The impact of particle size of cellulosic residue and solid loadings on enzymatic hydrolysis with a mass balance

Fuel 245 (2019) 514–520

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Full Length Article

The impact of particle size of cellulosic residue and solid loadings on enzymatic hydrolysis with a mass balance

T

Manali Kapoora, Surbhi Semwala, Alok Satlewala, Jayaraj Christopherb, Ravi P. Guptaa, ⁎ Ravindra Kumara, , Suresh K. Puria, S.S.V. Ramakumarc a

DBT-IOC Centre for Advance Bioenergy Research, Research & Development Centre, Indian Oil Corporation Limited, Sector-13, Faridabad 121007, India Analytical Division, Research & Development Centre, Indian Oil Corporation Limited, Sector-13, Faridabad 121007, India c Research & Development Centre, Indian Oil Corporation Limited, Sector-13, Faridabad 121007, India b

A R T I C LE I N FO

A B S T R A C T

Keywords: Rice straw Dilute acid pretreatment Particle size Glucan hydrolysis IR spectroscopy

Rice straw has a great potential for ethanol production due to its richness in polysaccharides and abundant availability, however, for efficient utilization of these polysaccharides, size reduction is a prerequisite step. Therefore, biomass particle size plays a vital role for cellulosic ethanol commercialization. In this study, the effects of rice straw particle size on dilute acid pretreatment efficiency and enzymatic hydrolysis are investigated. Different sizes; 5, 10 and 20 mm were subjected to dilute acid pretreatment in a continuous pilot scale system with a horizontal screw feeder reactor followed by enzymatic hydrolysis at varying solids (10 and 15%) and enzyme dosages (5 and 10 FPU/g of pretreated residue). The glucan hydrolysis for 5, 10 and 20 mm are 65.6, 80.0 and 60.0% using 5 FPU and 79.5, 93.4 and 72.8% using 10 FPU/g pretreated residue respectively at 10% loading, whereas, at 15% it is significantly lower in respective experiment. Overall sugar recovery with 10 mm is 63.8 and 72.9% with 5 and 10 FPU respectively. RS with 10 mm biomass particle size at both solid loadings and enzyme dosages resulted in much higher enzymatic hydrolysis than others and in turn the overall sugar recovery and this was found to be due to the variation in the degradation products and pseudolignin contents in the pretreated biomass. The insight into the structural intricacies of biomass after pretreatment are studied using FT-IR and SEM revealing significant changes in biomass properties responsible for improved sugar recovery.

1. Introduction The interests in alternative energy sources have increased due to energy security and environmental concerns [1–3]. Lignocellulose based renewable energy, such as cellulosic ethanol has the potential to replace petroleum-based sources of energy [1,2]. Ethanol from agricultural residues has been considered as a potent option owing to its richness in fermentable sugars. However, harnessing these sugars poses a great challenge due to the cell wall complexity and rigidness. Therefore, to overcome the recalcitrant nature of lignocellulosic biomass (LCB), pretreatment is an imperative step that breaks down the lignin-carbohydrate-complex (LCC) structure and disrupts the crystalline structure of cellulose thereby enhancing cellulose accessibility to cellulases in enzymatic hydrolysis [2,4]. Rice straw (RS) can be the potential feedstock for ethanol production, owing to its abundant availability and high sugar content [5]. An estimate shows that 700–800 million tons of rice straw is produced globally every year [5].

⁎

India has about 8.9 million tons of rice straw available annually as surplus that could be a potential feedstock for production of 3.7 billion litres of ethanol annually [6]. Among the most potential pretreatment methods, alkali, steam explosion and dilute acid pretreatment are being explored extensively. These methods have achieved varying levels of success. Alkali pretreatment is effective to disrupt and removal of lignin resulting to significantly improve the hydrolysis of LCB. However, the consumption of alkali is in molar ratio to hydroxyls present in the lignin, Moreover, recycling of alkali is very energy intense process leading to higher cost and is the major impediment for commercialization [7]. Steam explosion results in the formation of xylo-oligomers which are detrimental to cellulases, consequently leading to poor enzymatic hydrolysis. Due to these demerits in first two methods, dilute acid (DA) pretreatment is one of the most suitable methods to deconstruct the cell wall matrix by hemi-cellulose solubilisation [8]. Various factors such as particle size, temperature, pH and residence time have been reported to influence the

Corresponding author. E-mail address: [email protected] (R. Kumar).

https://doi.org/10.1016/j.fuel.2019.02.094 Received 11 November 2017; Received in revised form 15 February 2019; Accepted 18 February 2019 0016-2361/ © 2019 Published by Elsevier Ltd.

