Research Article pubs.acs.org/journal/ascecg
Structural Features of Dilute Acid Pretreated Acacia mangium and Impact of Sodium Sulfite Supplementation on Enzymatic Hydrolysis Surbhi Semwal,† Ruchi Gaur,† Suman Mukherjee,‡ Anju Chopra,‡ Ravi P. Gupta,† Ravindra Kumar,*,† and Deepak K. Tuli† †
DBT-IOC Centre for Advanced 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 S Supporting Information *
ABSTRACT: Lignocellulosic biomasses (LCB) differ in their chemical composition and cell wall architecture from one another and within the same LCB due to varying geographical conditions. Thus, pretreatment parameters need optimization for recovery of sugars across different biomasses. In the current study, Acacia mangium has been investigated at bench scale for its potential as a feedstock for fermentable sugar production. Attempts were first made to target hydrolysis of hemicellulose using dilute acid (DA) as a catalyst. Pretreatment at temperature of 160 °C, residence time of 30 min and H2SO4 concentration of 2% (w/w) yielded highest overall saccharification efficiency (50%) corresponding to a glucan conversion in enzymatic hydrolysis (57.8%). Further enhancement in glucan conversion and reduction in the formation of inhibitors was brought about by using sodium sulfite (SS). It was observed that SS caused a significant increase in overall sugar recovery. Interestingly, the order in which SS and DA were added to the pretreatment medium was an important strategy to improve enzymatic saccharification. The condition (SS→DA) where sodium sulfite (SS) was added right in the beginning along with the biomass followed by dilute acid (DA) addition at the desired temperature was more effective in improving the glucan conversion yield (77.0%). Highest BET surface area of SS→DA residue (3.7 m2/g) among all the pretreated residues is one of the factors contributing to this high conversion yield. To get further insight into the basis for improved saccharification, cellulase adsorption studies were conducted. The results showed that the solid residue obtained by SS→DA despite having the lowest maximum adsorption capacity (σmax) resulted into highest saccharification yield which was supported by its highest affinity constant (Ka = 0.25 mL/mg) for the enzyme. Among the pretreated residual solids, SS→DA residue showed lignin modification and cellulose peak alterations by FT-IR spectroscopy and increased surface area by BET measurement revealing implication in improved enzymatic saccharification and overall sugar recovery. KEYWORDS: Acacia mangium, Pretreatment, Delignification, Cellulase adsorption, Enzymatic saccharification, Fermentable sugars
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facilitate the release of fermentable sugars.2 Interestingly, plant cell-wall structural construction and composition varies across different biomasses and hence, variation in their recalcitrant nature. These unique features of different biomass required optimization of process parameters to maximize the recovery of sugars for individual biomass. Different pretreatment methods target hemicelluloses and/or lignin and result in various physicochemical changes in the LCB, e.g., chemical composition, surface area, biomass crystallinity and degree of polymerization, etc. Alteration in these properties affect cellulose accessibility and consequently, influence the enzymatic hydrolysis and overall sugar recovery.3 Pretreatment methods can broadly be divided into physical (milling and grinding), physicochemical (steam pretreatment/
INTRODUCTION
Finite availability of fossil fuels and their adverse impact on the environment has been the main reasons for the development of biofuels like ethanol and biodiesel across the globe. Presently, most of the ethanol is produced either from corn or sugarcane which can provide only limited quantities. Therefore, alternate sources for producing ethanol are being researched. Lignocellulosic biomass (LCB) is being explored as a feedstock for mass production of ethanol and other value added chemicals.1 LCB contain a considerable amount of carbohydrates which can potentially be utilized for producing green fuels like bioethanol and biobutanol. However, in LCB the intimate association among three major structural polymers, viz. cellulose, hemicellulose and lignin, results into complex and rigid cell-wall architecture. This imparts high resistance to the biomass toward microbial and biochemical deconstruction. To overcome it, various pretreatment methods have been employed that alter the structural architecture of LCB to © XXXX American Chemical Society
