Impact of Conditioning Prior to Dilute Acid Deconstruction of Biomass

Mar 23, 2017 - Hence, it would be interesting to calculate the enzyme dose and cost of alkali and enzyme required to produce 1 kg of sugar. Enzyme Cos...
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Research Article pubs.acs.org/journal/ascecg

Impact of Conditioning Prior to Dilute Acid Deconstruction of Biomass for the Production of Fermentable Sugars Manali Kapoor, Shveta Soam, Surbhi Semwal, Ravi P. Gupta, Ravindra Kumar,* and Deepak K. Tuli DBT-IOC Centre for Advance Bioenergy Research, Research and Development Centre, Indian Oil Corporation Limited, Sector-13, Faridabad-121007, India S Supporting Information *

ABSTRACT: Cost of cellulases is a major impediment in commercialization of cellulosic ethanol. To reduce the enzyme doses for the production of fermentable sugars from rice straw (RS), a series of alkali conditioning experiments were conducted prior to dilute acid (DA) pretreatment. This approach resulted in removal of a majority of extractives, ash, acetic acid, and part lignin, and thus resulted in lowering pseudolignin formation thereby increasing enzymatic hydrolysis yields. Glucan hydrolysis of 69.8%, 74.0%, and 83.5% was obtained at 10 wt % water insoluble solid (WIS) using 8 FPU enzyme/g WIS of biomass conditioned using 0.2, 0.4, and 0.5 wt % alkali prior to pretreatment, which is 14−37% higher than the control (61.0%). The overall sugar recovery in these experiments were 69.2%, 70.2%, and 68.5% at 15 wt % WIS resulting in a sugar concentration greater than 120 g/L, which in turn can produce approximately 5−6% w/v ethanol concentration in fermentation broth. It was found that this approach resulted in a decrease of the enzyme consumption vis-a-vis the conventional process by 46.4% to recover the same amount of sugars. This lowering of enzyme consumption has resulted in net savings, after taking into account the cost of alkali used in the conditioning steps. KEYWORDS: Rice straw, Dilute acid pretreatment, Alkali conditioning, Enzymatic hydrolysis, Economic analysis

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INTRODUCTION Dwindling energy resources and environmental sustainability prompted researchers to shift focus on producing biofuels.1 The U.S. Department of Energy’s (DOE) Office of Energy Efficiency and Renewable Energy (EERE) has set up a goal to reduce greenhouse gas (GHG) emissions in the range of 17% and oil imports by 50% by 2020.2 The European Commission also set the specific target of 10% renewable energy use in the transportation sector by 2020 and reducing GHG emissions in the transportation sector by 6%.3 Similarly, the Indian government has mandated use of 20% blends of ethanol across the country by 2017.3 In order to meet these targets for sustainable energy, sources need to be explored such as biodiesel and cellulosic ethanol. Lignocellulosic (LC) biomass has the potential to be used for the production of biofuels as is the most abundant and low cost renewable material available across the globe.4 LC biomass is mainly composed of carbohydrate polymers (cellulose and hemicellulose) and lignin apart from extractives and ash. © 2017 American Chemical Society

Cellulose is intimately bound to lignin and hemicellulose through hydrogen and covalent bonds in biomass cell wall matrix.4 This structural complexity strongly restricts the cellulase accessibility to cellulose to release fermentable sugars. Therefore, in order to make the enzymes accessible to biomass, an efficient pretreatment method is required to cleave the lignin−carbohydrate matrix and to disrupt plant cell wall structures for its conversion to sugars.4,5 A number of pretreatment methods, i.e., dilute acid, steam explosion, alkaline, ammonia, and biological processes, have been explored.2−6 Among all, dilute acid (DA) pretreatment has been perceived as one of the most promising due to relatively low cost and economically relevant scales.7 DA pretreatment effectively hydrolyzes hemicelluloses and causes favorable structural transformation of other biomass compoReceived: January 30, 2017 Revised: March 16, 2017 Published: March 23, 2017 4285

DOI: 10.1021/acssuschemeng.7b00302 ACS Sustainable Chem. Eng. 2017, 5, 4285−4292

Research Article

ACS Sustainable Chemistry & Engineering

g pretreated WIS, the final sugar concentration of the enzymatic hydrolysate was more than 120 g/L which could result in approximately 5−6% w/v ethanol concentration after fermentation. The enzyme consumption per unit of sugar production was also calculated considering the cost of alkali used. This approach resulted in the reduction of enzyme dose up to 46.4% and a significant cost reduction to produce the same sugar, thereby paving the way forward for scale-up of cellulosic ethanol. This approach has been made based on the design of a 10 ton per day demonstration plant.

