Structural and Functional Characterization of Two Pennisetum sp

Research Article pubs.acs.org/journal/ascecg

Structural and Functional Characterization of Two Pennisetum sp. Biomass during Ultrasono-Assisted Alkali Pretreatment and Enzymatic Hydrolysis for Understanding the Mechanism of Targeted Delignification and Enhanced Saccharification Sonali Mohapatra,† Sivakumar Pattathil,*,‡ and Hrudayanath Thatoi*,§ †

Department of Biotechnology, College of Engineering and Technology, Biju Patnaik University of Technology, Bhubaneswar-751003, India ‡ Complex Carbohydrate Research Centre, University of Georgia, Georgia 30602 USA and BioEnergy Science Center (BESC), Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831, United States § Department of Biotechnology, North Orissa University, Sriram Chandra Vihar, Takatpur, Baripada-757003, Odisha India S Supporting Information *

ABSTRACT: The recalcitrance offered by lignocellulose to get converted into simple sugars makes its conversion process complicated, hence pretreatment is required prior to enzymatic hydrolysis and fermentation. Ultrasonication-assisted alkali pretreatment (UA-NaOH) was found to be an effective pretreatment for delignification and enzymatic hydrolysis of denanath grass (DG) and hybrid napier grasses (HNG) in terms of maximum delignification and reducing sugar production. To determine the mechanism of pretreatment and enzymatic hydrolysis, the structural and functional characterization of native and pretreated grass biomass were investigated using SEM (scanning electron microscope), FT-IR (Fourier transformation infrared) spectroscopy, TGA (thermal gravimetric analysis), DSC (differential scanning) spectroscopy, and solid state 13C CP/MAS NMR (cross-polarization magic angle spinning nuclear magnetic resonance) spectroscopy. The surface erosions, distorted surface morphology and deconstruction of the cell wall components of the Pennisetum sp. were detected by SEM. The differences in the intra- and intermolecular hydrogen bonds that make the crystalline and amorphous regions in the cellulose were detected by FT-IR. While TGA studies revealed higher phenolic content in untreated grass biomass, DSC patterns indicated the formation of laevoglucose in DG pretreated samples. Interestingly, the NMR studies revealed the presence of maximum aliphatic lignin components with absence of the aromatic lignins in both DG and HNG samples. NMR results also showed the presence of maximum hexosans and xylans revealing that the presence of aliphatic lignin components could be a helpful way of retaining the monosaccharides. KEYWORDS: Ultrasonication assisted-NaOH pretreatment, Delignification, Enzymatic hydrolysis, Reducing sugar, Grass biomass

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INTRODUCTION Diminishing availability of fossil fuel resources and environmental concerns associated with the CO2 accumulations in the atmosphere have prompted researchers to develop renewable energy resource like lignocellulosic biomass for biofuel production.1 Lignocelluloses are most abundant biopolymer in the world and are promising materials for biofuel production which are available in plenty and are cheaper in comparison to other raw materials. To produce biofuel, the lignocellulosic materials must undergo a bioconversion process such as enzymatic hydrolysis into fermentable sugars which could be then fermented to ethanol by fermenting microorganisms. Lignocellulosic biomass primarily consists of cellulose, hemicellulose, and lignin, which makeup the complex cell wall structure of this biomass.2 However, the recalcitrance of the © 2017 American Chemical Society

lignocellulosic biomass limits significantly the bioconversion process. The three major factors of the plant cell-wall, that is, crystallinity of cellulose, hydrophobicity of lignin, and encapsulation of cellulose by the lignin-hemicellulose matrix form a barricade restricting the accessibility of enzymes in the hydrolysis step.3 Because of rigid association between lignin, hemicellulose, and cellulose less conversion of cellulose into simple sugar takes place due to reduced accessibility of cellulose to enzymes and microorganisms.4 Pretreatment is a vital step to overcome recalcitrance of feedstocks and improve the bioconversion process of Received: February 24, 2017 Revised: June 20, 2017 Published: July 4, 2017 6486

DOI: 10.1021/acssuschemeng.7b00596 ACS Sustainable Chem. Eng. 2017, 5, 6486−6497

Research Article

ACS Sustainable Chemistry & Engineering

and ultrastructural changes that occur during the ultrasonication pretreatment and subsequent enzymatic hydrolysis in grass biomass. In the present study two Pennisetum varieties, namely, denanath grass and hybrid napier grass (C0-3) grasses were pretreated using UA-NaOH (ultrasonication assisted sodium hydroxide) and were enzymatically hydrolyzed using two enzyme sets. Out of two enzyme sets, the first set consisted of Celluclast 1.5L with xylanase added to it and the second set was an enzyme having both cellulase and hemicellulase activity (Palkonol MBW). The goal was to investigate the influence of bioconversion process on the structure and different proportion of cellulose, hemicellulose, and lignin of two different grass varieties. These grass varieties were chosen because of their high biomass production with less input in tropical soil and are most suitable for bioethanol production for their high cellulose content.24 The effectiveness of the pretreatment, specifically on the removal of S-subunits on the grass biomass and its linking properties for enhanced enzymatic hydrolysis was characterized using NMR. The thermal properties of the grass biomass that are related to enzyme adsorption were also studied using TGA and DSC. The changes in the surface morphology and the functional groups were analyzed using SEM and FTIR. The results from this study should give a better understanding of the partial delignification process of the grass biomass, rather than elimination of entire lignin content which consumes both time and energy. Along with this the observed phenomenon of depolymerization and repolymerization of the linkages that occurs during ultrasonication assisted alkali pretreatment could give substantial insights into the enhancement of enzymatic hydrolysis in grasses.