Received: April 13, 2016 Revised: July 26, 2016
A
DOI: 10.1021/acssuschemeng.6b00758 ACS Sustainable Chem. Eng. XXXX, XXX, XXX−XXX
ACS Sustainable Chemistry & Engineering
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auto hydrolysis, hydrothermolysis, wet oxidation and AFEX), chemical (alkali, dilute acid, ammonia percolation, organosolv, oxidizing agents, organic solvents), biological (white rot fungi) and combination of these.4−6 Different methods have achieved varying levels of success but choice of single pretreatment method still remains as one of the major impediments in cellulosic ethanol commercialization. Dilute acid (DA) pretreatment is one of the most investigated methods, typically conducted using dilute sulfuric acid at moderate to high temperatures.7−9 DA enhances the digestibility of lignocelluloses mainly by hydrolyzing hemicelluloses. Satisfactory levels of cellulose saccharification have been reported for agricultural residues and some hardwood species. For the befoul process to be cost-effective, ample and inexpensive availability of the feedstock with little alternative uses is necessitated. Woody biomass fits well in this regard due to low cost and global availability. However, lignin is one of the major recalcitrants present in biomass reducing cellulose accessibility for biomass conversion. It retards the glucan hydrolysis either by acting as physical barrier between cellulase and cellulose or by nonproductively binding with the cellulase due to its hydrophobic nature (Gu et al. 2013).10 Sulfite pretreatment has lately received attention as effective pretreatment method for wood species.11−13 During sulfite pretreatment, possibly lignin gets sulfonated at C-α of the side chain leading to breaking of α-O-4 and β-O-4 linkages between phenyl propane units to create new free phenolic hydroxyl groups. This results in the formation of lignosulfonate (LS) fragments and insoluble residual sulfonated lignin with increased hydrophilicity.14 The reduced electrostatic interaction caused by the increased negative charge on lignin reduces the nonproductive binding between lignin and cellulases (Del Rio et al. 2011).13−15 This increases the likelihood of cellulosecellulase binding thereby improving the glucan conversion. Shuai et al. (2010)12 employed SPORL and DA pretreatment on spruce and achieved 91% glucose conversion at 24 h for the former and 55% at 48 h for later at same enzyme loading. Improved sugar and ethanol yields were obtained by SPORL pretreatment over DA pretreatment when applied on four wood poplar genotypes (Wang et al., 2012).16 Acacia mangium, native of Australia, is one such fast growing woody biomass largely available in tropical regions, particularly South Asia, Africa and America.17 Limited literature is available on A. mangium as a feedstock for producing fermentable sugars. Kaida et al. (2009)18 examined ultrasonic pretreatment and saccharification of A. mangium trunk resulting into very low sugar yields. Boondaeng et al. (2015)19 employed alkaline pulping as the pretreatment process (steam explosion at 190− 200 °C for 5 min) followed by alkaline peroxide delignification (80 °C/45 min) for A. mangium and A. hybrid. In the current study, a systematic investigation on pretreatment of A. mangium has been conducted in one pot with the following objectives, (i) screening and selection of optimum pretreatment conditions for A. mangium using dilute acid (DA), (ii) evaluating the impact of supplementing sodium sulfite (SS) in improving enzymatic saccharification, (iii) an insight into the physicochemical alterations occurring in native and DA/SS pretreated A. mangium (by BET method, FT-IR and SEM), (iv) correlation of enzymatic hydrolysability with the physicochemical features and cellulase adsorption kinetic parameters of pretreated biomass to decipher the reasons for improved enzyme saccharification.