nents leading to increasing enzyme accessibility and consequently increased release of fermentable sugars.4 However, DA pretreatment results in the coproduction of some inhibitory compounds formed from the degradation of monomeric sugars which influence the downstream processing.8 The amount of lignin present in the pretreated biomass has also been found to be higher than present in the native biomass quantitatively due to formation of pseudolignin and hence inhibits the accessibility of enzyme to cellulose either by physical barrier or ligninenzyme binding or both.9,10 Different theories have been postulated in the literature to explain the increase in lignin content. For example, carbohydrate degradation products such as furfural and 5hydroxylmethyl furfural (HMF) could condense within themselves or with lignin resulting in a spike of lignin contents.9 Additionally, nonstructural sugars and other low molecular weight compounds present in plant extractives also polymerize and form insoluble solids during pretreatment and have also been found to be responsible for a surge in lignin content.11 Moreover, ash has also found to inhibit enzyme either physically or by deactivating the enzyme or both.12,13 Due to the presence of these unwanted substances in slurry, higher quantities of enzymes are required, which in turn can lead to higher cost of processing and thus reduces the overall efficiency of ethanol production. The cost of cellulase is still one of the major bottlenecks in the commercialization of biomass conversion to ethanol. Some studies have shown that enzyme contributes to about 20−25% of the total cost of the production of fermentable sugars from biomass;14 hence, it is desirable to restrict the production of pseudolignin and reduction of ash. Therefore, the ash and the material responsible for production of pseudolignin need to be removed before pretreatment so as to reduce the enzyme consumption. Various strategies have been applied for enzyme cost reduction which mainly focuses on increasing enzyme efficiency, enhancing enzyme-specific activity, fed batch approach, surfactant addition, and enzyme recycling for successive hydrolysis.15 Cosolvent-based biomass fractionation has also been put forward to reduce enzyme cost.16 However, if pretreatment is modified in such a way that the majority of extractives, ash, and lignin and their condensed products are removed beforehand, the inhibition to the cellulose accessibility could be reduced, which in turn can lower the requirement of the enzyme. In this aspect, modification of the pretreatment step in such a way that the biomass becomes enriched in carbohydrates and made more amenable for hydrolysis appears to be an attractive option as it would not only decrease enzyme dosage but would also improve pretreatment efficiency. An aqueous alkali soaking method has been used to remove acetyl groups present in the corn stover to improve enzymatic hydrolysis; however, no efforts have been made on evaluating its effect on extractives, ash, and lignin removal.17 Therefore, a systematic approach, a series of varying amounts of alkali conditioning experiments, were conducted prior to DA pretreatment at a pilot plant of the Indian Oil Corporation Limited, Research and Development Centre, Faridabad, India, and its effect on glucose and xylose recovery after pretreatment was examined. All these experiments were conducted at a unit (250 kg/day) so as to get high solid loading (>10% w/w) which is not possible to generate in laboratory set-ups. The pretreated slurry was thereafter subjected to enzymatic hydrolysis at 10 and 15 wt % water insoluble solid (WIS) loading without washing and separation. Using 8 FPU enzyme/

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EXPERIMENTAL SECTION

Rice (Oryza sativa) straw (RS) was collected from Mathura (27.28° N 77.41° E) in Uttar Pradesh (North India) at the time of harvesting (October 2015), and all experiments were conducted using a single lot. RS was air-dried and shredded to the particle size ∼10 mm by knife mill. The cellulase enzyme was supplied by M/s Advanced Enzymes Technologies, Ltd. (Mumbai, India). Glucose, arabinose, xylose, acetic acid, 5-hydroxymethylfurfural (HMF), furfural, sulfuric acid, ammonium hydroxide, and sodium hydroxide were obtained from M/s Merck, India. All the chemicals were of analytical grade and used without further purification. Analysis Methods. Moisture content of the RS was determined using NREL LAP.4 Compositional analysis of native and pretreated RS was conducted by two-stage acid hydrolysis using NREL LAP.4 The water insoluble solids (WIS) content of the pretreated slurry was determined by NREL LAP.18 Sugars and inhibitors concentration in the pretreatment hydrolysate were measured by HPLC (Waters Gesellschaft Gmbg, Austria) following NREL LAP.4 Inhibitors analysis was performed using a Biorad Aminex HPX-87H column (Biorad, USA) connected with a UV detector. The mobile phase was 0.005N H2SO4 at a flow rate of 0.6 mL/min, with a 50 °C column temperature. Sugar analysis was conducted using an Aminex HPX-87P column (Biorad, USA) connected with refractive index detector. The mobile phase was milli-Q water with a flow rate of 0.6 mL/min, at a column temperature of 75 °C. Both the columns were equipped with recommended guard columns. Alkali Conditioning of RS. Rice straw, RS (10 kg, moisture content 5%) was soaked in 120 L of alkali solution (0.2, 0.4, and 0.5 wt %). The RS was continuously mixed by using a recirculating pump for 1 h at 60 °C. After 1 h, liquid was drained out, and the biomass was again suspended in recirculating water (80 L) in the same chamber for 30 min. The liquid was again drained out, and washing was repeated once again followed by DA soaking of (1% aqueous H2SO4) alkaliconditioned biomass. A control experiment was set up by soaking RS in water only and heating at 60 °C for 1 h and then subjecting to similar conditions for acid soaking as mentioned above. Pilot-Scale DA Pretreatment. The four batches of (RS) obtained after water/alkali conditioning were then soaked in the acid solution (1−1.1 wt %) for 30 min in a soaking chamber at room temperature. The soaking chamber was equipped with a recirculating pump. Pretreatment of RS was conducted in a 250 kg biomass per day processing unit comprising a horizontal pretreatment reactor. The wet RS after soaking was drained under gravity for 2 h and further pressed using a hydraulic filter press to dewater the solids to 63−65 wt % (total solids) for 15 min at 100 bar. The soaked and pressed rice straw (SPRS) was then fed into the reactor at a rate of 10 kg h−1 and treated at a temperature of 162 °C at 5.4 bar pressure with a residence time of 10 min.18 After the pretreated slurry was cooled to room temperature, the weight and moisture content of the slurry was determined. WIS, composition of pretreated biomass, and sugar and inhibitors concentration in the pretreatment hydrolysate were determined by the HPLC method as described above. The pretreated slurry was then kept in a polybag at 4 °C until further use. Enzyme Assays and Enzymatic Hydrolysis. Filter paper units (FPU) and endoglucalanase (CMCase) activity was determined according to the method described earlier.19 β-Glucosidase activity 4286

