Effect of Mixed Acid Catalysis on Pretreatment and Enzymatic

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Effect of mixed acid catalysis on pretreatment and enzymatic digestibility of sugarcane bagasse Siddhartha Pal, Shereena Joy, Pramod S. Kumbhar, Kalpana D Trimukhe, Anjani J. Varma, and Sasisanker Padmanabhan Energy Fuels, Just Accepted Manuscript • DOI: 10.1021/acs.energyfuels.6b01011 • Publication Date (Web): 09 Aug 2016 Downloaded from http://pubs.acs.org on August 12, 2016

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Effect of mixed acid catalysis on pretreatment and enzymatic digestibility of sugarcane bagasse

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Siddhartha Pal, †, # Shereena Joy,† Pramod Kumbhar, †, # K.D.Trimukhe,‡ A.J.Varma, ‡, #, §,* and Sasisanker Padmanabhan, †,*

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Praj Matrix –The Innovation Center, Urawade, 412115, Pune, India

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Polymer Science & Engineering Division, CSIR-National Chemical Laboratory, 411008, Pune, India

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Central University of Haryana, Post-Pali, Dist. Mahendergarh, 123029, Haryana, India

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Department of Technology, Savitribai Phule Pune University, 411007, Pune, India

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ACS Paragon Plus Environment

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Abstract. Aqueous pretreatment using homogenous acid catalyst is considered as a low-cost technology in the production of lignocellulosic bioethanol. To establish the synergism of mixed acids, pilot-level aqueous pretreatments of bagasse covering a wide range of combined severity (CS) were carried out. To investigate the effect of application of mixture of acids on xylose hydrolysis as well as glucose hydrolysis via pretreatment and enzymatic hydrolysis, the following three combinations of acids were explored: (1) oxalic acid + sulfuric acid (organic + mineral acid), (2) phosphoric + sulfuric acid (mineral acids), and (3) ferric chloride + sulfuric acid (Lewis acid with a mineral acid). Of the pretreatments evaluated, the synergism was most pronounced for the combination of sulfuric and phosphoric acid, which resulted in more than 90% conversion of hemicellulose to xylose and 70% conversion of cellulose to glucose through enzymatic hydrolysis. Fourier transform infrared (FTIR) studies of pretreated samples showed higher syringyl/guaiacyl (S/G) ratio for sulfuric and phosphoric acid combination pretreatment, leading to higher enzymatic conversion. FTIR and dynamic light scattering (DLS) experiments conducted on pretreated sugarcane bagasse provided useful correlation with regard to the pretreatment type, particle size and enzymatic hydrolysis.

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Keywords: Pilot-scale pretreatment, dilute mixed acid treatment, enzymatic hydrolysis, acid catalysis of biomass.

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1. Introduction

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The depletion of fossil-fuel resources combined with the carbon emissions has stimulated worldwide interest in the utilization of sustainable energy resources. Sugarcane bagasse (SB), a fiber-based agricultural residue left after the juice has been extracted from the sugarcane, represents one of the abundant and low-cost renewable feedstock for production of biofuels and biochemicals.1-5 However, the effective utilization of SB for the production of ethanol and other platform chemicals requires a pretreatment unit operation which involves combination of chemical, physical, thermal and mechanical methods. This is because an effective pretreatment can overcome the recalcitrant nature of biomass structure and provide amenable substrate that can be hydrolyzed through biochemical routes to yield fermentable sugars for further fermentation to ethanol or biochemicals. In the last few decades, various pretreatment technologies have been investigated for the production of bioethanol and biochemicals from SB.6-13 Several of the reported technologies show good yield of C6 sugars through biological conversion. In addition, some of the pretreatment technologies reported good removal of hemicellulose and lignin.8,9 Of the various pretreatment methods, dilute acid (DA) pretreatment, which typically employs sulfuric acid as catalyst, show good promise towards commercialization.6,12,13 This is because DA pretreatment has important features such process simplicity, shorter residence time and low-cost. DA treatment is widely employed for production of monomeric sugars (pentose and hexose) from lignocellulose. The use of DA also provides co-utilization of pentose sugars for the full optimization of biorefinery. In addition to hydrolysis of hemicellulose, DA pretreatment disrupts the ultra-structure of biomass to yield a solid portion that contains cellulose and the acid insoluble lignin. Cellulose in the pretreated solid is further hydrolyzed to C6 (glucose) sugars using cellulase enzymes and the lignin remaining in the solid is further available either for the production of value-add chemicals or for the power generation in the boilers.11 Because DA treatment is very promising for bioethanol production, evidently, there are plenty of literature reports on the use of single dilute acid for the conversion of hemicelluloses to its monomeric sugar components. For the pretreatment of lignocellulosic biomass (LC), most of the studies have utilized mineral acids such as hydrochloric, nitric, sulfuric and phosphoric acid.6,13-18 Of the above mineral acids, sulfuric acid has been used predominantly due to its low cost and availability.13 In addition to minerals acid, organic acids like maleic and oxalic acids were also employed.19-22 Although there is a good amount of technical maturity with regard to the use of single acid for the treatment of SB, there are not many reports on the use of mixed acid concept which synergistically combines the complementary acid hydrolysis. Especially, there are no studies on the use of mixed acids for the treatment of sugarcane bagasse at pilot-scale level. Most of the DA pretreatment studies have tested various concentrations of single acid (sulfuric), different retention time and temperature. All factors can have significant influence on the yield of xylose, by products formation mainly phenolics which affects the enzymatic hydrolysis efficiency. 23-25 and the material of construction used in the pretreatment. Hence, it would be advantageous explore the concept of binary acid and avoid inherent drawbacks that basically come through the