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solids of 60–63 wt% (total solids) for 10 min at 100 bar and was then fed to the reactor. The pilot plant consists of a hopper (weight and loss feeder) followed by pug mill for forward movement of biomass and a continuous screw feeder based horizontal reactor. Before adding soaked rice straw into the reactor, the pretreatment reactor was preheated using steam jacket for about 1 h and allowed to reach 162 °C. Rice straw with about ∼65% moisture was fed to hopper at a rate of 10 kg h−1 to deliver at a constant flow through hopper to the pug mill. After discharge from the pug mill, the biomass was subjected to plug screw feeder and into the pressurized zone of the reactor. The material was heated by direct steam injection at 162 °C for residence time 10 min while keeping the flow of steam into the reactor [9]. Screw auger was used to convey rice straw through the horizontal tube and residence time was controlled by the rotational speeds of the screw. To prevent rapid depressurization of the reactor, the pretreated slurry was collected in the slurry tank through a pair of automated, air-actuated ball valves opened and closed in an alternative pattern. First 1 h pretreated slurry was rejected and the slurry received while a consistent run of 4 h was considered for analysis and further processing. After cooling the pretreated slurry to room temperature, weight, TS, WIS, sugar and inhibitor concentration of the slurry was determined. The pretreated slurry was then kept in a poly bag at 4 °C until further use without any solid-liquid separation or washing.

efficiency of DA pretreatment [9–11]. Biomass particle size is a crucial factor which can notably influence the efficiency of the pretreatment process owing to potent heat and mass transfer effects [12]. It is considered as one of the most important variables that influences process yield and production cost for a heterogeneous reaction systems like DA pretreatment of biomass [13]. Particle size has been found to not only affect the cost of equipments but also the diffusion kinetics and effectiveness of pretreatment, sugar yield, lignin removal and rate of reaction [14]. Larger biomass particles may suffer from incomplete pretreatment of the interior part, furthermore heat and mass transfer problems may result in over-hydrolysis of the outer part [10]. In contrast, smaller particles give a larger surface area but are difficult to handle and more likely to degradation into byproducts [10]. Prior to DA pretreatment each biomass essentially has to undergo particle size reduction. Due to high energy requirements of the milling step in order to get smaller particles, the overall process becomes expensive. Therefore, optimization of biomass particle size is essential for achieving high sugar conversion and low production cost [12]. Numerous studies of pretreatment using DA have been conducted with variety of biomass of particle size varying from 0.85 to 3 mm on laboratory scale using low solid loading and in a high pressure reactor CSTR type [15] and replicating these parameters across different biomass may not be technically viable at a commercial scale. Therefore, for scale up studies each biomass need to be optimized so as to have high sugar recovery. There are limited studies available on evaluating the effects of rice straw particle size on DA pretreatment efficiency on a pilot scale. Therefore, different particle size of rice straw was subjected DA pretreatment conducted in a continuous pilot scale system of 250 kg per day with a horizontal screw feeder reactor at Faridabad, India. The paper envisages the impact of particle size of rice straw using 5, 10 and 20 mm at pre-optimized condition, i.e., 162 °C and 10 min with acid concentration of 0.35% w/w of the biomass residue [9]. As it is necessary to get high titer of ethanol in fermenting broth, hence high solid loadings of 10 and 15% at two enzyme dosages are subjected to enzymatic hydrolysis leading to get more than 100 g/L sugars in the hydrolysate having a potential of producing the 5% v/v ethanol concentration in the final broth. To understand the structural intricacies responsible for pretreatment efficiency and sugar recovery, FT-IR and SEM are also discussed.

2.3. Enzymatic hydrolysis The pH of the pretreated slurry was raised to 5.2 by using aqueous ammonia and enzymatic hydrolysis of the pretreated slurry was performed at 10 and 15% WIS. For 10% WIS, 20 g pretreated rice straw (oven dry weight, ODW) on WIS basis was suspended in 100 ml of 0.1 M sodium citrate buffer making final volume up to 200 ml. The mixture was pre-incubated at 50 °C for 30 min at 200 rpm followed by addition of 5 and 10 FPU cellulases/g WIS. Samples were withdrawn at varying intervals and enzyme was denatured by immediately keeping in boiling water bath for 5 min. The supernatant was collected by centrifugation at 8000×g for 10 min and filtered using 0.2 µm nylon filters for sugars analysis by HPLC. Likewise, experiments were performed at 15% WIS using 5 and 10 FPU cellulases /g WIS.

2. Materials and methods 2.4. Conversion and enzymatic hydrolysis yield Rice (Oryza sativa) straw was collected at the harvesting time (October 2016) from Mathura (27.28 °N 77.41 °E) in Uttar Pradesh (India) and was air dried and shredded to 5, 10, 20 mm by knife mill. All experiments were conducted using a single lot of rice straw. Cellulases preparation was obtained from M/s Novozymes (India). Acetic acid, arabinose, glucose 5-hydroxymethylfurfural (HMF), furfural, sodium hydroxide, sulfuric acid and xylose were procured from M/s Merck, India. All chemicals were analytical grade and were used without any further purification.

The conversion of pretreatment and enzymatic hydrolysis was calculated by the equations given in Supporting information.

2.5. Physicochemical characterization FT-IR spectrometer (IR Prestige-21) was used to generate the IR spectra of the samples in an absorbance mode with a resolution of 4 cm−1 and 200 scans per sample in the region of 400–4000 cm−1. Samples were analyzed by grinding with KBr (1:100, w/w) and pressing into pellets in drift mode. SEM images were conducted at 2000× magnifications using HITACHI S-3400 (US). The specimens were mounted on a conductive tape and sputtered with gold using a fine coater (4 nm) and observed at an accelerating voltage of 2.0 kV.