lignocellulosic biomass into ethanol. This step can be utilized for efficiently increasing the porosity of the biomass with partial removal of lignin and hemicellulose that act as the main barriers for cellulose conversion.4 Various pretreatment technologies, such as physical/physicochemical (mechanical disruption, ammonia fiber expansion, etc.) or chemical (alkali, dilute acid, ionic liquids, etc.), have been developed for effective bioconversion of lignocellulosic biomass. However, much of the reported literatures show several limitations. Therefore, efforts have been made to develop an efficient pretreatment system that contributes to the reduced recalcitrance and maximum release of reducing sugars. The alkaline pretreatment studies by the author and others with sodium hydroxide (NaOH) have been reported to exhibit enhanced bioconversion process.5,6 The alkaline treatments as compared to acid treatments remove most of the lignin along with some amount of hemicellulose from the biomass.6 The various reactions such as formation of alkali stable groups, disbanding of nondegradable polysaccharides, hydrolysis of glyosidic bonds, during alkaline pretreatment contribute to the better pretreatment of the biomass.7 However, alkaline pretreatment processes can be improved by the application of ultrasound, as the oxidizing radicals produced during ultrasonication increase the mass transfer within the solution.8 The process is helpful in that it can improve the contact between biomass and alkaline reagents leading to shortened pretreatment time and higher final sugar yields.8 Recent research on ultrasound-assisted pretreatment focuses on combination treatments of sonication with chemicals, such as alkali,9−12 dilute acid,9,13,14 and ionic liquids,9,15 to effectively deconstruct cell wall components in lignocellulosic biomasses. Velmurugan and Muthukumar evaluated the effect of ultrasound on the production of reducing sugars from sugar cane bagasse and found promising results with its application.12 Ultrasonication-assisted alkali pretreatment has been shown to significantly alter the components of grass biomass for enhanced enzymatic hydrolysis and bioethanol production.16−18 During pretreatment and enzymatic hydrolysis changes in the pore size and accessible surface area of the substrate are likely to occur.1 Pretreatment studies on grasses have been mostly targeted for complete delignification of syringyl (S), guaicyl (G), and p-hydroxyphenyl (H) subunits because assessment of grass lignins is particularly difficult because of the presence of high fraction of p-hydroxycinnamates.19 But reports suggest that modification of S subunits can be targeted as its presence plays a major role in recalcitrance of the biomass rather than the latter two subunits.20 Recently, Singh et al., found that oxidation of S subunits with sLac (a small laccase from Amycolatopsis sp.) substantially alters the interunit linkage distributions which will eventually help in enhanced delignification.21 On the other hand reports suggesting G subunits having higher adsorption capacity to the enzymes have also been observed.22 Further, the weakening effect of the pretreatment on cleavage of certain bonds, leads to higher formation of toxic products. Similarly, the observations on the untwisting of cellulose microfibrils during the initial reactions of enzymatic hydrolysis and the channel formation along the cellulose microfibrils on the later stages, were significant findings made through structural analysis.23 While the positive effects of ultrasound pretreatments in combination with ionic liquids or alkaline solutions are now clear, the specific roles of the pretreatment on alteration of the morphology and structure of biomass is still not studied in detail. Hence, it is rational to hypothesize the morphological

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MATERIALS AND METHODS

Materials. Hybrid Napier grass (CO-3 variety) [HNG] [Pennisetum purpureum] and Denanath grass [DG] [Pennisetum pedicellatum], which were three months old were collected from the Directorate of Research for Women in Agriculture (DRWA), Bhubaneswar, Odisha, India, during the month of March, 2015. Further processing of the grasses, except the roots, was done by bringing it to the microbiology laboratory of Department of Biotechnology, College of Engineering and Technology, Bhubaneswar. Pretreatment and Enzymatic Hydrolysis Methods. Preparation of Samples. The two Pennisetum sp. were dried in the shade and monitored for their moisture content. It was powdered using a ball mill at the Herbarium section of the Institute of Minerals and Material Technology, Bhubaneswar, Odisha. To achieve uniform size of the grounded particles it was passed through a sieve of mesh size of 2 mm and immediately stored in airtight containers at room temperature. Ultrasonication-Assisted NaOH (UA-NaOH) Pretreatment. Milled grass (10 g) sample was suspended in of 100 mL a 1% NaOH solution and incubated for 22 h at 55 °C and 18 h at 60 °C for DG and HNG respectively.25 The resulting samples were further subjected to treatment with ultrasonication with power supply of 60 W, 40 °C for 40 min (DG) and 70 W, 40 °C with an incubation of 50 min for HNG. Following the pretreatment, double-layered muslin cloth was used for filtering the biomass. The filtrate was kept at 4 °C and the biomass was washed continuously to attain the neutral pH so that no residual alkali was left on the surface of biomass particles. The moisture content was kept below 10% by incubating it at 105 °C in hot air oven for 24 h, and then further used for lignin determination. Triplicate samples were employed for all the experiments. Enzymatic Hydrolysis. The enzymatic hydrolysis was performed using two sets of enzymes i.e. a mixture of cellulase and hemicellulase [Palkonol MBW (Maps Enzymes Ltd.)] and Celluclast 1.5L (Sigma) and xylanase enzyme (Novozymes) [in combination] on the UANaOH samples. Palkonol MBW had a cellulase activity of 12 000 6487


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Table 1. Summary of Total Delignification Caused and Reducing Sugar Produced after UA NaOH Pretreatment and Reducing Sugars Produced after Saccharification Sl no.

grass biomass

total delignification after UA-NaOH pretreatment (%)

total reducing sugar released after UA-NaOH pretreatment (mg/g)

1

DG

89.3

227.2 ± 0.2

2

HNG

86.7

242.8 ± 0.6

enzymes used for saccharification of UA-NaOH pretreatment

xylan (mg/g)

glucan (mg/g)

total reducing sugar released after saccharification (mg/g)