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use of single acid. Pretreatment process that utilizes the benefits provided by individual acid by combining them with the other acids may negate the disadvantages that single acid use provide, such as partial replacement of sulfuric acid with other acids can provide benefits in terms of disposal of gypsum and material of construction. In terms of reaction severity, the binary acid pretreatment can also provide wider severity range required for the pretreatment, possibly offering reduction in the combined severity as compared to the use single acids alone.20 The reactions conditions are chosen in such a way that the a wide range of combined severity factors are studied . The study of mixing of type of acids is intriguing as they can reveal the impact of the different types of acids on the pretreatment and enzymatic hydrolysis following the pretreatment. Further, how different types of mixed acid pretreatment impact the structure, morphology and chemistry of biomass is an interesting study.

The mixed acids chosen for this study were a combination of a mineral acid with a mineral acid (sulfuric and phosphoric acid or SA+PA), mineral acid with a biomimetic organic acid (sulfuric and oxalic acid or SA+OA) and finally a combination of mineral acid with a Lewis acid (sulfuric and ferric chloride or SA+FeCl3). To study the impact on glucose/xylose production, this study also investigated the role of individual acid treatment. One mineral acid that was common to all mixed acid strategy was sulfuric acid due to its high acidity, low cost and common use in most of the DA pretreatment studies. 11-13Oxalic acid is a strong organic acid and due to its dicarboxylic properties it is used for xylan hydrolysis. Oxalic acid is also shown to be biocompatible acid. 19-21Ferric chloride is an inexpensive Lewis acid catalyst and it selectively attacks on cell wall polymer networks, including C-O-C and C-H bonds in cellulose. 22, 26 The Lewis acidity in ferric chloride emerges from the ability of ferric ion to attract electron pairs from neighboring water. With regard to phosphoric acid, it has been used for partial decrystallization of cellulose which will increase the rate and efficiency of enzyme hydrolysis. The use of H3PO4 is that after neutralization of the slurry with NaOH, a salt of sodium phosphate is formed which helps the fermenting strain for effectively producing bioethanol with reduced nutrient consumption.16, 17 To understand the effect of mixed acids pretreatment and its effect on enzymatic hydrolysis, this work employed FTIR, DLS experiments on the pretreated bagasse and untreated bagasse. These studies enable better structural and chemical understanding of various pretreated substrates before and after pretreatment and the impact of the pretreatment on the enzymatic hydrolysis. The use of FTIR studies offers chemical changes in SB, in particular, syringyl/guaiacyl ratios of lignin, which can have influence on the hydrolytic behavior of enzymes.

2. Experimental Methods and Analytical Procedures 2.1. Materials SB was provided by Om Sai Chemicals, Maharashtra, India. It had ~30-35% moisture and this SB was stored under ambient conditions and was used as such without any washing. Before feeding into the pilot-scale reactor, bagasse was shredded in a hammer mill in the range of 15-25 mm.

2.2. Compositional Analysis Methods The total solids, carbohydrate content and acid insoluble lignin content of untreated bagasse and the solid fraction remaining after various pretreatments was determined using National Renewable Energy Laboratory Analytical (NREL) procedure. 27-29 The composition of pretreated slurry and enzymatic hydrolysis slurry was determined by measuring glucose, xylose, arabinose, acetic acid, HMF and furfural using Bio-Rad Aminex HPX-87H ion exchange column with a flow rate of 0.6mL/min, 55 C and a mobile phase of 0.005 M Sulfuric acid . The oligomer sugar analysis was based on NREL analytical procedure.29 The filtered samples of hydrolyzate was brought to a final sulfuric acid concentration of 4%w/w, autoclaved at 121 C for 1 h, and then centrifuged for HPLC determination of monomers hydrolyzed from the oligomer. The total oligomeric content was determined as the difference between the amount of monomers obtained after post hydrolysis after correcting the losses in post hydrolysis and measuring the amount before hydrolysis.