2.1. Analysis methods Compositional analysis of native and pretreated rice straw, total solids (TS), water insoluble solid (WIS) of the pretreated slurry and inhibitors and sugars concentration in the pretreatment hydrolysate were analyzed as per NREL method [9]. 2.2. Pilot-scale DA pretreatment

2.6. Statistical analysis

Rice straw was soaked in the acid solution (1.0%) for 30 min at room temperature in a soaking chamber which was equipped with water recirculation pump. The wet rice straw after soaking was drained under gravity for 1 h and dewatered using hydraulic press to get the

The JMP software trial version (SAS, US) was used to perform statistical analysis by one-way ANOVA followed by Tukey's HSD post hoc tests and statistical significance was determined at the 0.01 level (P ≤ 0.01) and 0.05 level (P ≤ 0.05). 515

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Table 1 Chemical composition of native and pretreated rice straw.

Native rice straw Pretreated rice straw

Pretreatment experiments

Glucan (%)

– A (5 mm) B (10 mm) C (20 mm)

37.8 52.3 52.9 50.2

± ± ± ±

0.2a 0.2b,* 0.2c* 0.1d

Xylan (%)

Arabinan (%)

Lignina (%)

18.3 ± 0.2a 3.2 ± 0.3b 3.6 ± 0.2b 6.9 ± 0.3c

3.4 ± 0.2a 0 0 0.6 ± 0.3b

12.9 28.5 25.9 26.6

± ± ± ±

0.2a 0.2b 0.1c 0.2d

Ash (%)

Acetic acid (%)

Extractivesb (%)

6.3 ± 0.4a 16.9 ± 0.2b 16.6 ± 0.2b 18.2 ± 0.2c

2.0 0 0 0.8

19.8 ± 0.3 0 0 0

* Pretreatment experiments A, B and C corresponds to the pretreatment of rice straw of initial sizes 5, 10 and 20 mm respectively at 162 °C, 10 min. Lignin includes both acid soluble and acid insoluble lignin. Extractives include water and ethanol extractives. All experiments were conducted in triplicate and each value is expressed as mean ± S.D. Values in the same column with different superscripts letters indicate significant difference at P ≤ 0.01. *Corresponds to significant difference at P ≤ 0.05.

3. Results and discussion 3.1. Compositional analysis of native and pretreated residue

%

Table 1 provides the chemical composition of native and pretreated rice straw, wherein native is found to contain 37.8% glucan, 18.3% xylan and 3.4% arabinan. These values are comparable with the ones reported in the literature [16]. Upon pretreatment of rice straw of 5, 10 and 20 mm, glucan content surged from 37.8% in native rice straw to 52.3, 52.9 and 50.2% whereas, xylan contents decimated to 3.2, 3.6 and 6.9% respectively in the pretreated residues. Lignin is increased from 12.9% in native rice straw to 28.5, 25.9 and 26.6% in pretreated rice straw with respective biomass particle size. Ash contents are also increased qualitatively in the pretreated rice straw. During dilute acid pretreatment, solubilization of xylan results in the increase in other biomass components and hence glucan and lignin contents of the pretreated residues are higher than the native. There is no significant difference in glucan and xylan contents of the pretreated rice straw on using either 5 or 10 mm particle size. However, glucan content is relatively lower (50.2%) and xylan content (6.9%) is relatively higher for 20 mm residue than 5 and 10 mm leading to presumption that pretreatment severity is lower for 20 mm as relatively lower xylan is hydrolyzed. Small amount of residual acetic acid (0.8%) is also present in the 20 mm pretreated residue which further supports the argument of lower pretreatment severity.

90 80 70 60 50 40 30 20 10 0

a

b c

a

b

c

Glucan

A (5 mm) B (10 mm) a* b* c C (20 mm)

a* b* c

Xylan

Solubilization

Glucose

Xylose

Yield

Fig. 1. Effect of initial biomass sizes of rice straw on glucan/xylan solubilization and glucose/xylose yields during pretreatment. All experiments were conducted in triplicate and the mean is reported. Means within the same group with different superscripts letters are significantly different at P ≤ 0.01. * Corresponds to significant difference at P ≤ 0.05. The solubilization and yields are calculated by the equations reported in Table S1.

higher, i.e. 56.4–62.0% than glucose yield, i.e. 4.1–6.2% according to the solubilisation patterns for glucan and xylan in pretreated residue (Fig. 1). It is noticed that the glucose yield initially increases slightly from 5.6 to 6.2% with increase in biomass particle size from 5 to 10 mm and then decreases for 20 mm (4.1%) (Fig. 1). Similarly, xylose yields are found to increase from 5 to 10 mm (60.9 to 62.0%) and then decrease to 56.4% (Fig. 1). The lowest glucose and xylose yields are produced using 20 mm whereas, highest are obtained at 10 mm. For 5 mm, even though the glucan and xylan solubilization are highest among all (Fig. 1), however, glucose and xylose yields are lower than the 10 mm due to conversion of these sugars to higher amounts of HMF (0.49 g/L) and furfural (2.06 g/L) (Table 2). Other degradation products, i.e. formic acid (1.15 g/L) and acetic acid (3.55 g/L) are also found to be the highest with 5 mm. Lower yields with 5 mm could be accredited to the increased susceptibility to acid hydrolysis leading to the higher degradation of sugars.