Palkonol MBW Celluclast1.5l + xylanase Palkonol MBW Celluclast1.5l + xylanase

157.6 45.9 107.6 94.4

504.4 387.5 323.4 276.4

662.0 433.4 431.2 370.8

MCU/mL (Maps cellulase unit/mL) and hemicellulase activity of 5000 MHU/mL (Maps hemicellulase unit/mL), while Celluclast 1.5L and xylanase had 700 EGU/g (endoglucanase unit/g) and 8450 U/mL (unit/mL) respectively. The experiments were performed in aqueous solution containing sodium acetate buffer (0.1M) in 150 mL Erlenmeyer flask with total reaction volume of 5−15 mL. The flasks were incubated in an incubator cum shaker (INNOVA’ 40). The parametric conditions for DG and HNG were maintained at temperature-50 and 45 °C, pH-5.25 and 5, substrate concentration0.75 and 0.5 g, enzyme loadings −250 and 200 μL and incubation time of 30 and 35 h respectively for Palkonol MBW. In case of combination of Celluclast 1.5 L and xylanase enzyme, temperature of 52 and 54 °C, pH of 6 and 6.5, substrate concentration of 1 and 0.75 g, xylanase enzyme loading of 4 and 2.5 μL/200 μL of Celluclast 1.5L, and incubation time of 40 and 48 h was maintained for DG and HNG, respectively. Analysis. Total Lignin Estimation. Acid insoluble lignin (AIL) and acid soluble lignin (ASL) were analyzed using the solid and liquid fraction respectively, of the UA-NaOH pretreated sample. The UANaOH samples were kept in a pressure tube with 72% H2SO4 and incubated for 1 h in a cold water bath maintained at 25 °C. Stirring of the samples was done with a glass rod in an interval of 15 min. The samples were vacuum filtered to separate the solid residue and liquid hydrolysate. The solid residue which were kept for 24 ± 6 h in the muffle furnace at 575 ± 25 °C was used to determine the AIL and ASL was determined from the liquid hydrolysate by spectrophotometric method at 260 nm. Both the AIL and ASL were determined as per the NREL protocol.26 Total Reducing Sugar (TRS) Analysis. The liquid fraction obtained after the ultrasonication assisted alkali pretreatment was analyzed for the presence of the total reducing sugars using the standard DNS protocol.27 The liquid hydrozylate obtained after enzymatic hydrolysis of the pretreated samples was analyzed for individual hexose and pentose sugars, using HPLC with a refractive index (RI) detector (Shimadzu Corporation, Japan) along with a Aminex HPX-87H column (Bio-Rad). Elution of the sugars was obtained using 5 mM H2SO4 as a mobile phase with the flow rate maintained at 0.6 mL/min and temperature set at 60 °C. Before HPLC analysis the samples were diluted suitably (i.e., the concentrated samples were diluted) and were filtered in a 0.2 μm membrane. SEM Analysis. The untreated, alkali pretreated, ultrasonicationassisted alkali-pretreated and enzyme-hydrolyzed samples were investigated for alterations in their structures using scanning electron microscopy (SEM) [Carlzeiss EVO-18, USA]. The samples were mounted in very thin layers over the copper sample holder, which was gold coated and then analyzed for the morphological changes.28 FTIR Analysis. For Fourier transformation infrared spectroscopy (FTIR) analyses, the spectra of the untreated, ultrasonication assisted alkali pretreated and enzyme hydrolzed samples in the KBr phase (i.e., pellets formed using potassium bromide) were recorded in a PerkinElmer FTIR spectrophotometer, with a nominal resolution of 2 cm−1averaging 44 scans.5 TGA Analysis. The thermal characterization of untreated, alkali pretreated, ultrasonication assisted alkali pretreated and enzymatic hydrolyzed biomass was conducted using a thermogravimetric analysis (TGA) of type PerkinElmer STA-6000. It has a weighing capacity of 1500 mg with resolution around 0.1 μg. This technique helps in determining the decomposition behavior of the biomass. All the

experiments were carried out at a programmed temperature range of 20−500 °C within nitrogen environment with constant heating at 10 °C min−1. For analysis of TGA a biomass weight of 5 ± 1 mg was used. NMR Analysis. NMR samples were prepared with ground biomass packed into a 4 mm cylindrical Zirconia MAS rotor. Solid-state NMR measurements were carried out on a Bruker DSX 300 High Resolution Multinuclear FT-NMR Spectrometer. The spinning speeds were taken at different frequencies. For some bands it was recorded at 4 kHz, for some it was at 6 kHz and some at 10 kHz. The spinning speed was increased so as to make the bands disappear. The 13C NMR experiments were operated at relaxation delay of 5s, temperature of 20.6 °C and 1024 total scans.

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RESULTS AND DISCUSSION

The purpose of this study is to focus on characterization of UANaOH and enzyme treated grass biomass to access the mechanism of delignification and enzymatic hydrolysis in grasses. Therefore, the structural and functional characterization of the optimized UA-NaOH treated and enzyme treated lignocellulosic biomass from two Pennisetum sp.(DG & HNG) were investigated through SEM, FTIR, TGA, and NMR with a view to understand the mechanism of pretreatment and enzymatic hydrolysis during delignification and sugar production from grass biomass. UA-NaOH Pretreatment and Enzymatic Hydrolysis of Biomass. Experiments were conducted for optimization of pretreatment of DG and HNG with different parameters such as power, temperature and on−off time of ultrasonicator and revealed maximum delignification at 60 W, 40 °C for 40 min (DG), and 70 W, 40 °C with an incubation of 50 min for HNG (data not shown). Maximum lignin removal of 89.3% and 86.7% was observed for DG and HNG respectively. At the same conditions of pretreatment the reducing sugars for DG and HNG were found to be 227.2 ± 0.2 and 242.8 ± 0.6 mg/g of raw biomass, respectively. The small differences between the delignification and production of reducing sugar of DG and HNG samples might be the result of slight differences in their acid soluble lignin (ASL) and cellulose content of the untreated biomass.5 Further, enzymatic hydrolysis of the two Pennisetum sp. with two sets of enzymes was performed for enhanced release of reducing sugars. Palkonol MBW enzyme showed enhanced enzymatic hydrolysis with maximum TRS release of 662.0 ± 0.5 mg/g and 433.4 ± 0.3 mg/g in DG and HNG variety, respectively. However, attractive results were not exhibited in case of DG and HNG varieties for enzymatic hydrolysis with the mixture of Celluclast1.5L + xylanase as compared to Palkonol MBW. The maximum TRS obtained was 433.4 ± 0.3 mg/g in DG samples while for HNG reduced TRS of 370.8 ± 0.5 mg/g was observed. The total delignification caused and reducing sugar produced (DNS method) after UANaOH pretreatment and reducing sugars produced (HPLC method) after saccharification is summarized in Table 1. 6488


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Figure 1. SEM analysis of untreated DG and HNG (A and F), 1.5% NaOH-treated DG (B), 1% NaOH treated HNG (G), UA-NaOH DG(C), UANaOH Pretreated HNG(H), Celluclast 1.5L + xylanase hydrolyzed DG (D), Celluclast 1.5L + xylanase hydrolyzed HNG (I), Palkonol MBW hydrolyzed DG (E), and Palkonol MBW hydrolyzed HNG (J).