DOI: 10.1021/acssuschemeng.7b00302 ACS Sustainable Chem. Eng. 2017, 5, 4285−4292

Research Article

ACS Sustainable Chemistry & Engineering Table 1. Effect of Conditioning on RS Components alkali in water (wt %) component removala (wt % with respect to native biomass)

water (control)

0.2

extractive acetic acid ash lignin glucose xylose

46.7 ± 0.4 43.8 ± 0.2A 5.4 ± 0.4A 1.3 ± 0.5A 0A 0A

± ± ± ± ± ±

A

56.3 56.1 19.7 5.2 1.2 1.6

0.4 B

0.1 0.4B 0.3B 0.2B 0.2B* 0.3B

62.2 61.8 23.1 8.7 2.4 6.6

± ± ± ± ± ±

0.5 C

0.3 0.3C 0.3C 0.2C 0.4C* 0.2C

74.9 70.4 25.2 12.9 5.7 10.1

± ± ± ± ± ±

0.3D 0.2D 0.2D 0.3D 0.2D 0.4D

Component removal (%) = 100 × [1 − {component in conditioned biomass (g) ÷ component in native RS (g)}]. Component is calculated on the basis of chemical composition and weight of the biomass. Chemical composition of the conditioned biomass is not presented. All the experiments were done in triplicate, and each value is expressed as mean ± SD. Values in the same row with different superscript letters indicate significant difference at P ≤ 0.01. *Corresponds to significant difference at P ≤ 0.05.

a

was determined as described previously.20 The cellulase SacchariSEBC6 used in the study was found to contain β-glucosidase activity: 5930 IU/g; filter paper unit: 250 FPU/g; endocellulases activity: 4625 IU/g; endoxylanase activity: 64867 IU/g preparation; and protein: 268 mg/g. Pretreated slurry was subjected to enzymatic hydrolysis without washing/separation by adjusting to a pH of 5.2 prior to the experiments by the addition of aqueous ammonia. Twenty grams of 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 so as to have 10 wt % WIS. The mixture was preincubated at 50 °C for 30 min at 200 rpm. The hydrolysis was started by adding 8 FPU of SacchariSEBC6/g WIS and incubated for 48 h. After 48 h, the reaction was stopped, and the final liquid sample was denatured and filtered using 0.2 μm nylon filter for analysis of sugars concentration by HPLC.4 Experiments were also performed similarly at 15 wt % WIS. Conversion and Enzymatic Hydrolysis Yield. The conversion of pretreatment (P) and enzymatic hydrolysis (E) was calculated by the equations as given by Kapoor et al.18 Statistical Analysis. Statistical analysis was performed by one-way ANOVA followed by Tukey’s HSD post hoc tests using a trial version of JMP software (SAS, USA), and statistical significance was determined at the 0.01 level (P ≤ 0.01) and 0.05 level (P ≤ 0.05).

most pronounced which upon using 0.2, 0.4, and 0.5 wt % alkali was 56.3%, 62.2%, and 74.9%, respectively, followed by acetic acid removal of 56.1%, 61.8%, and 70.4%, respectively, whereas, water conditioning as a control experiment could result in reduction of 46.7% extractives and 43.8% acetic acid. Lignin is a polymer of phenolics which are highly reactive with alkali to form sodium phenates. These sodium phenates are water soluble and hence removed by extraction and separation, and this may be attributed to the cleavage of hydrolyzable linkages such as α- and β-aryl ethers in lignin leading to the dissolution of some lignin under alkaline conditions. As the alkali concentration increased from 0.2 to 0.5 wt %, the lignin removal increased from 5.2% to 12.9%; however, with water conditioning, only 1.3% lignin removal was observed (Table 1). Therefore, it may be argued that the higher alkali concentration in the conditioning solution results in higher lignin removal. Silverstein et al. reported that the lignin content of the cotton stalk decreased from 23.3% to 21.9% on increasing alkali concentration from 0.5% to 1%.21 Kim and Han reported 29.8% to 42.4% lignin removal when alkali concentration varied from 1% to 4%, respectively.22 Increase in lignin removal with an increase in alkali concentration has also been observed by other researchers.4,23 Similarly, there was a reduction in ash content by 19.7%, 23.1%, and 25.2% while using 0.2, 0.4, and 0.5 wt % alkali, respectively, whereas a minor reduction (5.4%) in ash content was observed in the control experiment. Since RS contains silica as a majority amount as an ash content,24,25 reduction in ash content using alkali may be attributed to the formation of sodium silicate, which is soluble in water and removed while washing and separating. However, while applying only hot water as a control, this reaction could not proceed much, hence a lower removal of ash. This also indicates that the metal oxides present in RS are structural in nature and hence could not be washed out using hot water. Other authors also reported silica as a structural material in RS.24,25 Alkali conditioning along with removal of lignin also cleaves glycosidic bonds in carbohydrates leading to the hydrolysis of carbohydrate, and hence, some amount of glucose and xylose losses were also observed using 0.2, 0.4, and 0.5 wt % alkali solutions. However, no loss of sugar was observed with water (Table 1). Since the majority of the unwanted materials like extractives, acetic acid, ash, and part lignin are removed during conditioning, less biomass load is carried forward for DA pretreatment which in turn decreases the load on the pretreatment reactor. The loss in weight of biomass was 16−24% across different conditioning experiments. Subsequently, this reduces the load on the enzymatic hydrolysis reactor, amount of chemicals, buffers, and enzymes,