3.2. Glucan/xylan solubilization and glucose/xylose yields after pretreatment Glucan and xylan solubilization implies the glucan and xylan solubilized from the native rice straw during pretreatment to glucose/xylose and other degradation products. This solubilization presented an interesting picture as with the increase in biomass particle size from 5 to 20 mm, the glucan and xylan solubilization decreases from 17.7 to 8.5% and 89.7 to 73.9% respectively (Fig. 1). This could be attributed to the decrease in susceptibility to dilute acid hydrolysis with an increase in biomass particle size as diffusion of the acid to the biomass could have reduced with the increase in particle size. Qi et al. [11] reported that the longer particle size of biomass makes acid diffusion more difficult, so the hydrolyzing rate of polysaccharides is lower in larger particle size than that of a shorter. Solubilization of glucan and xylan results in the monomeric glucose and xylose in the pretreatment hydrolysate. It is worth mentioning that the yield of the monomeric sugars is significantly lower than that of glucan and xylan solubilization which may be due to degradation reactions that result in production of inhibitors such as HMF, furfural, acetic acid etc. (Fig. 1 and Table 2). During acid pretreatment, sugar release in hydrolysate is observed mainly due to breakdown/or hydrolysis of hemicellulose to xylose and small amount of glucose, which undergoes further dehydration to form furfural from xylose and HMF from glucose. Hence, Due to degradation of monomeric sugars the sugar recovery is lower than the solubilization of the sugars present in the biomass. The xylose yield in pretreatment hydrolysate is significantly

3.3. Enzymatic hydrolysis DA pretreated rice straw without any separation or washing is hydrolyzed using cellulases at two WIS loadings (10 and 15%) and two enzyme dosages (5 and 10 FPU/g WIS). Fig. 2(a) and (b) shows the time course of glucan hydrolysis at 10 and 15% WIS respectively using 5 FPU as a function of different biomass particle sizes. It can be noticed from the Fig. 2 that, for all the cases initial 8 h hydrolysis is faster, but then it slows down. This may be attributed to various factors such as substrateproduct ratio, product inhibition. The glucan hydrolysis for 5, 10 and 20 mm within 2 h is 12, 20 and 8% respectively at 10% WIS whereas with 15% WIS, these are 10, 15 and 6% respectively. From the beginning itself, the hydrolysis of 10 mm surmounts the others, i.e., 5 and 20 mm. As expected, glucan hydrolysis is lower for higher solid loading, i.e., 516

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Table 2 Composition of pretreatment hydrolysate. Pretreatment experiments

Formic acid (g/L)

A (5 mm) B (10 mm) C (20 mm) * *

Acetic Acid (g/L)

a*

HMF (g/L)

a

3.55 ± 0.03 2.56 ± 0.05b 1.39 ± 0.04c

1.15 ± 0.02 1.05 ± 0.06a* 0.60 ± 0.03b*

Furfural (g/L) a*

Total (g/L)

a

0.49 ± 0.03 0.41 ± 0.02b* 0.35 ± 0.04c

2.06 ± 0.04 1.83 ± 0.03b 1.44 ± 0.02c

7.25 5.85 3.78

Pretreatment experiments A, B and C as given in Table 1. Values in the same column with different superscripts letters indicate significant difference at P ≤ 0.01. Corresponds to significant difference at P ≤ 0.05.

15% WIS. It has been shown earlier that higher solid loading result in lower enzymatic hydrolysis due to increase in viscosity, concentrations of inhibitors and mass transfer limitations [17]. Though, the glucose hydrolysis for 10 mm is significantly higher than 5 and 20 mm during the course of hydrolysis for both 10 and 15% WIS, however, the effect of biomass particle size on enzymatic hydrolysis is more pronounced at lower solid loading, 10% WIS as compared to 15% WIS (Fig. 2). The apparent reason could be that at higher loading, more factors like viscosity, inhibitors might have overcome the impact of particle size. Solubilization of higher amount of xylan has been directly related to higher enzymatic digestibility as it exposes the cellulose thereby increasing the accessibility of cellulose [18] but the glucan hydrolysis achieved for 5 and 10 mm is in contrary to what is anticipated (Fig. 2). The order for xylan solubilization is 5 mm (89.7%) > 10 mm (87.6%) > 20 mm (73.8%), whereas glucan hydrolysis follows the order 10 mm (80.0%) > 5 mm (65.6%) > 20 mm (60.0%). The plausible reason for lower glucan hydrolysis of 5 mm as compared to 10 mm can be accredited to the differential diffusion of acid during pretreatment which in turn varied the susceptibility to hydrolysis of different particles size ultimately leading to variation in the degradation products and pseudolignin formation while pretreatment. The total degradation products for 5, 10 and 20 mm are 7.25, 5.85 and 3.78 g/L respectively (Table 2) and increase in lignin contents are 32, 24 and 22% respectively (Table 3) and thus both follows the order A (5 mm) > B (10 mm) > C (20 mm). The increased lignin content in the dilute acid pretreated biomass apparently can be explained by the formation of pseudolignin, which is formed by the condensation of sugar degradation products such as furfural and HMF within themselves or with lignin. It has been reported that the pseudolignin inhibits the accessibility of cellulases to cellulose either by physical barrier or nonproductive lignin-enzyme binding or both thereby leading to reduce enzymatic hydrolysis [9,19–21]. Increase in enzyme dosages from 5 to 10 FPU results in an increase in hydrolysis. The glucan hydrolysis for 5, 10 and 20 mm are 65.6, 80.0 and 60.0% respectively at 5 FPU and increases to 79.5, 93.4 and 72.8% using 10 FPU (Fig. 3). The effect is much prominent for 10 mm as by increasing enzyme dose from 5 to 10 FPU; hydrolysis increased from