Assessment of Morphology and Topography of Biomass. Modifications on the surface after alkaline pretreatment, ultrasonication-assisted alkaline pretreated and enzymatic hydrolysis (both Palkonol MBW and combination of Celluclast 1.5L + xylanase) of DG and HNG were investigated using SEM. SEM images of untreated DG and HNG grasses [Figure 1A and F] showed smooth surface with somewhat ruptured cells that is covered with residues. This is attributed to the milling of the grass biomass which has lightly ruptured the cell wall. The alkaline pretreated samples of DG and HNG [Figure 1B and G] have considerably low surface residues and discrete lignin droplets, which indicate removal of lignin from the surface of biomass. Similar effects were also observed in Parthenium hysterophorus, with lower concentrations of NaOH.29 However, after UA-NaOH pretreatment [Figure 1C and H] the surface topology for DG was comparatively different from HNG. In DG, the unidirectional separation of the vascular bundles are prominent, whereas in HNG opening of the pores is more noticeable. Though the delignification is profound in both DG and HNG, the removal of hemicellulose is not intense in HNG. This difference can be attributed to the different lignin composition of both the biomasses. In reports by Lima et al., disaggregation of cell wall bundles in eucalyptus bark have been seen to have a positive correlation to greater delignification.30 Further, in the present study a considerable removal of middle lamella is observed. The removal of middle lamella can be attributed to removal of maximal lignin content from the cell wall. Literature review have revealed that around 50% of the lignin content in lignocellulosic biomasses is concentrated in the middle lamella.31 The surface roughness for UA-NaOH pretreated was also observed in DG, which was due to erosion, which is produced by physical effects of acoustic waves and the micro turbulence caused by transitory cavitation bubbles. Further enzymatic hydrolysis of DG hydrolyzed by the mixture of Celluclast 1.5L+ xylanase enzymes (Figure 1D) did

not demonstrate any significant morphological changes. However, in HNG (Figure 1I) scaling of layers and tiny pores were observed. This layering up was attributed to the loss of hemicellulose and cellulose fibers, thereby leading to low reducing sugar yields. DG and HNG with Palkonol MBW [Figure 1E and J], illustrate cracks and hollow areas in DG with swollen areas in the internal fibrillar structure. The cracks caused might have resulted by the removal of the hemicellulose, and thereby increasing the accessibility of cellulase enzymes and hydrolysis efficiencies. These observations were supported by the high glucose yields during enzymatic hydrolysis of the DG. The observed results are also in agreement with the previous studies on perennial ryegrass (Lolium perenne L.), tall fescue (Festuca arundinacea Schreb), and bentgrass (Agrostis sp.).32 Ultrasound assisted surfactant pretreatment on sugar cane tops also exhibited similar results.33 In case of Palkonol MBW hydrolyzed HNG, the decrease in the swollen structure as compared to the UA-NaOH pretreated HNG was observed, which was not anticipated. Assessment of Molecular Components and Structures. Then technique of FTIR is widely used for analysis of the chemical shifts that occur in a biomass. In the present study this technique has been applied for analyzing the difference between untreated, UA-NaOH treated, Palkonol MBW saccharified, and Celluclast1.5L + xylanase saccharified DG and HNG respectively as shown in Figure 2a and b. The band positions along with their respective assignments are also illustrated in Table 2. Characteristic assignment of cellulose was observed at positions 3510 and 3480 cm−1 in untreated DG and HNG respectively. Chemical shifts at this position were observed in UA-NaOH pretreated and enzyme hydrolyzed samples of DG and HNG suggesting the rupture of intramolecular hydrogen bonds between phenolic and aliphatic structures.34 Peaks in between 1000 and 1200 cm−1 which is attributed to breakage of the β-glycosidic linkages in between the sugars, stretching of C−O−C−O−C bonds in cellulose and 6489


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deformation in methoxyl groups, cellulose and hemicellulose respectively in all the pretreated and enzymatic saccharified biomasses of DG and HNG. Further the prominent band at 1098 and 900 cm−1 in UA-NaOH treated DG and HNG, Palkonol MBW treated DG and HNG and Celluclast 1.5L + xylanase-treated DG was a clear indication of the conversion of crystalline to amorphous cellulose. However, the absence of this band in Celluclast 1.5L + xylanase-treated HNG indicated the low conversion of crystalline cellulose to amorphous cellulose, restricting its further conversion to reducing sugars. Assessment of Thermal Analysis of Biomass. To apprehend the changes in thermal properties of biomass in native, NaOH-treated, UA-NaOH-treated, Palkonol MBWtreated, and Celluclast 1.5L + xylanase-treated DG and HNG samples were analyzed using TGA. The behavior of biomass materials during devolatilization was related to the presence of chemical constituents such as cellulose, hemicellulose and lignin. The overall TGA distribution profiles of untreated pretreated and enzyme hydrolyzed biomass, were found to be distinct from each other in both DG and HNG. Figure 3a and b shows the TGA curves for DG and HNG samples. As observed from the figures the initial weight loss step which occurs at 30− 120 °C because of the evaporation of the water absorbed, is absent in all the samples of DG except NaOH and UA-NaOH treated DG.39 In case of HNG, untreated, NaOH and UANaOH treated HNG exhibit the weight loss at around 120 °C. In the second step, onset temperature of untreated DG and HNG was found to be around 260 and 250 °C respectively. NaOH treated and UA-NaOH DG degraded at 300 °C, while NaOH treated and UA-NaOH HNG degraded at around 310 °C. UA-NaOH pretreated DG hydrolyzed with Palkonol MBW and cocktail of Celluclast 1.5L + xylanase, exhibited degradation at 180 °C, while samples of similar conditions in HNG degraded at 200 °C. Degradation of compounds in the range of 180−350 °C is attributed to the degradation of components of carbohydrates in the lignin samples, which are converted to volatile gases, such as CO, CO2, and CH4.40 The final stage of degradation in case of untreated, NaOH and UA-NaOH treated biomass of DG and HNG was observed to occur at temperatures above 350 °C. Degradation temperatures above 350 °C is attributed for degradation of volatile products derived from lignin including phenolic, alcohols, aldehyde acids along with the formation of gaseous products which gets removed.41 In case of DG and HNG saccharified with Palkonol MBW and mixture of Celluclast 1.5L + xylanase, the final degradation step occurred at a temperature of around 300 °C, which clearly indicates the absence of phenolics, aldehydes and alcohols in the enzyme saccharified samples. The thermal decomposition studies on sugar cane bagasse demonstrated similar results.42,43 Flattening of tail beyond 500 °C was observed in both the perennial grass biomass which indicates removal of lignin after UA-NaOH pretreatment.44 Hence with TGA analysis of native and pretreated biomass, substantial alteration in the lignocellulosic structure is observed, which is responsible for the improvement in enzymatic hydrolysis as depicted in HPLC results. Further, the absence of byproducts of lignin in the enzyme saccharified biomass of DG and HNG indicates the potential of the substrate and enzyme combination for further bioethanol production. The differential scanning curves (DSC) for the untreated, NaOH pretreated, UA-NaOH-assisted pretreated and enzymehydrolyzed DG and HNG are as shown in Figure 3c and d respectively. The DSC curves can be broadly divided into three