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2.3 Pretreatment Methods 2.3.1. Continuous pretreatment Pretreatments were done using one tonne per day (TPD) continuous pilot-scale horizontal screw-type reactor. This reactor set up is similar to the one described in the work of Schell et al.13 The total solid handling capacity of this reactor is up to 22% (w/w). SB was first chopped down to 15-25 mm and was fed to the pretreatment reactor using a belt conveyor. The pretreatment reactor is a part of a reactor feeder blow tank system operating at a feed rate of approximately 30 kg on dry mass basis/h. The system is provided with constant supplies of acid, steam pressure, temperature. Both the acids were mixed at room temperature in an acid tank and diluted to 5-6 % (w/w) solution using process water .This dilute acid solution was then pumped with a metering type of dosing pump under operating steam pressure. The pretreatment reactor can be operated at a temperature range of 140-200 °C, pressure of 4-20 bar and retention time between 15 to 30 min. The whole pretreatment reactor is made of SS 316 L steel. Different dilute mixed acid concentrations (shown in Tables 1-4) were dosed on dry biomass basis and at a temperature range of 150-170° C for a holding time of 15 min. Due to the addition of steam and acid the final solids concentration after pretreatment was 20-22%w/w. The slurry is flashed under atmospheric pressure and the vapours are recovered as a condensate. The pretreated slurry was filtered in a filter press(Andritz , Model PRFP 118 ) with 11 plates (470 × 470 mm) using a hydraulic cylinder serving as a pressing of filter plates provides a filtration pressure of 8-9 bar (g). A filter area of 3 m2 and a cake thickness of 30 mm were achieved. Post filtration, a solid phase (wet cake) and aqueous phase (xylose-rich stream) were obtained.

2.3.2 Pretreatment procedure The pretreatment trials were performed after attaining steady state operations. The feed rate of bagasse, acid flow and temperature were maintained for 15-20 min post which all the pretreatment readings were taken. A total of 29 experiments were conducted at pilot scale. The pretreatment conditions were varied from temperature of 150-170 °C and acid dose between 2 -3 % (w/w) on dry biomass basis for single acids and 1-2.5 % (w/w) on dry basis for mixed acid dose. All the pretreatment experiments were conducted at a total solids loading of 20%w/w. Table 1-4 shows the detailed compositional analysis (sugars and by products) and pH of the pretreated slurry.

2.4. Enzymatic Hydrolysis Enzyme hydrolysis is carried out using commercial Novozymes Cellic® CTec3 enzyme in a 10L reactor with anchor impeller and at 14-15% total solids and 13-14% total insoluble solids. The enzyme dose used was 30 mg/g glucan (8 FPU/g glucan) .The enzyme activities were determined according to the IUPAC method described by Ghose.30.The specific activities of this commercial enzyme are not provided here as it is the proprietary information of the manufacturer. The samples were incubated at 50oC for 120 h and the pH was maintained in the range of 5.0-5.2. Samples were collected every 24 h for sugar and by-products analysis using HPLC, as mentioned in analysis method section. Enzymatic hydrolysis efficiency was calculated by the ratio of glucose yield obtained after 120 h to the theoretical possible glucose yield after pretreatment in the solid fraction.

2.5 Fourier Transform Infrared (FTIR) A Perkin Elmer Spectrum 1 FTIR was used. The samples were used in the form of KBR discs, which were prepared by grinding 1 mg sample/150 mg pre-dried KBR. The spectra were recorded in the range of 450–4000 cm-1.

2.6 Combined Severity Factor (CSF) The CSF is defined as

. in which t and T are residence time (min) and temperature (°C), respectively.13

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2.7 Dynamic light scattering Dynamic light scattering (DLS) studies were performed with instrumentation from Brookhaven Instruments Corporation using 90 Plus Particle Sizing Software Version (Version 3.94) was utilized. Samples (1 % w/w) were dispersed in water by sonication, and the uppermost 1 mL of the dispersion was taken and diluted 100 times for the DLS studies. DLS was performed on all pretreated slurries obtained from different types of pretreatment mentioned earlier

3. Results and Discussion 3.1 Effect of different mixed acid pretreatments on xylose yield and inhibitors The present study investigated mixed acid pretreatments at various combined severity factors (CSF) and its effect on xylose hydrolysis and cellulose hydrolysis with the help of enzymes. The monosaccharide concentrations and pH of the hydrolyzate by acid pretreatment of bagasse are shown in Table 1. The composition of SB on dry basis was found to consist of: cellulose (40.3 ± 2%), xylan (21.3 ± 0.7%), arabinan (2.2% ± 0.1), acid insoluble lignin (19.3% ± 0.8), acid soluble lignin (3.5± 0.2%), ash (2.6 ± 0.15%), proteins (2.7 ± 0.1%), acetate (2.9 ± 0.1%), water extractives (3.50 ± 0.2%) and ethanol extractives (1.5± 0.2%).