90

90 (a) 10% WIS

a

70

b

60

c

50 40

A (5mm)

30

B (10mm)

20

C (20mm)

10

Pretreatment Conditions A(5 mm)

5FPU

5.73 2.83 1.76

Pretreated residue (kg)

G X L

4.74 0.29 2.32

4.93 0.34 2.19

5.23 0.74 2.16

Pretreatment hydrolysate (kg)

G X

0.32 1.73

0.36 1.76

0.23 1.60

Pretreatment recovery (%)

G X L

88.8 71.4 132 (32)

92.2 74.2 124 (24)

95.4 82.5 122 (22)

Enzymatic hydrolysate* (kg)

G X

2.88 0.03 58.2

3.31 0.03 63.8

2.83 0.03 54.9

G X

3.67 0.04 67.5

4.09 0.04 72.9

3.56 0.04 63.6

Enzymatic hydrolysate (kg) Overall sugar recovery %

Initial rice straw was 15 kg containing < 10% moisture. Where G, X and L stand for glucose, xylose and lignin respectively. Glucose/xylose in pretreated residue is calculated on the basis of total solids (TS 20.3, 25.0, 23.01%), insoluble solid contents, WIS (70.4, 65.7 and 73.2%) and chemical composition obtained after pretreatment in experiments A, B, and C respectively. Values in the brackets indicate % increase in lignin content. * Enzymatic hydrolysis conducted at 15% WIS. Pretreatment recovery and overall sugar recovery were calculated using equations given in Table S1.

80.0 to 93.4% within 24 h and thus results in the saving of time of enzymatic hydrolysis. Thus, rice straw with 10 mm particle size achieves the better enzymatic hydrolysis, which emulates the better pretreatment efficiency. Similarly, for 15% WIS, from 5 to 10 mm, glucan hydrolysis increases and then it decreases for 20 mm. The glucan

(b) 15% WIS a

70 60

b

50

c

40

A (5mm)

30

B (10mm)

20

C (20mm)

10

0

0 0

10

20

30

40

50

0

C(20 mm)

G X L

Overall sugar recovery % 10 FPU

B(10 mm)

Native (kg)

80

Glucan hydrolysis (%)

Glucan hydrolysis (%)

80

Table 3 Component based mass balance for pretreatment of rice straw and overall sugar recovery obtained from pretreatment and enzymatic hydrolysis.

10

20

30

Time (h)

Time (h) 517

40

50

Fig. 2. Time course of glucan hydrolysis of the pretreated biomass at (a) 10 and (b) 15% WIS loading with enzyme dosage of 5 FPU. Glucan hydrolysis was calculated using Eq. (1) (f) (Table S1). All experiments were conducted in triplicate and the mean is reported. Means with different superscripts letters are significantly different at P ≤ 0.01.

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160

b' a' a

a"

b

c' b"

c c"

140 120 100 80 60 40 20 0

5FPU 10FPU 5FPU 10FPU 5FPU 10FPU A (5mm)

B (10mm)

Total sugar concentration (g/L)

Glucan hydrolysis (%)

100 90 80 70 60 50 40 30 20 10 0

GH (10% WIS) GH (15% WIS) TSC (10 % WIS) TSC (15% WIS)

C (20mm)

Fig. 3. Glucan hydrolysis of pretreated biomass and total sugar concentration obtained at 10 and 15% WIS loading for 5 and 10 FPU enzyme loadings after 48 h. All experiments were conducted in triplicate and the mean is reported. Means with within the same group with different superscripts letters are significantly different at P ≤ 0.01.

hydrolysis for 5, 10 and 20 mm are 60.7, 67.2 and 54.0% using 5 FPU which surges to 77.4, 83.0 and 68.0% respectively using 10 FPU (Fig. 3). For 10% WIS, the maximum total sugar concentration achieved with B (10 mm) is 95.4 g/L using 10 FPU (Fig. 3). Hydrolysis for 15% WIS is lower than 10% but overall sugar concentration is relatively higher due to higher amount of glucan present in the WIS and higher amount of sugars present in the pretreatment hydrolysate at the beginning of the enzymatic hydrolysis. For 15% WIS, the 10 mm results in maximum total sugar concentration of 120 and 135.4 g/L with 5 and 10 FPU respectively which could lead to generate > 5% v/v ethanol on fermentation.