Figure 2. (a) FTIR images of untreated DG, UA-NaOH pretreated− ultrasonication assisted NaOH DG, MDG-Palkonol MBW-hydrolyzed DG and CXDG-Celluclast 1.5L + xylanase hydrolyzed DG. (b) FTIR images of untreated HNG, UA-NaOH pretreated−ultrasonication assisted NaOH HNG, MHNG-Palkonol MBW-hydrolyzed HNG and CXDG-Celluclast 1.5L + xylanase-hydrolyzed HNG.

vibration of crystalline cellulose were prominently observed in pretreated and Palkonol MBW treated DG and HNG. In case of Celluclast 1.5L + xylanase treated DG the breakage of βglycosidic linkages was less evident, while for Celluclast 1.5L + xylanase treated HNG the peak representing the same was absent. This can be attributed to the fact that though the β-1,4 glycosidic bonds themselves are not too difficult to break but because of these hydrogen bonds, cellulose can form very tightly packed crystallites.35 These crystals are sometimes so tight that neither water nor enzyme can penetrate them, suggesting the importance of β-glycosidase in the enzyme mixture for complete hydrolysis of cellulose. Bands from 1731 to 1742 cm−1 are attributed to removal of conjugates of xylan.36 These were not observed in any of the DG samples while only one peak was observed in UA-NaOH pretreated HNG. The absence of this band in the pretreated DG samples confirms the removal of hemicellulose and thereby the efficiency of the pretreatment technique. A gradual decease in bands at 1638, 1575, and 1508 cm−1 corresponding to carboxylic groups of the ferulic and p-coumeric acids, CO stretch in p-substituted aryl ketones and aromatic skeletal vibration in lignin was observed in DG and HNG, with greater decrease in the former one.37,38 Decrease in the peak at 1375 and 1318 cm−1 indicated the 6490


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Table 2. FTIR Spectral Assignments for UDG-Untreated Denannath Grass, UA-NaOH DG-Ultrasonication-Assisted NaOH Pretreatment of Denannath Grass, Palkonol MBW DG-Palkonol MBW Saccharified DG, Celluclast1.5L + Xylanase− Celluclast1.5L + Xylanase Saccharified DG, UHNG-Untreated Hybrid Napier Grass, UA-NaOH HNG-Ultrasonication-Assisted NaOH Pretreatment of Hybrid Napier Grass, Palkonol MBW HNG-Palkonol MBW Saccharified HNG, Celluclast1.5L + Xylanase−Celluclast1.5L + Xylanase Saccharified HNGa DG

band position (cm−1) 3348/3414 2900 2840−2870 1742 1731 1720 1638 1575 1508 1450−1473 1375 1318 1161 1053 1098 900 a

HNG Palkonol MBW HNG

Celluclast 1.5L + xylanase HNG

assignment

UDG

UANaOH DG

UHNG

UANaOH HNG

O−H stretching (related to rupture of cellulose hydrogen bonds) C−H stretching (related to rupture of methyl/ methylene group of cellulose) −OCH3 group of aromatics Carbonyl bonds (related to removal of hemicelluloses) Complex linkages between hemicellulose and lignin, such as ester-linked acetyl, feruloyl and p-coumaroyl groups Carboxylic acids/ester groups Aromatic stretch Aromatic ring stretch (related to lignin removal) Aromatic ring vibration (related to lignin removal) C−H deformation of the methoxyl group C−H deformation in cellulose and hemicellulose C−H vibration in cellulose and C1−O vibration in syringyl ring derivatives C−O−C stretching at the β-glycosidic linkage between the sugar units Stretching of C−O−C−O−C bonds in cellulose C−O vibration of crystalline cellulose Band of cellulose

3510

3380

3414

3422

3480

3432

3380

3444

2948

2932

2913

2924

2959

2925

2955

2935

2953 NOB

2950 NOB

2950 NOB

2857 1749

2884 1744

2831 NOB

2985 NOB

1730

1739

1756

1734

1766

1740

1751

1730

1723 1638 1572 1508

NOB 1623 1568 1502

NOB 1621 NOB 1502

NOB 1622 NOB 1501

1725 1638 1578 1510

1723 1631 1562 1508

1721 1630 NOB 1506

1723 1628 1562 1506

NOB 1382 NOB

1473 1373 1315

1467 NOB 1315

1469 1370 1314

NOB 1385 NOB

1466 1372 1316

1418 1374 1315

14 1375 1316

1171

1161

1163

1159

1174

1168

1164

NOB

NOB NOB NOB

1053 1095 925

1063 1093 910

1072 1081 903

1068 NOB NOB

1055 1088 901

1062 1086 912

1068 1083 926

2860 1755

b

Palkonol MBW DG

Celluclast 1.5L + xylanase DG

Band position assignments acquired from Lima et al.30 and Singh et al.45 bNOB: Not observed.

