11–17. Kim, M., Day, D.F., 2013. Enhancement of the enzymatic digestibility and ethanol production from sugarcane bagasse by moderate temperature-dilute ammonia treatment. Appl. Biochem. Biotechnol. 171 (5), 1108–1117. Kim, M., Aita, G., Day, D.F., 2010. Compositional changes in sugarcane bagasse on low temperature, long-term diluted ammonia treatment. Appl. Biochem. Biotechnol. 161 (1–8), 34–40. Krishnan, C., Sousa, L.D., Jin, M., Chang, L., Dale, B.E., Balan, V., 2010. Alkali based AFEX pretreatment for the conversion of sugarcane bagasse and cane leaf residues to ethanol. Biotechnol. Bioeng. 107 (3), 441–450. Kunamneni, A., Camarero, S., García-Burgos, C., Plou, F.J., Ballesteros, A., Alcalde, M., 2008. Engineering and applications of fungal laccases for organic synthesis. Microb. Cell Fact. 7 (1), 32. Moilanen, U., Kellock, M., Galkin, S., Viikari, L., 2011. The laccase-catalyzed modification of lignin for enzymatic hydrolysis. Enzyme Microb. Technol. 49 (6), 492–498. Moilanen, U., Kellock, M., Várnai, A., Andberg, M., Viikari, L., 2014. Mechanisms of laccase-mediator treatments improving the enzymatic hydrolysis of pre-treated spruce. Biotechnol. Biofuels 7 (1), 177. Mood, S.H., Golfeshan, A.H., Tabatabaei, M., Jouzani, G.S., Najafi, G.H., Gholami, M.,

3.4. Fermentation of hydrolyzate from laccase-HBT assisted hydrolysis The hydrolysate containing glucose and xylose obtained from enzymatic laccase-HBT assisted hydrolysis of pretreated SCB was evaluated for fermentation of glucose and xylose to ethanol and xylitol respectively by C. tropicalis. The hydrolysate consisted of 8.5 g/L of glucose and 5.8 g/L of xylose. The profiles of growth, utilization of glucose and xylose, and production of ethanol and xylitol are shown in Fig. 8. C. tropicalis completely consumed glucose in 12 h and simultaneously synthesized ethanol. The concentration of ethanol after 12 h was 4.13 g/L with metabolic yield of 95.5%. The volumetric productivity of ethanol was 0.31 g/L/h. The ethanol productivity (Yp/s) was 0.43 g/g. Xylose was utilized at lower rate than glucose and after 16 h of growth 73% of initial xylose was utilized. The concentration of xylitol synthesized was very low at 0.33 g/L. These results clearly showed the absence of toxicity of HBT on yeast for metabolism of glucose and xylose. There have been no reports on fermentation of biomass hydrolyzate consisting of HBT. The mass balance for conversion of sugars to ethanol using lowtemperature AAS and laccase-mediator assisted hydrolysis followed by fermentation with C. tropicalis is given in Fig. 9. There was no loss of sugars during pretreatment. This is evident from the absence of sugars in the liquid fraction after pretreatment. A very small difference in sugar contents between raw and pretreated SCB is within the error level. The enzymatic hydrolysis and metabolic yield of ethanol were also high suggesting the compatibility of laccase-mediator system with the process. It is evident from the present study that laccase-mediator assisted enzymatic hydrolysis of biomass results in enhanced sugar yield and supports fermentation of sugars to ethanol by C. tropicalis. 4. Conclusions The conditions for low-temperature AAS pretreatment were optimized for SCB based on sugar yields during enzymatic hydrolysis of pretreated SCB. The low temperature pretreatment caused partial delignification and structural disintegration of SCB facilitating enzymatic hydrolysis of glucan. Addition of laccase to cellulase increased the sugar yield. The presence of laccase-mediator in the cellulase laccase mixture further enhanced the sugar yield. The mediator increased the catalytic efficiency of laccase since the amount of laccase required was reduced by the presence of mediator. The observed sugar yield from the low 682

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Ardjmand, M., 2013. Lignocellulosic biomass to bioethanol, a comprehensive review with a focus on pretreatment. Renew. Sustain. Energy Rev. 27, 77–93. Myers, R.H., Montgomery, D.C., Anderson-Cook, C.M., 2009. Response Surface Methodology: Process and Product Optimization Using Designed Experiments, Third ed.). Wiley, New York. Oliva-Taravilla, A., Moreno, A.D., Demuez, M., Ibarra, D., Tomás-Pejó, E., GonzálezFernández, C., Ballesteros, M., 2015. Unraveling the effects of laccase treatment on enzymatic hydrolysis of steam-exploded wheat straw. Bioresour. Technol. 175, 209–215. Oliva-Taravilla, A., Tomás-Pejó, E., Demuez, M., González-Fernández, C., Ballesteros, M., 2016. Phenols and lignin: key players in reducing enzymatic hydrolysis yields of steam-pretreated biomass in presence of laccase. J. Biotechnol. 218, 94–101. Palonen, H., Viikari, L., 2004. Role of oxidative enzymatic treatments on enzymatic hydrolysis of softwood. Biotechnol. Bioeng. 86 (5), 550–557. Rico, A., Rencoret, J., del Río, J.C., Martínez, A.T., Gutiérrez, A., 2014. Pretreatment with laccase and a phenolic mediator degrades lignin and enhances saccharification of Eucalyptus feedstock. Biotechnol. Biofuels 7 (1), 6. Rodakiewicz-Nowak, J., Haber, J., Pozdnyakova, N., Leontievsky, A., Golovleva, L.A.,

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