Fig. 4. FTIR spectra of different pretreated biomass.

the spectra (Fig. 4). Aromatic skeletal vibrations in lignin are noticed at 1639 and 1514 cm−1 whereas peak at 1429 cm−1 results from the aromatic C–H deformations and increase in the intensity of these peaks in the pretreated biomass samples as compared to native arise due to enhanced lignin concentration. The increase in intensity of the peak in the pretreated samples at around 1369 and 1321 cm−1 (syringyl and guaiacyl condensed lignin) also corresponds to the increase in the lignin content. Higher amount of lignin in biomass after pretreatment is also supported by IR which was presented in Table 3. In all samples, the absorption bands centered at 3375 cm−1 (OH stretching) and 2916 cm−1 (CH stretching) are observed [16]. As pretreatment brings out changes in structure of cellulose, viz. lateral order index (LOI), hydrogen bond index (HBI) and properties of lignin, viz. S/G ratio are calculated by the method described by Oh et al. [23]. Lateral order index is correlated to the overall degree of order in cellulose and can be calculated by using the absorbance (A) ratio A1429/A901 [24]. The CH2 scissoring motion in cellulose associated with the amount of crystalline structure is ascribed by the band at 1429 cm−1, while CH deformation assigned to the amorphous region in cellulose is accredited to the band at 901 cm−1. Since, lower LOI indicates less ordered structure of cellulose, therefore, lower the LOI, lower is the crystallinity and by virtue of this higher should be the enzymatic hydrolysis. As expected, on plotting the graph between LOI and glucan hydrolysis of different pretreated biomass, negative correlation is observed (Fig. 5). Thus, maximum enzymatic hydrolysis achieved by pretreated biomass (10 mm) can be explained by the lowest LOI values (Table 4). An improved digestibility with a decrease in LOI values has been observed upon phosphoric acid pretreatment [25]. The nature of the lignin changes during pretreatment and is found to have a profound impact on enzymatic hydrolysis of the biomass [27]. Syringyl (S) and guaiacyl (G) lignin are the major constituents of lignin

Sugar recovery during pretreatment was calculated by considering amount of sugars present in pretreatment hydrolysate and the sugars recovered in water insoluble solid of pretreated slurry and are presented in Table 3. Xylose recovery has been found to be lower than the glucose recovery due to higher susceptibility to degradation of xylose. Similar results are reported in the literature [22]. Glucose recovery increases from 88.8% to 95.4% using 5 and 20 mm particle size respectively (Table 3). Similarly, xylose recovery increases from 71.4% to 82.5% with 5 and 20 mm residues respectively. The lignin recovery for 5, 10 and 20 mm is 132, 124 and 122%. Rice straw with 5 mm has lowest glucose and xylose recovery during pretreatment as it was subjected to a more severe reaction than 10 and 20 mm due to higher acid diffusion which results in higher concentration of degradation products, formation of higher amount of pseudolignin (Table 3). As pretreatment efficiency of the process can only be determined from the overall sugars obtained during pretreatment and enzymatic hydrolysis, overall sugar recovery was calculated and reported in Table 3. Overall sugar recovery for 5, 10 and 20 mm are 58.2, 63.8 and 54.9% respectively with 5 FPU of enzyme which increased to 67.5, 72.9 and 63.6% respectively on increasing the enzyme dosage to 10 FPU (Table 3). In spite of changing the enzyme dosages, the sugar recovery obtained with 10 mm is found to be the maximum as compared to other biomass particle sizes. The sugar recovery thus obtained could further be improved by increasing enzyme dose but cost of enzyme would have serious implications on the overall economics. Therefore, there would always be of a tradeoff between enzyme cost and overall sugar recovery.

1.05

R² = 0.9682

1

LOI

2.54

0.95

R² = 0.8638

0.9

2.49

S/G

3.4. Mass balance of the sugars after pretreatment and enzymatic hydrolysis

0.85 3.5. Biomass structural features and impact on enzymatic hydrolysis

0.8

2.44 50

Insight into the structural transformations during pretreatment of different particle size biomass is correlated with the enzymatic hydrolysis. Alteration in cellulose, hemicelluloses and lignin are established by using IR spectroscopy and characteristic peaks have been marked in

55

60

65

Glucan hydrolysis (%)

70

Fig. 5. Correlation of S/G with glucan hydrolysis (%) corresponding to pretreated biomass A (5 mm), B (10 mm) and C (20 mm). 518

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the study. Therefore, the utilization of 10 mm particle during dilute acid pretreatment of rice straw would be more appropriate for both operational and capital costs and would have great implications in the scale-up. However, it is recommended to carefully optimize the process parameters for each biomass as it may change with change of design of plant and the nature of biomass. Scale-up of the process for 10 ton per day plant is underway.