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RESULTS AND DISCUSSIONS Recently, DA pretreatment experiments (without conditioning) were optimized for rice straw (RS), i.e., 0.35 wt % acid concentration in the reactor, 162 °C reaction temperature, and 10 min residence time on the basis of maximum overall sugar recovery (pretreatment hydrolysate + enzymatic hydrolysate).18 However, the amount of enzyme required for the enzymatic hydrolysis was very high (10 FPU/g pretreated biomass). The pseudolignin could have adsorbed some of the enzyme resulting in its unavailability for hydrolysis. The presence of the unwanted substances like extractives, ash, and lignin and their condensed products in pretreated slurry results in increased formation of pseudolignin and consequently decreases the efficiency of cellulases. Accordingly, alkali conditioning experiments were performed in a pilot plant at 0.2−0.5 wt %, alkali at 60 °C for 1 h, and were limited to 0.5 wt % as sugar losses (∼6% glucose and 10% xylose) were observed at this concentration. One control experiment was also set up in which conditioning was performed using water at 60 °C for 1 h. All these biomass were then subjected to pretreatment in a screw type pilot plant at previously optimized conditions.18 Reduction in Extractives, Ash, and Lignin after Conditioning. Table 1 shows the reduction in unwanted RS components after alkali conditioning. Extractives removal was 4287

DOI: 10.1021/acssuschemeng.7b00302 ACS Sustainable Chem. Eng. 2017, 5, 4285−4292

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ACS Sustainable Chemistry & Engineering Table 2. Chemical Composition of Native and Pretreated RS pretreatment experimentsb a

components (wt %)

native

glucan xylan arabinan ligninc ash acetic acid extractivesd

± ± ± ± ± ± ±

37.8 18.3 3.4 12.9 6.3 2.0 19.8

A

0.3 0.2A 0.2 0.4A 0.2A 0.1 0.5

A (control)

B

56.9 ± 0.3 8.8 ± 0.3B 0 24.3 ± 0.3B 10.6 ± 0.3B 0 0

59 ± 0.3 7 ± 0.2C 0 22 ± 0.2C* 11.9 ± 0.4C 0 0

B

C

D

66.3 ± 0.4 6.4 ± 0.3C* 0 20 ± 0.3D 8 ± 0.3D 0 0

C

70.6 ± 0.4E 5.4 ± 0.2D* 0 21.1 ± 0.2E* 4.8 ± 0.2E 0 0

D

a

Components constitute water insoluble solids of pretreated biomass. bExperiments A, B, C, and D represent the pretreatment of the biomass which has been preconditioned using 0, 0.2, 0.4, and 0.5 wt % alkali at 60 °C for 1 h, respectively, at 0.35 wt % acid concentration in the reactor and 162 °C for 10 min. cLignin includes both acid soluble and acid insoluble lignin. dExtractives include water and ethanol extractives. All experiments were done in triplicate, and each value is expressed as mean ± SD. Values in the same row with different superscript letters indicate significant difference at P ≤ 0.01. *Corresponds to significant difference at P ≤ 0.05.

lignin content was observed after DA pretreatment (0.35 wt % acid, 162 °C, 10 min) due to pseudolignin formation.18 Samuel et al. reported a 10% increase in lignin content in pretreated switchgrass after DA pretreatment at 190 °C with a residence time of 1 min.26 Sannigrahi et al. reported lignin content of the acid-treated biomass to be 17% higher than the native biomass.27 Thus, the benefit of using a conditioning method prior to DA pretreatment is apparent as it resulted in the reduction of the lignin content in the pretreated RS. Analysis of pretreatment hydrolysate shows that alkali conditioning prior to pretreatment resulted in a decrease in degradation products like HMF, furfural, and acetic acid. In control experiment A, HMF (0.5 g/L), furfural (1.1 g/L), and acetic acid (1.8 g/L) were detected, whereas in experiments B to D, HMF concentration ranged from 0.4 to 0.2 g/L, furfural from 0.9 to 0.7 g/L, and acetic acid from 1.2 to 0.2 g/L, respectively (Table 3). Lower degradation products may be