regions. The peaks from 245−290 °C can be attributed to hemicelluloses zone while the peaks from 290−350 and 350− 500 °C can be attributed to zone of cellulose and lignin, respectively.45 In Figure 3c and d peaks in between 400 and 450 °C is observed for untreated DG and only NaOH-treated DG. A sharp peak in this region is also observed for Celluclast 1.5L + xylanase-treated DG, showing the presence of some lignin linkages in the biomass. But, clear endothermic peaks in the region of 290−350 °C were observed in Celluclast 1.5L +xylanase and Palkonol MBW treated DG with higher peak intensity in the later. This pattern is observed when the breakage of the glucosidic bonds, with formation of laevoglucose has taken place.46 The breakage of the glucosidic bonds can also be explained by the fact that the thermal degradation reaction starts in the amorphous domain of the cellulosic materials, in a quick devolatization reaction which eventually leads to very little solid residue.47 The findings suggest that partial elimination of lignin can also help in good enzymatic digestibility of cellulose. This was well demonstrated by Lu et al. where liquid hot water (LHW) pretreatment was seen to effectively improve the enzymatic hydrolysis of reed canary grass using cellulase after pretreatment, while only partial lignin in biomass was dissolved.48 In Figure 3d the lignin peaks were observed for untreated, NaOH treated, as well as UA-NaOH substrates. This indicates that the pretreatment was not much efficient in case of HNG. The cellulose decomposing peaks, though were present in Celluclast 1.5L + xylanase and Palkonol MBW treated HNG, but the pattern indicates incomplete decomposition of the same. Further, unlike DG,

better decomposition was observed in Celluclast 1.5L + xylanase treated HNG. Solid-State NMR Analysis. Solid-state NMR of the solid fractions of the untreated, UA-NaOH pretreated, Palkonol MBW, and Celluclast 1.5L + xylanase saccharified samples. The chemical shift assignments are based on the comparison of the 13 C NMR spectra from the switch grass and sugar cane bagasse.30,49 The chemical shifts of lignin, cellulose, and hemicelluloses are summed up in Table 3. Accordingly the presented table data is divided and presented in three parts, that is, lignin, hemicellulose, and cellulose in this section. Lignin. Lignin-related peaks showed variable tendencies in both DG and HNG. The lignin signals in DG and HNG were either related to aliphatic or aromatic components of the lignin. In untreated DG, peaks at 21.6 and 54.6 ppm corresponding to acetyl groups and oxygenated Cα, Cβ, and Cγ carbons of the phenyl propane in lignin respectively were observed. For untreated HNG, lignin aliphatic regions at 22.2, 41.4, 53.7, 57.3, and 82.9 (ppm) corresponding to acetyl group, minor contributions of phenyl propane lignin structure, aryl methoxyl carbons, and guaiacyl units of lignin, respectively, were observed. Further, in UA-NaOH and enzyme-saccharified (Palkonol MBW and Celluclast 1.5L + xylanase) biomass of DG and HNG, the aliphatic lignin components were mostly absent, indicating the demolition of the same. Decrease in aryl methoxyl carbon of lignin has been observed to decrease the inhibition of cellulase interaction with cellulose.50 The peaks corresponding to phenyl-propane lignin structure units were observed in both UA-NaOH treated DG and HNG grass 6491


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Figure 3. (a, b) TGA graphs representing UDG and UNG, (pink line) untreated denanath grass and hybrid napier grass, NaOH DG, and NG, (green line) NaOH pretreated DG and NG, NaOH + sonicated DG, and NG, (deep blue line) ultrasonication-assisted NaOH pretreated denanath grass and hybrid napier grass, M DG and M NG, (black line) Palkonol MBW hydrolyzed denanath grass and hybrid napier grass, CX DG and NG, and (light blue line) Celluclast1.5L + xylanase enzyme hydrolyzed denanath grass and hybrid napier grass. (c, d) Heat flow graphs representing UDG and UNG, (black line) untreated denanath grass and hybrid napier grass, NaOH DG and NG, (deep blue line) NaOH-pretreated DG and NG, NaOH + sonicated DG and NG, (red line) ultrasonication-assisted NaOH pretreated denanath grass and hybrid napier grass, M DG and NG, (green line) palkonol MBW enzyme hydrolyzed denanath grass and hybrid napier grass, CX DG and NG, and (light blue line) Celluclast1.5L + xylanase enzyme hydrolyzed denanath grass and hybrid napier grass.

phobicity of the cell wall leading to decreased digestibility by enzymes.51 The decrease in the reducing sugar observed in the enzyme hydrolyzed HNG might be due to the presence of

biomass. The peak at 46.5 ppm was also observed in Celluclast 1.5L + xylanase saccharified HNG. The presence of phenyl propane units, have been reported to increase the hydro6492


6493

lignin aromatic lignin aromatic lignin aromatic lignin and hemicellulose lignin aromatic lignin aromatic

saccharide saccharide lignin aromatic lignin aromatic lignin aromatic lignin aromatic lignin aromatic

lignin aliphatic lignin and polysaccharides lignin aliphatic lignin aliphatic lignin aliphatic saccharide lignin and saccharide saccharide lignin aliphatic saccharide and Lignin saccharide saccharide saccharide saccharide saccharide saccharide

acetyl groups of in hemicelluloses and lignin

large category

C3 and C5 aromatic carbons of syringyl in lignin carboxyl groups in lignin ester-linked fatty/hydroxyl acids esters and carboxylic acids and carbonyl groups in lignin and carboxyl carbons in hemicellulose carboxyl groups of lignin carboxyl groups of lignin