Table 4 Cellulose and lignin related properties of pretreated biomass. Pretreated biomass

LOI (1423/898)

S/G (1329/1268)

HBI (3400/1323)

A (5 mm) B (10 mm) C (20 mm)

2.52 2.45 2.55

0.89 1.02 0.87

1.28 1.32 1.35

Acknowledgements

and a characteristic peak at 1321 cm−1 corresponds to the breathing of the syringyl ring with C–O stretching and the bands at 1233 cm1 corresponds to the breathing of the guaiacyl ring with C–O stretching [28]. Since, S-has two cross-linking sites while G-has three such sites, these two differ from each other with respect to the available sites for further substitution and, hence, lignin polymerization. Thus, “G” having relatively more sites than “S” leads to the higher degree of cross-linking or polymerization [26]. Therefore, it can be assumed that higher the ratio of syringyl (S) and guaiacyl (G) units, i.e., measured by the ratio of peak intensities at 1321 and 1233 cm−1 in the pretreated residue, higher the enzymatic hydrolysis. This is well defensible by considering the S/G values of different pretreated biomass and the glucan hydrolysis as positive correlation is observed when graph is plotted between them (Fig. 5). S/G ratio increases from 0.89 to 1.02, for A (5 mm) to B (10 mm), and then decreases to 0.87 for C (20 mm) and so is the enzymatic hydrolysis which increased from 60.7 to 67.2% and then decreased to 54.0% (Table 4 and Fig. 3). Enzymatic hydrolysis yield has been reported to increase with increasing S/G ratio for a given pretreatment method [29]. The hydrogen bond index (HBI) which is determined by the ratio of the absorbance bands at 3400 cm−1 (H-bonded absorption) and 1321 cm−1 (CH2 rocking vibration) is also measured. The HBI of cellulose is closely related to the crystal system and the degree of intermolecular regularity, i.e. crystallinity [23]. HBI values are found to be increasing from 1.28 to 1.35 for A (5 mm) to C (20 mm) whereas, glucan hydrolysis increased from A (5 mm) to B (10 mm) but then decreased (Table 4). Therefore, clear cut relationship between HBI values and glucan hydrolysis cannot be established. Thus, by considering LOI and S/G, higher glucan hydrolysis of pretreated biomass B (10 mm) as compared to others can also be justified. SEM of native rice straw (image a) show very uniform and seamless surface whereas that of pretreated rice straw represented by images b-d show very disrupted and disorganized surface which exposes the cellulose fibrils for easy accessibility to cellulases (Fig. S1). Pretreatment of rice straw of 5 mm results in a severe destruction of LCB matrix (image b). Large number of lignin droplets can also be seen on the surface. Disruption of biomass is also observed with 10 mm particle size, i.e. B (10 mm) but lesser number of lignin droplets can be seen (image c) and hence better enzymatic hydrolysis is observed than A (5 mm) due to lower non-productive binding of cellulase on lignin. Further with C (20 mm), least disruption occurred as seen from the SEM image d which results in the lowest enzymatic hydrolysis of the biomass as compared to others.

The authors would like to thank Indian Oil Corporation Limited (IOCL) and Department of Biotechnology (DBT) for supporting this work conducted at DBT-IOC Advance Bio Energy Research Centre. DBT grant number is BT/PB/08/03/2007. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.fuel.2019.02.094. References [1] Kazi FK, Fortman JA, Anex RP, Hsu DD, Aden A, Dutta A, et al. Techno-economic comparison of process technologies for biochemical ethanol production from corn stover. Fuel 2010;89:S20–8. [2] Kapoor M, Raj T, Vijayaraj M, Chopra A, Gupta RP, Tuli DK, et al. Structural features of dilute acid, steam exploded, and alkali pretreated mustard stalk and their impact on enzymatic hydrolysis. Carbohydr Polym 2015;124:265–73. [3] Eliana C, Jorge R, Juan P, Luis R. Effects of the pretreatment method on enzymatic hydrolysis and ethanol fermentability of the cellulosic fraction from elephant grass. Fuel 2014;118:41–7. [4] Kapoor M, Soam S, Semwal S, Gupta RP, Kumar R, Tuli DK. Impact of conditioning prior to dilute acid deconstruction of biomass for the production of fermentable sugars. ACS Sustainable Chem Eng 2017;5(5):4285–92. [5] Lim JS, Manan ZA, Alwi SR, Hashim H. A review on utilisation of biomass from rice industry as a source of renewable energy. Renewable Sustainable Energy Rev 2012;16(5):3084–94. [6] Sukumaran RK, Surender VJ, Sindhu R, Binod P, Janu KU, Sajna KV, et al. Lignocellulosic ethanol in India: prospects, challenges and feedstock availability. Bioresour Technol 2010;101(13):4826–33. [7] Dai X, Li X, Zhang D, Chen Y, Dai L. Simultaneous enhancement of methane production and methane content in biogas from waste activated sludge and perennial ryegrass anaerobic co-digestion: the effects of pH and C/N ratio. Bioresour Technol 2016;216:323–30. [8] Wang M, Zhou D, Wang Y, Wei S, Yang W, Kuang M, et al. Bioethanol production from cotton stalk: a comparative study of various pretreatments. Fuel 2016;184:527–32. [9] Kapoor M, Soam S, Agrawal R, Gupta RP, Tuli DK, Kumar R. Pilot scale dilute acid pretreatment of rice straw and fermentable sugar recovery at high solid loadings. Bioresour Technol 2017;224:688–93. [10] Liu ZH, Qin L, Pang F, Jin MJ, Li BZ, Kang Y, et al. Effects of biomass particle size on steam explosion pretreatment performance for improving the enzyme digestibility of corn stover. Ind Crops Prod 2013;44:176–84. [11] Qi W, Zhuang X, Zhang Y, Wang Q, Yu Q, Yuan Z. Effect of particle microstructure on sulfuric acid distribution during biomass hydrolysis. Energy Sources Part A 2015;37(23):2524–34. [12] Zhu JY, Pan XJ, Wang GS, Gleisner R. Sulfite pretreatment (SPORL) for robust enzymatic saccharification of spruce and red pine. Bioresour Technol 2009;100(8):2411–8. [13] Vidal BC, Dien BS, Ting KC, Singh V. Influence of feedstock particle size on lignocellulose conversion—a review. Appl Biochem Biotechnol 2011;164(8):1405–21. [14] Gu T, editor. Green biomass pretreatment for biofuels production. Springer Science & Business Media; 2013. [15] Kim SB, Lee YY. Diffusion of sulfuric acid within lignocellulosic biomass particles and its impact on dilute-acid pretreatment. Bioresour Technol 2002;83(2):165–71. [16] Hsu T-C, Guo G-L, Chen W-H, Hwang W-S. Effect of dilute acid pretreatment of rice straw on structural properties and enzymatic hydrolysis. Bioresour Technol 2010;101(13):4907–13. [17] Di Risio S, Hu C, Saville B, Liao D, Lortie J. Large-scale, high-solids enzymatic hydrolysis of steam-exploded poplar. Biofuels Bioprod Biorefining 2011;5(6):609–20. [18] Pu Y, Hu F, Huang F, Davison BH, Ragauskas AJ. Assessing the molecular structure basis for biomass recalcitrance during dilute acid and hydrothermal pretreatments. Biotechnol Biofuels 2013;6(1):1. [19] Jönsson LJ, Alriksson B, Nilvebrant NO. Bioconversion of lignocellulose: inhibitors and detoxification. Biotechnol Biofuels 2013;6(1):16. [20] Yu Y, Christopher LP. Detoxification of hemicellulose-rich poplar hydrolysate by polymeric resins for improved ethanol fermentability. Fuel 2017;203:187–96. [21] Samuel R, Pu Y, Raman B, Ragauskas AJ. Structural characterization and