and size of vessel. Thus, the overall process of conditioning results in enrichment of carbohydrates in RS as given in Table 2. Analysis of Pretreated Biomass and Pretreatment Hydrolysate. Glucan and xylan contents of the native RS were 37.8 and 18.3 wt % which altered to 56.9 and 8.8 wt %, respectively, in pretreatment experiment A (Table 2). In experiments B, C, and D, glucan contents further increased to 59.0, 66.3, and 70.6 wt %, respectively, owing to hemicellulose solubilization during pretreatment and removal of significant amounts of extractives, lignin, and ash during conditioning, whereas xylan contents subsided to 7.0, 6.4, and 4.2 wt %, respectively (Table 2). Lignin content in native RS was 12.9 wt %, which increased qualitatively to 24.3, 22.0, 20.0, and 21.1 wt % in experiments A, B, C, and D, respectively. In experiment A (control), a 16.6 wt % increase in lignin content of the pretreated RS was observed quantitatively with respect to native RS which may be attributed to the formation of pseudolignin. In experiment B, relatively lower pseudolignin was formed as compared to the control experiment, and consequently, there was only a 5.6 wt % increase in the lignin content of pretreated RS (Figure 1), whereas in experiments C and D lignin content decreased notably by 12.8 and 23.1 wt %, thereby leading to speculation that this could be due to the complete exclusion of the formation of pseudolignin due to huge reduction in extractive, ash, and lignin contents during alkali conditioning (Table 1). About a 35.0 wt % increase in

Table 3. Chemical Analysis of Pretreatment Hydrolysate pretreatment experimentsa degradation Products (g/L)

A

B

C

D

HMF furfural acetic acid

0.5 ± 0.02A 1.1 ± 0.03A 1.8 ± 0.06A

0.4 ± 0.04B 0.9 ± 0.02B 1.2 ± 0.05B

0.3 ± 0.03C 0.8 ± 0.02C 0.8 ± 0.05C

0.2 ± 0.05D 0.7 ± 0.03D 0.2 ± 0.04D

a

Experiments A, B, C, and D represent the pretreatment of the biomass which has been preconditioned using 0, 0.2, 0.4, and 0.5 wt % alkali at 60 °C for 1 h, respectively, at 0.35 wt % acid concentration in the reactor and 162 °C for 10 min. Analysis of the pretreatment hydrolysate was carried out in triplicate, and each value is expressed as mean ± SD. Values in the same row with different superscript letters indicate significant difference at P ≤ 0.05 for the first row and P ≤ 0.01 for the second and third rows.

accredited to the removal of extractives and nonstructural carbohydrates prior to pretreatment in alkali conditioning (Table 1). A decrease in acetic acid in the pretreatment hydrolysate was apparent as it was partially removed in the conditioning process (56.1−70.4%). Chen et al. reported about 75% acetyl groups removal from raw corn stover on the application of a dilute alkaline extraction step prior to pretreatment.28 The decreased amount of these inhibitory products is beneficial as the slurry can be directly used for enzymatic hydrolysis without washing and thereafter for fermentation. Therefore, it may be concluded that the

Figure 1. Reduction in lignin, ash, and extractives contents of pretreated RS vis-a-vis native RS. Component reduction (%) = 100 × [1 − {component in pretreated solids (g)/component in native RS (g)}]. Pretreatment experiments A, B, C, and D as explained in Table 2. 4288

DOI: 10.1021/acssuschemeng.7b00302 ACS Sustainable Chem. Eng. 2017, 5, 4285−4292

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ACS Sustainable Chemistry & Engineering Table 4. Mass Balance of Glucose and Xylose for Pretreatment of RS pretreatment components pretreatment experiments A B C D

glucose xylose glucose xylose glucose xylose glucose xylose

glucose/xylose in native RS (kg)a

xylose-rich hydrolysate (kg) (1)

glucan-rich residueb (kg) (2)

total (kg) (1 + 2)

sugar yield in hydrolysate (P)c (wt %)

sugar recovery in pretreatment (P)d (wt %)

4.19 2.07 4.19 2.07 4.19 2.07 4.19 2.07

0.36 1.25 0.10 1.13 0.14 1.09 0.17 0.96

3.58 0.21 4.04 0.49 3.98 0.35 3.68 0.24

3.95 1.45 4.14 1.63 4.12 1.44 3.85 1.19

8.59 60.25 2.38 5.47 3.34 52.42 4.08 46.44

94.08 70.12 98.72 78.63 98.27 69.52 91.69 57.26

a Initial rice straw was 10 kg containing 5% moisture. bGlucose/xylose in glucan-rich residue is reported on the basis of total solids (TS: 20.3, 24.0, 25.0, 25.0 wt %), water insoluble solids (68.5, 76.0, 73.4, 67.4 wt %), and chemical composition of the RS obtained after pretreatment experiments A, B, C, and D, respectively. cGlucose/xylose yield was calculated using equations as given in ref 18. dGlucose/xylose recovery (P) calculated by dividing (glucose/xylose obtained in the pretreatment hydrolysate + glucose/xylose retained in the water insoluble solid residue after pretreatment) by the glucose/xylose present in the native rice straw.