3.1.1 SA+OA Pretreatment Pretreatment involving combination of sulfuric plus oxalic acid led to physical and chemical changes of the material, including the opening of the fibre bundles in bagasse. Table 1 provides the slurry composition after pretreatment at various conditions for SA, OA and SA +OA treatment. Dilute SA (Table 1,run no.1) pretreated slurry provided ~83 % xylose monomer yield, 10% higher than dilute OA (3 wt % on dry basis) alone treatment (Table 1, run no.2). This is due to higher pKa1 of H2SO4 (-3) compared to that of oxalic acid (1.4). 19, 20, 31,32The use of higher doses of oxalic acid may not be economical due to its higher cost as compared to cheaper mineral acids. Although better yields of C5 sugars are obtained through sulfuric acid treatment, the yield of byproducts like acetic acid (4200 ppm), HMF (200ppm) and furfural (800 ppm) are much higher in this pretreatment (Table1) as compared to OA pretreatment. The pH of SA treated slurry is 0.9, the lowest among all the pretreatments reported in this work. This low pH may not be beneficial for reactors as it can cause corrosion, resulting in reduction of the life span of reactors and increase of capital investment. Therefore, in order to reduce the degradation effects and the possibly the cost of the pretreatment process, dilute mixed acid pretreatment was conducted at different pretreatment conditions. Table 2 provides the results of mixed dilute acid treatment involving sulfuric acid and oxalic acid. The yields of monomeric xylose is maximum (83%) for run no. 9 with inhibitors (0.36%acetic acid, 0.01% HMF and 0.01% furfural) at a pH of 1.2. Xylose yield drops beyond 160°C and there is an eight fold increase in furfural (0.08%) at higher temperature (Table 2, run no. 11) as compared to lower temperatures (0.01%). However, increase in temperature favors xylose hydrolysis for the lower mixed acid doses as shown in Table 2. For example, xylose conversion at SA (1%) and OA (1%), 160 °C provided 51.2% as compared to 54.1% at 170 °C, other conditions remaining same. No such correlation is observed for higher mixed acid dosages (run no 9, 11). As shown in Table 2 lower inhibitors viz. acetic acid, HMF and furfural are generated at a lesser dose of acids as compared to higher doses. At lower dosages of acids, higher pH (1.5-2.5) is obtained which is favorable for the pretreatment reactor. However, at lower doses of acids lesser xylose yields are obtained. The results shows that the monomeric xylose yield increases for SA+OA till a CSF in the range of (1.7-1.8) beyond which the xylose yield starts decreasing presumably due to the degradation of pentose sugars at higher severities. This shows that by use of SA+OA we can lower down the combined severity and still achieve similar xylose conversion efficiencies as compared to single acids (SA here). The inhibitors concentration is also minimized in mixed acids treatment as compared to the use of sulfuric acid alone. Lowering of sulfuric acid in the pretreatment is advantageous as this will result in lower gypsum formation.

3.1.2 SA+FeCl3 Pretreatment Dilute FeCl3 pretreatment (1.5%) provided xylose monomeric efficiency of 70.1% (Table 1, run no.3). Lower efficiencies have been observed at higher doses of ferric chloride (>2%w/w) possibly due to degradation of pentose sugars. Similarly, at lower ferric chloride dose ( SA+OA>SA+FeCl3.

3.2 Cellulose hydrolysis in Pretreatment In the acid pretreatment process part of the amorphous cellulose is solubilized and forms glucose in the pretreatment. 31, 6 Acid pretreatment also increases the porosity of the remaining cellulose in the solid phase. Glucose is found in low concentrations in the liquors, which is in agreement with the relatively low degree of hydrolysis observed for cellulose. The hydrolysis of cellulose in terms of glucose is estimated in the liquid hydrolyzate by HPLC. The samples pretreated with SA+PA has higher glucose concentrations in the liquid phase than other pretreatments studied here. With an increase in temperature higher glucose yields are obtained in the pretreatment. As shown in Table 1, the maximum concentration of free glucose (1.02%) is obtained at 170°C for 1.5%SA+1%PA pretreatment.

3.3 Effect of combined acids pretreatment on enzymatic hydrolysis efficiencies The effect of different mixed acid pretreatment methods employed here can have significant influence on the cellulose enzymatic hydrolysis. The results of these effects are discussed in the following section. The enzyme hydrolysis of all the pretreated solid phases were studied with a dose of 30 mg/g glucan Novozymes Cellic® CTec3 enzyme (equivalent to 8 FPU/g glucan) at a total solids of 15% and total insoluble solids of 14%. Higher solid concentration can provide fairly higher concentration of glucose for further fermentation to ethanol. Although there are many studies on the use of higher solid concentrations for the enzymatic hydrolysis, there are no studies for the different mixed acid pretreatment such as the one considered in this work. Hence, from the scale-up perspective, the results of this work are quite useful. Table S1 provides the composition of the pretreated bagasse cake that is used as a feed for enzymatic hydrolysis.