4. Conclusions Particle size of the rice straw affects the dilute acid pretreatment and subsequent enzymatic hydrolysis. Out of 5, 10 and 20 mm particle sizes, 10 mm results in significantly higher enzymatic hydrolysis. With 5 mm particle size, degradation is highest and this results in the highest amount of inhibitory compounds and pseudo-lignin formation leading to lower enzymatic hydrolysis, whereas 20 mm results in ineffective pretreatment. Higher enzyme dosages and lower solid loadings results in higher enzymatic hydrolysis. Glucan hydrolysis at 15% loading with 10 mm is 67.2% using 5 FPU which increases to 83.0% with 10 FPU. Overall sugar recovery with 10 mm is 63.8 and 72.9% with 5 and 10 FPU respectively. IR spectroscopy and SEM supports the outcome of 519

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[22]

[23]

[24]

[25]

[26] Teramura H, Sasaki K, Oshima T, Aikawa S, Matsuda F, Okamoto M, et al. Changes in lignin and polysaccharide components in 13 cultivars of rice straw following dilute acid pretreatment as studied by solution-state 2D 1H–13C NMR. PLoS One 2015;10(6):e0128417. [27] Friday ON, Muhammad MM. Isolation and physicochemical characterization of lignin from Chromolaena odorata and Tithonia diversifolia. J Appl Sci Environ Manage 2015;19(4):787–92. [28] Studer MH, DeMartini JD, Davis MF, Sykes RW, Davison B, Keller M, et al. Lignin content in natural Populus variants affects sugar release. Proc Natl Acad Sci USA 2011;108(15):6300–5. [29] Xiao LP, Lin Z, Peng WX, Yuan TQ, Xu F, Li NC, et al. Unraveling the structural characteristics of lignin in hydrothermal pretreated fibers and manufactured binderless boards from Eucalyptus grandis. Sustainable Chem Process 2014;2(1):9.

comparison of switchgrass ball-milled lignin before and after dilute acid pretreatment. Appl Biochem Biotechnol 2010;162(1):62–74. Canilha L, Santos VT, Rocha GJ, e Silva JB, Giulietti M, Silva SS, et al. A study on the pretreatment of a sugarcane bagasse sample with dilute sulfuric acid. J Ind Microbiol Biotechnol 2011;38(9):1467–75. Oh SY, Yoo DI, Shin Y, Kim HC, Kim HY, Chung YS, et al. Crystalline structure analysis of cellulose treated with sodium hydroxide and carbon dioxide by means of X-ray diffraction and FTIR spectroscopy. Carbohydr Res 2005;340(15):2376–91. Carrillo F, Colom X, Sunol JJ, Saurina J. Structural FTIR analysis and thermal characterisation of lyocell and viscose-type fibres. Eur Polym J 2004;40(9):2229–34. Ishola MM, Millati R, Syamsiah S, Cahyanto MN, Niklasson C, Taherzadeh MJ. Structural changes of oil palm empty fruit bunch (OPEFB) after fungal and phosphoric acid pretreatment. Molecules 2012;17(12):14995–5012.

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