conditioning results in reduction of biomass load and formation of inhibitors. This could be attributed to a decrease in glucose and xylose degradation reactions as a majority of unwanted material and nonstructural carbohydrates were removed prior to pretreatment in the alkali conditioning (Table 1). Glucose and Xylose Recovery during Pretreatment. Sugar recovery during pretreatment was calculated by considering the amount of sugars present in the pretreatment hydrolysate and WIS of the pretreated slurry (Table 4). In experiment A, 94.1 wt % glucose and 70.1 wt % xylose recovery was achieved which increased to 98.7 and 78.6 wt %, respectively, in experiment B. The xylose recovery obtained in the current process is higher than the conventional process reported earlier (i.e., without conditioning it was 59.1 wt %).18 The reason for higher sugar recovery could be due to formation of lower degradation products and lower/nil pseudolignin formation. From experiments B to D, glucose recovery decreased by 7.1% and xylose recovery by 27.2% due to losses of sugar during both alkali conditioning and pretreatment. In all the experiments (A to D), xylose recovery is much lower as compared to glucose, and this is by virtue of the higher extent of degradation reactions for pentoses than for hexoses. Canilha et al. found higher glucose recovery than xylose and reported this as due to the higher susceptibility of pentoses to degradation under acidic conditions than that of hexoses.29 Impact of Alkali Conditioning on Enzymatic Hydrolysis. Pretreated RS obtained after experiments A to D have been designated as PA, PB, PC, and PD, respectively, and was subjected to enzymatic hydrolysis at 10 and 15 wt % WIS loading without washing/separating using 8 FPU enzyme/g WIS (Figure 2). Glucan hydrolysis for PB, PC, and PD was 69.8%, 74.0%, and 83.5%, respectively, which is 14−37% higher than the control PA (61.0%) at 10 wt % WIS (Figure 2). Xylan hydrolysis was 54.1% for experiment A and increased to 58.2%, 64.2%, and 69.3% for PB, PC, and PD, respectively (Figure S1). The total sugar concentrations (glucose + xylose) obtained were in the range from 70 to 108 g/L from PA to PD, respectively (Figure 2). The significant improvement in hydrolysis in PB, PC, and PD may be attributed to alkali conditioning which resulted in a decrease in the lignin content and other unwanted materials in the pretreated RS resulting in a reduction in pseudolignin as explained earlier. Moreover, removal of the majority of extractives, ash, and acetic acid resulted in the better

Figure 2. Glucan hydrolysis and total sugar concentrations obtained at 10 and 15 wt % WIS loading of pretreated RS using 8 FPU/g WIS of biomass at 48 h. PA, PB, PC, and PD refers to pretreated RS obtained after experiments A, B, C, and D. Glucan hydrolysis (E) (%) was calculated by using the equation in ref 18. Total sugar concentration implies glucose + xylose concentrations obtained in the enzymatic hydrolysate. All experiments were done in triplicate, and the mean is reported. Means within the same group with different superscript letters are significantly different at P < 0.01.

accessibility of cellulose leading to higher cellulose conversion which in turn led to higher sugar concentrations. To establish this assumption, Figure 3 was plotted (ash and lignin contents vis-à-vis glucan hydrolysis). A negative correlation was observed between the ash content of the pretreated RS and glucan

Figure 3. Correlation of ash and lignin contents of pretreated RS with glucan hydrolysis at 10 wt % WIS using 8FPU/WIS of biomass. 4289

DOI: 10.1021/acssuschemeng.7b00302 ACS Sustainable Chem. Eng. 2017, 5, 4285−4292

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ACS Sustainable Chemistry & Engineering Table 5. Enzyme Dose Savings with Different Pretreatment Experiments pretreatment hydrolysate (kg) (1)

enzymatic hydrolysateb (kg) (2)

pretreatment experiments

glucose

xylose

glucose

WCa A B C D

0.28 0.36 0.10 0.14 0.17

0.85 1.25 1.13 1.08 0.96

2.40 2.18 2.83 2.95 2.86

xylose

total sugar recovered from 10 kg biomass (1 + 2)

total enzyme required (FPU)d (×104)

total enzyme required for 1 kg sugar recoverye (FPU) (×103)

total enzyme requiredf(g)

% reduction of enzyme

0.16 0.11 0.24 0.21 0.15

3.69 3.90 4.30 4.38 4.14

62.8 50.2 49.3 42.3 37.7

17.0 12.8 11.4 9.5 9.1

68.2 51.5 45.9 38.6 36.5

− 24.4A 32.5B 43.3C* 46.4D*

Pretreatment experiments conducted at 0.35 wt % acid and 162 °C for 10 min without conditioning. bExperiments A, B, C, and D represent the pretreatment of the biomass which has been preconditioned using 0, 0.2, 0.4, and 0.5 wt % alkali at 60 °C for 1 h, respectively, at 0.35 wt % acid concentration in the reactor and 162 °C for 10 min. cGlucose/xylose recovered in enzymatic hydrolysate = (glucan% × enzymatic hydrolysis% × 1.1)/10,000. dTotal enzyme FPU required for enzymatic hydrolysis is calculated by WIS % × enzyme FPU required/g WIS × 1000. eTotal enzyme FPU required for 1 kg sugar recovery = total enzyme FPU required/total sugar recovered. fTotal enzyme required calculated by considering that 1 g contains 250 FPU. % reduction of enzyme = [{(enzyme dose required without conditioning − enzyme dose required with conditioning)/enzyme dose required without conditioning} × 100]. Considering ±3% experimental uncertainty, statistical significance was determined for % reduction of enzyme. Values in the column with different superscript letters indicate significant difference at P ≤ 0.01. *Corresponds to significant difference at P ≤ 0.05. a