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3.3.1 SA+OA pretreatment The effect of different pretreatment conditions is assessed in terms of glucose yield from enzymatic hydrolysis of the pretreated solids. The two best glucose conversions obtained are 58% (Table 2, run no.9) and 60% (Table 2, run no.11). However; the latter shows decrease in xylose yield due to the degradation of pentose sugar at high temperatures. The enzymatic glucose yields are lesser for lower doses of acids as compared to higher acid doses (Table 2). The lower conversions could be due to the lower monomeric xylose conversion in pretreatment and also could be an effect of xylo oligomers released after the pretreatment. 35, 36 Another reason for lower conversions could be due to the fibrous nature of substrate at lower acid doses which negatively affects the enzyme hydrolysis. With increase in temperature glucose yield increases depending on the pretreatment conditions as shown in Table2 (run no. 5-11). Figure 2 shows cellulose digestibility as a function of CSF. Cellulose digestibility increases as the CSF increases, the highest being achieved at a CSF of 2.03. An increase in cellulose digestibility with CSF is also seen with corn stover dilute acid pretreated substrate. 11

3.3.2 SA+FeCl3 pretreatment Of the different pretreatment conditions tested, the highest glucose conversion is 55.8% (Table 3, run no.20) and 53% (Table 3, run no.16). Lower glucose conversions are achieved in this pretreatment as compared to the SA+OA pretreatment. This could be attributed to the higher amount of degradation products being formed in the pretreatment and also the inhibition of cellulases enzymes in presence of ferric ion. Non cellulosic metal ions like ferrous, ferric and cupric has a negative impact on the enzymatic hydrolysis efficiency. 37 For pretreatment of SB using SA+FeCl3 as catalyst, no clear correlation is observed between pretreatment temperature and enzymatic hydrolysis efficiencies.

3.3.3 SA+PA pretreatment Of the three pretreatment method explored in this study, the highest glucose conversion of 67.1% is achieved in run no. 28 (Table 4). However, the xylose conversion (82%) was lower as compared to run no. 26 (Table 4), presumably due to the degradation of pentose sugar. The latter resulted in highest xylose conversion (91%) and glucose conversion (63%). This is considered to be the optimum condition providing a reasonably good xylose and glucose hydrolysis from pretreatment and enzymatic hydrolysis. With increase in temperature the glucose conversion increases for same pretreatment condition of acid dose. Figure 2 shows the cellulose digestibility as a function of CSF. Cellulose digestibility increases as the CSF increases, the highest being achieved at a CSF of 2.04.

3.3.4 Overall enzymatic hydrolysis trend Overall, the effect of mixed acids on enzymatic hydrolysis follows the order: SA+PA> SA+OA>SA+FeCl3. The enzymatic hydrolysis trend is similar to what is observed for hemicellulose hydrolysis following acid treatment as discussed earlier. The enzymatic hydrolysis efficiency is related to many factors such as nature of substrate, nature of pretreatment and nature of enzymes in addition to other additives like surfactants or proteins like BSA .38 It will be difficult to compare all these factors here as the conditions may vary from one study to another. The enzyme dose chosen for our study is comparable to the state of the art studies on acid pretreated bagasse. The enzymatic hydrolysis efficiency observed here is comparable to that observed in other studies on single acid treatment of bagasse .38-39 Various enzyme doses ranging from 5 50 FPU/g glucan have been reported with acid pretreated bagasse. A 67% enzymatic efficiency was obtained with oxalic acid pretreated bagasse at 20 FPU/g glucan .38 Ramos et al. also reported 67% enzymatic hydrolysis efficiency at 8.25 FPU/g glucan dose for phosphoric acid-impregnated steam-exploded sugarcane bagasse.39 Further increase in the enzyme dose does not justify the incremental efficiency observed and impacts the production cost which makes the process uneconomical .40

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3.4 FTIR analysis and characterization FTIR spectroscopy was carried out to investigate the changes in hemicellulose and cellulose structure before and after pretreatment. FTIR also provides information on structure of lignin (syringyl, guaiacyl ratios) and hydrogen-bonding effect in cellulose. Table 5 shows the FTIR peaks for the following treatments: untreated, SA+OA, (Table 2, run no. 9) SA+FC (Table 3, run no. 20) and SA+PA (Table 4, run no.27) and their assignments, which are based on literature values for sugarcane bagasse. 41-45 FTIR spectra of untreated bagasse show a band at 890 cm–1, which represent β-(1–4) glycosidic linkages of cellulose. The

band at 890 cm-1 is attributed to amorphous cellulose. Compared to the untreated bagasse, absorbance of amorphous cellulose has increased following pretreatment. However, the absorbance is higher for SA+PA treatment and SA+ FC as compared to SA +OA treatment. This data supports the discussion in Section 3.2 where maximum glucose release was observed for SA+PA treatment. Literature reports indicated that values at 1430 cm–1 and 890 cm–1 are sensitive to the crystalline structure of cellulose. 41, 45 The absorbance ratio of these two values (A1430 / A890) is considered to be crystalline index or lateral index order (LOI). This can be used for the presence of cellulose I in the solid material. LOI for untreated bagasse is 3.36, while for SA+OA, SA+FC and SA+PA pretreated bagasse is 2.43, 3.23 and 2.31. The value is lower for SA+PA, which indicates enhanced changes in the cellulose structure. SA+FC show higher index value as compared to SA+OA and SA+PA. This probably indicates that more amorphous cellulose was removed from the SA+ FC pretreatment.