173.1

154.9 168.8

129.7

105.6

100.0

98.7

89.3

84.2

171.3

146.1 147.5

104.7

98.1

88.2

83.2

64.9 72.7 74.7

65.2 75.3 82.9

54.6

21.6

UDG

53.7 57.3

25.91 30.8 33.25 41.4

NK* −CH2-(C5-CH2-C5) aliphatic lignin carbons not bound to oxygen peaks typical of polysaccharides, while the side-chain groups (oxygenated Cα, Cβ, and Cγ carbons) of the phenyl-propane lignin structure units also provide a minor contribution methyl carbons in methoxy groups of lignin aryl methoxyl carbons in lignin methoxy groups in lignin C6 carbon atoms in cellulose and C5 carbon atoms of hemicellulose C2,C3 and C5 atoms in cellulose and carbons of lignin C2,C3 and C5 atoms in cellulose and hemicelluloses guaiacyl unit of lignin C4 carbon hemicelluloses and carbons of lignin C4 carbon of amorphous cellulose C4 carbon of noncrystalline cellulose C4 carbon of crystalline cellulose NK* NK* NK* NK* C1 carbon of hemicelluloses C1 carbon of cellulose aromatic carbons of lignin syringyl units C2 of aromatic carbons guaiacyl in lignin quartarnary aromatic carbon atoms in lignin unsubstituted aromatic carbon atoms in lignin C3 and C5 aromatic carbons of syringyl andC1 and C4 aromatic carbons of guaiacyl in lignin

UHNG 22.2

small category

CH3 in acetyl group

annotation

179.7

105.4

89.1 92.2

83.8

65.2 72.9 75.4

46.5,48.0

UA-NaOH NG

179.7

125.7 128.5

104.7

97.4

88.9

83.5

64.9 72.7 75.2

55.0

46.0,47.7

UA-NaOH DG

179.8

105.3

89.0

84.5

60.0 65.1 72.9 75.3 82.9

46.5

Celluclast1.5L + xylanase HNG

104.7 105.2

99.9

97.0

89.0

83.5

64.9 72.8 75.1

Celluclast1.5L + xylanase DG

CPMASS NMR band locations

105.2

88.5

65.0 72.9 75.2

Palkonol MBW NG

84.0 88.5

65.0 72.9 75.2

Palkonol MBW DG

Table 3. Assignments of NMR Lines from 22.5 to 183.8 for UHNG-Untreated Hybrid Napier Grass, UHDG-Untreated Denannath Grass, UA-NaOH HNG-UltrasonicationAssisted NaOH-Pretreated Hybrid Napier Grass, UA-NaOH DG-Ultrasonication-Assisted NaOH-Pretreated Denannath Grass, Palkonol MBW HNG−Palkonol MBW Saccharified Hybrid Napier Grass, Palkonol MBW DG−Palkonol MBW Hydrolyzed Denannath Grass, Celluclast 1.5L + Xylanase HNG−Celluclast 1.5L + Xylanase Hydrolyzed Hybrid Napier Grass, Celluclast 1.5L + Xylanase Hydrolyzed DG−Celluclast1.5L + Xylanase Saccharified Denannath Grass

ACS Sustainable Chemistry & Engineering Research Article


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assigned to xylan units were observed in untreated, UA-NaOH treated and Celluclast 1.5L + xylanase treated DG and HNG. For Palkonol MBW saccharified DG and HNG no such peaks were observed. The peak at 100 ppm attributed to the C1 carbon of hemicellulose was observed only for untreated HNG biomass. Although, the presence of hemicellulose has been evidenced to reduce the mean pore size of the substrate thereby reducing the accessibility of hydrolytic enzymes to cellulose, the chemical shifts observed in the UA-NaOH treated grass biomass hint the conformational changes in xylan units of the cell wall. This breakage of intermonomer linkages needs further assessment to evaluate the exact effect of the pretreatment on the xylan units of two Pennisetum varieties. Overall it can be inferred that though the pretreatment was not much effective in removing most of the hemicelluloses from both the grass varieties, but alterations in the topography of the xylan backbone have been observed. Further, the absence of the hemicellulose related peaks in the Palkonol MBW saccharified DG and HNG indicates the efficiency of the enzyme for better digestibility of pentose sugars. Cellulose. The C2, C3, C5, and C6 carbon atoms in cellulose generally have peak intensities corresponding to 64.3−65.2 ppm. For untreated, UA-NaOH treated and Celluclast 1.5L+ xylanase treated DG signals were observed at 64.9 ppm. In case of HNG, peaks corresponding to 65.2 ppm were observed in untreated and UA-NaOH treated HNG, while strong peak at 65.1 ppm was obtained for Celluclast 1.5L + xylanase treated HNG. For Palkonol MBW saccharified DG and HNG similar peak intensity at 65.0 ppm was observed. Peaks corresponding to 84.2−89.0 are attributed to C4 carbon of crystalline and noncrystalline cellulose regions.61Nevertheless, the same region is also contributed by signals from lignin and hemicellulose, but the latter one not contributing significantly in the same spectral regions.62 Further a subtraction approach reported by Bernardinelli and his co-workers can be applied to know the exact crystallinity of the biomass.62 It was observed that UANaOH treated and Palkonol MBW saccharified DG had strong signals at 88.9 and 88.5 (ppm) respectively. Peaks at 89.3, 89.1, and 89.0 (ppm) were observed in untreated, UA-NaOH treated and Celluclast 1.5L+ xylanase treated HNG, while peak corresponding to 88.5 ppm was detected for Palkonol MBW saccharified HNG. Bernardinelli et al. reported that for improving the accessibility of cellulase hydrolyzing enzymes to cellulose the crystallinity of cellulose acts as secondary aspect, the primary aspect being lignin/hemicellulose content and distribution followed by the porosity of the biomass.62 Strong signals were observed at 97.4 and 97.0 ppm in UANaOH pretreated and Palkonol MBW treated DG samples indicating the presence of saccharides. These signals were not observed in untreated DG samples. The results of higher reducing sugar content in Palkonol MBW saccharified DG biomass as mentioned earlier is in agreement with the obtained signals in CP-MAS studies. The intense peak at 104.7 for DG, which is attributed to C1 carbon of cellulose was not altered in any of the DG samples except for Palkonol MBW hydrolyzed DG, showing that the cellulose is more recalcitrant structure than hemicellulose.63 It has been reported that ultrasonication assisted chemical pretreatment largely effects the structure of hemicellulose and lignin, with no structural changes observed over cellulose.64 For example, Velmurugan et al. after sonoassisted alkaline pretreatment in sugar cane bagasse obtained 21% hemicellulose and 75.4% lignin in bagasse, while only 0.8% cellulose was removed.12 However, the same attribution for