hydrolysis (R2 = 0.71) and between lignin content and glucan hydrolysis (R2 = 0.64). The lower lignin content of the pretreated biomass had been shown to contribute to the improved cellulose digestibility.30,31 A similar trend for hydrolysis was also observed for both glucan and xylan at 15 wt % WIS (Figures 2 and S1); however, hydrolysis was better using 10% WIS due to lower viscosity and lower product inhibition. The total sugar concentrations obtained after enzymatic hydrolysis while using 15% WIS were 100, 126, 131, and 157 g/L for PA, PB, PC, and PD, respectively (Figure 2), which is relatively higher than 10% WIS due to higher concentrations of sugars present in the pretreatment hydrolysate at the beginning of the experiment and higher cellulose present in the WIS quantitatively. This sugar concentration could result in greater than 50 g/L ethanol on fermentation and could make the process economically viable. A sugar concentration of 115.2 g/L was reported using 10 FPU enzyme/g solid at 20 wt % total solid loading.18 Enzymatic conversion of cellulose to glucose (70%) was achieved using washed dilute acid-pretreated corn stover produced in pilot reactor at a cellulose loading of 6 wt % and enzyme loading of 15 FPU/g cellulose.32 The data presented here show a significant benefit of using alkali conditioning prior to pretreatment as even with relatively lower enzyme dosages (8 FPU/g) the enzymatic hydrolysis and total sugar concentrations achieved were better than reported by others.18,32 Thus, it may be argued that higher sugar concentration (∼150 g/L) can be obtained using a lower dosage of enzyme. Therefore, there is a distinct advantage of conditioning using alkali prior to pretreatment. Total Sugar Recovery after Enzymatic Hydrolysis. Total sugar recovery implies the total sugar obtained during pretreatment and enzymatic hydrolysis of the sugars present in the native biomass and was calculated for 15 wt % WIS as it resulted in higher concentration of sugar. In experiment A, glucose and xylose recovery obtained during pretreatment were 94.1 and 70.1 wt % (Table 4), respectively, and in enzymatic hydrolysis, it was 60.0 and 55.0 wt %, respectively, thus resulting in total sugar recovery of 62.3 wt %. In experiment B, due to alkali conditioning, there was an increase in sugar recovery in both pretreatment (98.7 wt % glucose and 78.3 wt

% xylose) and enzymatic hydrolysis (72.2 wt % glucose and 66.2 wt % xylose). Consequently, total sugar recovery increased to 69.2 wt % which is about 10% higher than obtained in experiment A. For experiments C and D, there was a decrease in both glucose (98.2 and 91.6 wt %, respectively) and xylose recovery (69.5 and 57.0 wt %, respectively) during pretreatment, but due to increased enzymatic hydrolysis, total sugar recovery was 70.2 and 68.5 wt %, respectively. Thus, owing to an increase in enzymatic accessibility of biomass due to alkali conditioning, better hydrolysis was achieved resulting in increased sugar recovery in experiments B to D. Therefore, it may be concluded that alkali conditioning results in an increase in sugar recovery compared to experiment A, i.e., control. Since all the alkali experiments B, C, and D resulted in almost the same recovery, it may not be possible to choose the best performer. Hence, it would be interesting to calculate the enzyme dose and cost of alkali and enzyme required to produce 1 kg of sugar. Enzyme Cost Analysis. Since the cost of enzymes is a major bottleneck for biofuel production, development of an effective pretreatment technology to reduce the enzyme loading during saccharification would be an obvious choice for improvement. In this regard, potential enzyme dose benefits on adopting alkali conditioning were investigated. Considering the sugars recovered in the pretreatment hydrolysate and enzymatic hydrolysate (15% WIS), the potential enzyme dose savings as a result of reduction in biomass loading using a range of alkali concentrations is shown in Table 5. In experiment A, 0.36 kg glucose and 1.25 kg xylose were recovered in the pretreatment hydrolysate and 2.18 kg glucose and 0.11 kg xylose in the enzymatic hydrolysate amounting to total sugar (glucose and xylose) recovery of 3.90 kg from 10 kg biomass. The FPU of enzyme required for recovery of 1 kg of sugar was 12,882 FPU corresponding to 51.5 g enzyme powder. With incorporation of alkali conditioning, an increase in the total sugar recovery from 3.90 kg in experiment A to 4.30, 4.38, and 4.13 kg in experiments B, C, and D, respectively, was observed. This resulted in a decrease in the FPU requirement for recovering 1 kg sugar from 11,489 FPU in experiment A to 9129 FPU in experiment D and thereby decreasing the enzyme doses from 45.9 to 36.5 g, respectively. In the pretreatment 4290