The frequency ranges of 1200–1000 cm-1 can be attributed to contributions of hemicellulose and cellulose having maxima at 1040cm–1 due to C-O stretching mode and 1165 cm–1 due to the asymmetrical stretching C-O-C. The band absorption at 1247 cm–1 arises due to C–O stretching. This absorption region indicates features of hemicellulose as well as that of lignin. .38, 46, 47 The band at 1247 cm-1 shows removal of hemicellulose as compared to untreated SB. The band intensity is lower for SA+FC as compared to the others. From composition analysis, it can be observed that SA+ FC pretreatment yielded maximum solubilization of hemicellulose. Therefore, the intensity was lowered. However, all the hemicellulose solubilized does not get converted to xylose due to the side reactions from this pretreatment. The products so formed could be not identified. Similar side reactions due to addition of FeCl3 to xylo-oligomers have been reported, where unidentified compounds increased with increasing inorganic salt, particularly with ferric chloride. 48 This is also evident from the solid phase analysis which indicated minimum residual xylan (Table S2, 170°C, 1%SA+1%FC). The region of 1400-1460 cm-1 reflects aromatic skeleton C-H plane deformations in lignin. Similarly, 1500-1650 cm–1 is reported to include aromatic skeletal vibrations. Lower absorbance values are observed for SA+FC pretreatment. The intensity around 1500 cm-1 is different for SA+FC pretreatment as compared to the other two mixed pretreatment methods. Therefore, it is possible to suggest that addition of FeCl3 to SA resulted in more lignin changes as compared to the addition of OA or PA. From the compositional analysis also, it can be observed that SA+FC pretreatment yielded slightly higher acid-insoluble lignin (Table S2, 170°C, 1%SA+1%FC). Perhaps, presence of this lignin as well as presence of ferric ions may perhaps be responsible for the lower yield of glucose following SA+FC pretreatment. An interesting feature of FTIR is that it provides useful information on syringol (S) to guaiacol (G) ratio as both S and G are considered as basic aromatic subunits that represent the lignin present in cell- wall. G-type lignin has branched structure whereas S type is linear. 43-45 Moreover G-type lignin can covalently link up to three other units while S-type lignin can link only up to two other units. A higher S/G ratio enhances the enzymatic hydrolysis of pretreated biomass. 47, 49, 50 From the FT-IR spectra the S/G ratio can be obtained from the wavelength 1260 cm-1 and 1330 cm-1. For untreated bagasse, S/G ratio is 0.83, while for the various pretreatments attempted here the S/G ratios are the following: 0.98 for SA+OA, 0.97 for SA+FC and 1.01 for SA+PA. An increasing trend of S/G ratio after pretreatment is correlated with the enzymatic hydrolysis efficiencies. The SA+PA pretreatment have the highest S/G ratio among the pretreatments studied. The SA+ PA pretreatment also provided maximum enzymatic hydrolysis efficiencies.

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3.5 Influence due to particle size The average particle sizes of the three pretreatments studied are estimated and correlated with enzymatic hydrolysis efficiencies. The SA+PA system provided the lowest particle size (1028.2 nm) followed by SA+FC (1061.7 nm) followed by SA+OA (1108.2nm) .It is also known from previous studies that due to lower particle size the enzyme accessibility to the substrate increases and thus enhancing the enzymatic conversions.51 Similar observations were made with liquid hot water pretreated hardwood which shows that as the particle size reduced from 3.0-2.0 mm the enzymatic efficiency enhances by 50%. Small particle size increases the specific surface area available for cellulolytic enzymes .52

4. Conclusion This work, to the best of our knowledge, reports first pilot scale mixed acids pretreatment for bagasse at various severities. Of the three different combinations of mixed acids pretreatment tested, positive effect was observed in terms of xylose and glucose yield for sulfuric acid plus phosphoric acid pretreatment. The order of mixed acid pretreatment and subsequent enzymatic hydrolysis shows following trend: PA+SA> SA+OA>SA+FC. Mixed acid pretreatment involving sulfuric acid and phosphoric acid yielded highest amount of xylose (91%). Sulfuric acid plus phosphoric acid pretreated bagasse also showed improved glucose conversion (~70%) following enzymatic hydrolysis conducted at higher total solid loadings (> 15 %). FTIR study revealed a higher S/G ratio for sulfuric and phosphoric acid combination, providing maximum enzymatic conversion. DLS data indicates lowering of particle size following sulfuric and phosphoric acid pretreatment, indicating better enzymatic digestibility as compared to other combinatorial systems studied here.