phenyl propane units. Prominent peak at 82.9 corresponding to the guaiacyl (G) unit was detected in untreated and UA-NaOH pretreated HNG but was absent in the enzyme hydrolyzed HNG biomass. It is reported that presence of G lignin in diverse types of lignocellulosic biomass (corncob, aspen, pine, kenaf, Arabidopsis, etc.) had a higher adsorption capacity on enzymes than syringyl (S) lignin.22,52 Peaks corresponding to aromatic components of lignin were observed at 146.1, 147.5, and 171.3 (ppm) for untreated DG, while for UA-NaOH treated DG peaks at 125.7, 128.5, 179.7, and 183.8 (ppm) was obtained. These results indicate that syringyl lignin components which was present in the untreated DG biomass was absent in the subsequent pretreated and enzyme hydrolyzed DG biomasses. Ramos et al. reported that the presence of syringyl lignin facilitates less absorption of enzymes compared to guaiacyl lignin.53 Further, very low intensity peaks were observed for ferulate and p-hydroxyphenyl only in UA-NaOH treated DG samples. Ferulates that form a major part in ester cross-linking of lignin with polysaccharides, such as glucuronoarabinoxylan (GAX) in the cell wall of grasses, are considered to limit the digestibility of polysaccharide in grass biomass.54 The absence of the hydroxyphenyl peak in untreated DG and their presence in the pretreated DG can be attributed to the oxidative cleavage of the side chain of phenyl propane derivatives, which leads to formation of hydroxyphenolics.55,56 However, the presence of p-hydroxyphenyl imparts unnecessary binding of the cellulase enzyme onto it.22 Rahikainen et al. found that steam exploded pretreated wheat straw which had higher contents of phenolic hydroxyphenyls showed higher affinity for cellulases on steam explosion pretreated biomass.57 In case of untreated HNG, aromatic components of lignin were observed at 105.6, 129.7, 154.9, 168.8, 173.1(ppm), and at 179.7 ppm for UA-NaOH pretreated HNG. It was observed that P-coumarate, aromatic carbons of syringyl, acetyl, and carbonyl groups of lignin were absent in UA-NaOH treated HNG. Li et al. reported that the presence of P-coumarate and carbonyl groups of lignin have a negative effect on glucan digestibility in grasses.58 For Palkonol MBW saccharified DG and HNG no aromatic lignin components were observed. For Celluclast 1.5L + xylanase saccharified DG peaks at 179.3 and 183.8 were observed while for Celluclast 1.5L + xylanase saccharified HNG, peaks at 175.7 and 179.8 ppm were obtained. The peaks are attributed to the carboxyl groups in DG and acetyl and carboxyl groups in HNG. This occurs as most of the cellulose remains as oligomeric compounds (containing carbonyl or carboxyl groups) rather than being converted into monomeric units. Similar findings using Celluclast 1.5L for enzymatic saccharification of cellulose were observed by Moilanen et al., who reported incomplete monomerization of cellulose into D-glucose is in complete agreement with the present study.59 The results are also in agreement with the HPLC results (Table 2) with higher glucose and xylose yields observed in Palkonol MBW saccharified DG and HNG as compared to Celluclast 1.5L + xylanase saccharified DG and HNG. Hemicellulose. Peak at 83.2 ppm was observed for untreated DG and 83.5 ppm was observed for UA-NaOH treated and Celluclast 1.5L + xylanase saccharified DG. The peak is attributed to xylose units of hemicellulose and to carbon units of lignin and hence a more intense study can exemplify a better confirmation of the same.60 For HNG, the peak at 83.8 ppm was observed only in UA-NaOH pretreated HNG. Low intensity peaks from 92.2 to 99.9 (ppm), that are generally 6494



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untreated HNG at 105.6 ppm was slightly altered at 105.4, 105.3, and 105.1 (ppm) for UA-NaOH treated, Celluclast 1.5L + xylanase saccharified, and Palkonol MBW saccharified HNG samples, respectively. Other Signal. Remarkably, most of the aromatic lignin components along with some important aliphatic components of lignin like ferulate have been degraded, in UA-NaOH treated and Palkonol MBW enzyme saccharified samples. Moreover, the results were prominent for DG samples. Ultrasonication assisted alkali pretreatments have been seen to open up he aromatic rings in the lignin at the α-position by cleavage of the C−C bonds leading to the formation of macro radicals which stimulate the depolymerization of the lignocellulosic material.65,66

CONCLUSION Enzymatic hydrolysis of lignocellulosic biomass requires effective pretreatment strategy depending upon the type of biomass used. The removal of most of the aromatic components, particularly the syringyl (S) groups of lignin along with partial elimination of hemicellulose exhibits UANaOH pretreatment to be a useful pretreatment technique for targeted delignification of grass biomass. This can be attributed to the cavitation process generated during ultrasonication pretreatment. Although UA-NaOH pretreatment was not effective for disintegration of ferulate but further modifications in parameters of optimization can be employed for the same. The enzymatic hydrolysis by Palkonol MBW, a newly reported enzyme in the present study was more effective than the conventional combination of Celluclast 1.5L and xylanase. Based on the results obtained from FT-IR, TGA, DSC, and NMR studies it was evident that the structural changes that occurs after pretreatment and saccharification plays an important role in determining the production of reducing sugar. Thus, consideration of this structural information’s can be beneficial for the delignification and enzymatic hydrolysis studies which would be helpful in generation of high bioethanol yields. ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssuschemeng.7b00596. The details of the data used to prepare Table 3 (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Tel./fax:+91-9437306696. *E-mail: [email protected]. ORCID

Hrudayanath Thatoi: 0000-0003-1345-9542 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Authors are grateful to the authorities of college of engineering and technology, BPUT, Bhubaneswar, for providing necessary laboratory facilities to carry out this experiment and to Dr. Anil Kumar, Senior scientist-DRWA, Bhubaneswar, for kindly providing the two grass varieties. 6495


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Structural and Functional Characterization of Two Pennisetum sp

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