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ACS Sustainable Chemistry & Engineering Table 6. Cost Estimate of Enzyme and Alkali for Production of 1 kg Sugar pretreatment experimentsa WC A B C D

cost of enzyme in Rs (A) 34.09 25.76 22.98 19.32 18.26

(0.51) (0.38) (0.34) (0.28) (0.27)

alkali (required for 1 kg sugar recovery) kg

cost of alkali in Rs (B)

0.00 0.00 0.06 0.11 0.15

0.00 0.00 2.23 (0.033) 4.38 (0.065) 5.80 (0.087)

total cost of enzyme and alkali for 1 kg of sugar (A + B) 34.09 25.76 25.21 23.71 24.06

enzyme cost savings (%)

(0.51) (0.38) (0.37) (0.35) (0.36)

− 24.4A 26.0A 30.5B 29.4B

Pretreatment experiments as explained in Table 5. Enzyme cost savings = [{(enzyme cost without conditioning − total cost of enzyme and alkali)/ enzyme cost without conditioning} × 100]. Price of enzyme: Rs 500/kg (7.5 USD/kg); Alkali: Rs 40/kg (0.6 USD/kg). The price in bracket is in USD. Considering ±3% experimental uncertainty, statistical significance was determined for % enzyme cost savings. Values in the column with different superscript letters indicate significant difference at P ≤ 0.01.

a

out. The highest cost savings of 30.5% could be achieved using 0.4 wt % alkali concentration. Thus, the methodology of conditioning prior to pretreatment is an effective contribution in the field of cellulosic ethanol production where biomass pretreatment effectiveness in liberating cellulosic material for subsequent hydrolysis to sugars is enhanced and thus could pave the way for commercialization of the process. On the basis of the current cost of enzyme and alkali, the 0.4 wt % alkali conditioning experiment was found to be cost effective.

experiment in which no conditioning was conducted (calculated from data of publication18), total sugar recovered was 3.69 kg corresponding to 17,045 FPU/g sugar recovery, and consequently, the enzyme powder required was 68.2 g. Therefore, enzyme reduction of 24.4%, 32.5%, 43.3%, and 46.4% was observed in experiments A, B, C, and D, respectively, in comparison to the one in which no conditioning was conducted. Considering the total costs of enzyme and alkali, the potential enzyme cost savings using a range of alkali concentrations was also determined (Table 6). The industrial cost of alkali was set to Rs 40/kg (0.6USD/kg) and enzymes Rs 500/kg (7.5USD/kg) (quoted price). In experiment A, i.e, water conditioning, the total initial cost of enzyme was Rs 25.76/kg (0.38USD/kg) produced sugar. With alkali conditioning, i.e., experiments B, C, and D, the cost of enzyme became Rs 22.98 (0.34 USD), 19.32 (0.28 USD), and 18.26 (0.27 USD), respectively. However, the cost of alkali also increases from Rs 2.23 to Rs 5.80 (0.033 to 0.087 USD) on increasing the alkali concentration from 0.2% to 0.5%, therefore making the total cost from Rs 25.21 (0.37 USD) to Rs 24.06 (0.36 USD). However, when no conditioning was employed, the cost of enzyme was Rs 34.09 (0.51 USD). Therefore, using 0.2−0.5 wt % alkali, the enzyme cost savings was in the range of 26.0−30.5%; however, with water conditioning, cost saving was 24.4%. Even though the maximum enzyme dose reduction was observed with 0.5% alkali (Table 5), the maximum enzyme cost savings benefits could be achieved using 0.4% alkali (30.5%) (Table 6). This shows that there is a trade-off between the amount of alkali required and the potential cost savings. Therefore, it is highly beneficial to condition the biomass before pretreatment which could help to reduce the overall cost of sugar production and hence ethanol production.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssuschemeng.7b00302. Figure S1: Xylan hydrolysis of unwashed pretreated RS obtained at different pretreatment experiments at 10 and 15 wt % WIS loading using 8FPU/g WIS of biomass. Xylan hydrolysis (E) (%) was calculated by using the equation in ref 18. (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Phone: 0129-2294463. Fax: 01292286221. E-mails: [email protected]; [email protected]. ORCID

Ravindra Kumar: 0000-0002-5987-7603 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank the Department of Biotechnology (DBT) and Indian Oil Corporation for supporting this work carried out at DBT-IOC Advance Bio Energy Research Centre.

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CONCLUSIONS Conditioning prior to DA pretreatment resulted in the removal of a majority of extractives, ash, lignin, and acetic acid and consequently reduced the pseudolignin content in the pretreated RS thereby improving the hydrolysis by reducing the recalcitrance of RS. Overall, sugar recovery obtained with 0.2, 0.4, and 0.5 wt % alkali was 69.2%, 70.2%, and 68.5% at 15 wt % WIS. High sugar greater than 120 g/L was achievable using lower enzyme dosage (8FPU/g WIS of biomass) which could lead to production of approximately 5−6% w/v ethanol. The impact of conditioning prior to pretreatment on overall reduction in enzyme doses was established, and enzyme reduction of 24.4%, 32.5%, 43.3% and 46.4% was achieved with water, 0.2, 0.4, and 0.5 wt % alkali, respectively, in comparison to the one in which no conditioning was carried

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REFERENCES

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