ASSOCIATED CONTENT Supporting Information Table S1 – S4 provides extended information on carbohydrate and lignin composition on dry mass basis of SA+OA, SA+ FeCl3, SA +PA pretreatment system. FTIR spectra of untreated SB, S SA+OA, SA+ FeCl3, SA +PA pretreatment system are provided in Figures S1-S4 This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION *Corresponding authors *Fax +91-2066754104. Email: [email protected], *Fax +91-20-25902618. Email: [email protected]; [email protected],

Acknowledgements All experimental samples were generated at M/s Praj Matrix. This latter part of the work was funded by M/s Praj Matrix R&D. DLS and FTIR studies were carried out at CSIR-NCL by KDT. AJV and KDT thank Director CSIR-NCL for providing the instrument facilities.

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. The work presented here constitutes the doctoral research of SP under the supervision of AJV (academic institution) and PK and SSP (Praj Matrix industrial research). SJ provided technical inputs (compositional analysis, HPLC, experimental inputs) while KDT provided FTIR and DLS experiments. SP, AJV, PK and SSP conceptualized the research work, analyzed and interpreted the data. SP, SSP and AJV wrote the manuscript.

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Figure 1. Monomeric xylose conversion as function of combined severity factor for different mixed acid combinations: (A) SA+OA (B) SA+FeCl3 (C) SA+PA. Lines acts as a guide to the eye. Error in efficiency is ±2 %.

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Energy & Fuels

Figure 2. Enzymatic efficiency as a function of combined severity factor for different mixed acid combinations: (A) SA+OA (B) SA+FeCl3 (C) SA+PA. Lines acts as a guide to the eye. Error in efficiency is ±2 %.

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Table1. Sugars, inhibitors product profile after pretreatment, C5 conversion efficiencies and enzymatic efficiencies (C6) for single acids pretreatment a

b

acid dose on dry biomass basis, release of xylose monomeric sugars, * absolute error in composition ±5 %.

Run No.

Temperature

Acid dose a (%w/w)

Glucose

Xylose

(%)*

(%)*

Xylose oligomer

Acetic acid (%)*

HMF

Severity

pH

(%)

Furfural (%)

C5 Hydrolysis b efficiency (%)

C6 Enzymatic efficiency (%)

0.02

0.08

2.01

0.9

83.2

49.2

0.01

0.01

1.9

1.49

73.1

50.2

0.01

0.02

1.6

2.1

74.07

38.1

0.0

0.01

1.8

2.1

32.1

45

(%)*

SA Pretreatment 1

150

2

0.85

4.91

ND

0.42

OA Pretreatment 2

170

3

0.51

4.36

0.15

0.34

Fecl3 pretreatment 3

160

1.5

0.59

4.21

1.13

0.38

PA pretreatment 4

170

2

0.25

1.52

2.15

0.25

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Table2. Sugars, inhibitors product profile after pretreatment, C5 conversion efficiencies and enzymatic efficiencies (C6) SA+OA pretreatment a

b

*

acid dose on dry biomass basis, release of xylose monomeric sugars, absolute error in composition ±5 %.

Run No.

Temperature

Acid dose a( %w/w)

Glucose

Xylose

(%)*

(%)*

Xylose oligomer

Acetic acid (%)*

HMF

Severity

pH

(%)

Furfural (%)


C6 Enzymatic efficiency(%)

(%)*

5

150

0.5+0.5

0.23

2.22

0.94

0.15

0.00

0.00

0.73

1.91

44.1

43

6

160

0.5+0.5

0.22

2.55

1.62

0.21

0.00

0.00

1.06

1.88

48.1

45

7

160

1+0.5

0.65

3.89

0.48

0.30

0.01

0.01

1.49

1.45

59.1

51.7

8

160

1+1

0.47

4.12

0.23

0.36

0.01

0.01

1.61

1.35

65.1

51.2

9

160

1.5+1

0.52

4.58

0.78

0.36

0.01

0.01

1.70

1.25

83.4

58.1

10

170

1+1

0.61

4.21

0.22

0.25

0.01

0.01

1.93

1.30

71.4

54.1

11

170

1.5+1

0.78

4.38

0.20

0.46

0.03

0.08

2.03

1.29

78.2

60.1

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Table3. Sugars, inhibitors product profile after pretreatment, C5 conversion efficiencies and enzymatic efficiencies (C6) for SA+Fecl3 pretreatment a

acid dose on dry biomass basis, b release of xylose monomeric sugars, *absolute error in composition ±5 %.

Run No.

Temperature

Acid dose a( %w/w)

Glucose

Xylose

(%)*

(%)*

Xylose oligomer

Acetic acid (%)*

HMF

Severity

pH

(%)

Furfural (%)



(%)*

12

150

0.5+0.5

0.04

0.72

1.77

0.11

0.00

0.00

0.44

2.2

11.2

40.2

13

160

0.5+0.5

0.04

1.12

1.53

0.13

0.00

0.00

0.82

2.12

22.9

43.4

14

160

1+0.5

0.22

2.18

0.63

0.11

0.00

0.00

1.14

1.80

44.7

43.1

15

150

1+1

0.44

3.90

0.34

0.32

0.00

0.01

1.29

1.35

62.7

44..9

16

150

1.5+1

0.39

3.21

0.83

0.16

0.01

0.01

1.40

1.24

65.3

53.4

17

160

1+1

0.47

4.12

0.23

0.36

0.01

0.01

1.62

1.35

65.12

47.2

18

160

1.5+1

0.54

3.27

0.54

0.18

0.01

0.01

1.73

1.22

67.2

52.1

19

170

1+1

0.61

4.21

0.22

0.25

0.01

0.01

1.93

1.30

69.4

48.1

20

170

1.5+1

0.83

3.12

0.39

0.23

0.10

0.02

2.05

1.21

63.2

55.9

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Table4. Sugars, inhibitors product profile after pretreatment, C5 conversion efficiencies and enzymatic efficiencies (C6) for SA+PA pretreatment a

b

acid dose on dry biomass basis , release of xylose monomeric sugars, *absolute error in composition ±5 %.

Run No.

Temperature

Acid dose a( %w/w)

Glucose

Xylose

(%)*

(%)*

Xylose oligomer

Acetic acid (%)*

HMF

Severity

pH

(%)

Furfural (%)



(%)* 21

150

0.5+0.5

0.25

2.54

0.77

0.23

0.00

0.00

0.69

1.95

52.2

43.2

22

160

0.5+0.5

0.28

2.70

0.56

0.25

0.00

0.00

1.09

1.85

55.2

46.1

23

150

1+1

0.44

3.90

0.34

0.32

0.00

0.01

1.29

1.35

64..7

53.9

24

160

1+0.5

0.39

3.99

0.51

0.29

0.00

0.01

1.46

1.48

62.9

55.3

25

160

1+1

0.47

4.12

0.23

0.36

0.01

0.01

1.62

1.32

68.1

57.2

26

160

1.5+1

0.85

5.02

0.20

0.53

0.00

0.00

1.74

1.22

91.2

63.2

27

170

1+1

0.61

4.21

0.22

0.25

0.01

0.01

1.93

1.30

72.4

59

28

170

1.5+1

1.02

4.52

0.00

0.57

0.01

0.04

2.04

1.19

82.5

67.1

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Table 5. Assignments of IR spectrum with relative band intensity absorption for various pretreatments studied here Band no.

Band Region in Wave number (Cm-1)

Assignments

1

~835

2

~897

Relative absorbance of each different pretreatment and untreated bagasse UT

SA+OA

SA+FeCl3

SA+PA

C-H out of plane vibration in lignin

0.036

0.05

0.05

0.13

C-H deformation in cellulose

0.11

0.16

0.07

0.22

3

1040-1060

C-O stretch in cellulose and hemicellulose

0.69

0.55

0.38

0.92

4

1160-1170

C-O-C vibration in cellulose and hemicellulose

0.57

0.58

0.34

0.78

5

1240-1260

Guaiacyl ring breathing, C-O stretch in lignin,

0.4

0.52

0.27

0.5

6

1320-1330

Syringyl ring breathing in lignin

0.38

0.45

0.29

0.51

7

1370-1380

C-H deformation in cellulose and hemicellulose

0.37

0.39

0.27

0.50

8

1420-1430

Aromatic skeleton vibration (methyl) in lignin combined with C-H plane deformation in carbohydrates

0.34

0.43

0.25

0.51

9

1450-60

Aromatic C-H deformation; asymmetric in CH3, and –CH2

0.33

0.49

0.27

0.46

10

1510-1520

Aromatic C=C stretch from aromatic lignin

0.31

0.9

0.24

0.42

11

1600-1610

Aromatic skeletal vibration plus C=O stretch

0.21

0.48

0.25

0.43

12

1630-1640

Absorbed O-H, Conjugate C=O , ketone

0.46

0.37

0.21

0.34

13

~1705

C=O stretch unconjugated ketone, esters in xylan

0.22

0.89

0.20

0.33

14

2900-2910

C-H stretching, from methyl, methylene groups

0.33

0.42

0.38

0.49

15

3300-3400

O-H vibration from aromatic and aliphatic groups

0.57

0.76

0.54

0.95

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Effect of Mixed Acid Catalysis on Pretreatment and Enzymatic

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