Research Article
EXPERIMENTAL SECTION
Materials. A. mangium was collected from Maharashtra (18°55′ lattitude, 72°54′ longitude, India) in the month of July (2014). It was air-dried, shredded and sieved to the particle size ∼2 mm by Willy knife mill and stored in airtight containers at 25 °C until further use. All experiments were conducted using single lot of biomass. Pure cellulose (Avicel PH101) was obtained from Sigma-Aldrich (India). Sulfanilic acid, xylose, glucose, cellobiose, galactose, arabinose, acetic acid, 5-hydroxymethylfurfural (HMF), furfural, sulfuric acid, sodium sulfite, calcium carbonate and formic acid were obtained from Merck (India). Cellulase enzyme (SacchariSEBC6) was obtained from M/S Advanced Enzymes Technologies Ltd. (Mumbai, India) and contained the following activities: β-glucosidase (5930 IU/g), filter paper activity (265 FPU/g), endoglucanase (4625 IU/g) and endoxylanase (64867 IU/g). Protein content as determined by Bradford dye binding method20 was 115 mg/g preparation. βglucosidase activity, FPU and endoglucanase (CMCase) activities were determined as described by Jägar et al. (2001)21 and Panda et al. (1987)22 respectively. Design of Experiments for Pretreatment of A. mangium. To arrive at an optimum pretreatment condition, a set of seven experiments varying conditions such as acid concentration, residence time and operational temperature were conducted. The temperature range was varied between 150 and 180 °C, acid concentration as 1 and 2% (w/w) and residence time as 20−40 min at a constant biomass loading of 10% (w/w). The 2 L reactor system made up of hastelloy consisted of PID temperature controller, digital pressure control, nitrogen pressure and sample inlet/outlet along with stirrer. The heating up time of reactor was ∼20 min. The system had an additional feature to add liquid catalyst to the biomass at a desired temperature by pressure equalizing mechanism and a rapid cooling system by circulating chilled water in reactor internal tubes. After the completion of the reaction, the slurry was separated into “cellulose-rich solid” and “xylose-rich liquid” streams. The solid residue was washed with deionized water (2−3 times) using 10 times the weight of the residue each time. These washing liquids were analyzed with respect to sugar concentrations and degradation products and the respective solid residues were perused for enzymatic saccharification as described below. On the basis of the highest enzymatic saccharification efficiency, the basal pretreatment condition was chosen. Analysis of Xylose Rich Hydrolysate. The xylose rich hydrolysates obtained were neutralized using calcium carbonate to pH 5−6, clarified through 0.22 μm filter and subjected to sugar analysis using HPLC (Waters, Switzerland) fitted with Biorad Aminex HPX-87H column at 50 °C equipped with a guard column. Sulfuric acid (0.005M) was used as mobile phase at 0.6 mL/min. Sugars, acetic acid and formic acid were analyzed by Refractive Index detector and other degradation products, HMF and furfural by UV detector.23 Enzymatic Saccharification. Five grams (dry basis) of pretreated cellulose rich biomass was suspended in 45 mL of 0.05 M sodium citrate buffer (pH 4.8) containing 0.02% sodium azide. The final solid loading of 10% (w/w) was maintained. Mixture preincubated at 50 °C for 10 min, 20 FPU (per g solid residue) of cellulase preparation was added and incubation was perused at 50 °C and 200 rpm up to 72 h. Samples were withdrawn at varying intervals and analyzed for sugars through HPLC as described above. Compositional Analysis of Cellulose Rich Residue. Moisture content of samples was determined according to NREL LAP24 using an infrared drier from Sartorius MA-150C (Model No. 000230 V1), Germany. The compositional analysis of native and pretreated A.mangium was carried out by two stage acid hydrolysis following the standard protocol of NREL.25 Supplementation of Sodium Sulfite. After selection of basal pretreatment condition, six set of experiments were conducted varying with respect to the sequence of the chemicals viz. H2SO4 and Na2SO3, added during the pretreatment. The reactor containing 10% (w/w) A. mangium (AM) biomass with final slurry volume of 1 kg was heated to the desired temperature with or without H2SO4/Na2SO3. These were either added together along with the biomass or in sequence, one B
DOI: 10.1021/acssuschemeng.6b00758 ACS Sustainable Chem. Eng. XXXX, XXX, XXX−XXX
Research Article
ACS Sustainable Chemistry & Engineering Table 1. Overall Saccharification Efficiency of Pretreatment and Enzymatic Hydrolysis components of xylose rich hydrolysateb (g)
conditions
[T/t/C]
a
150/20/1 150/30/1 160/30/1 160/30/2 160/40/2 180/30/1 180/30/2
CSF
glucose
xylose
2.8 2.9 3.2 3.5 3.7 3.8 4.1
3.3 ± 0.1A 2.9 ± 0.2A 5.1 ± 0.1B 9.4 ± 0.2C 10.9 ± 0.2D 14.6 ± 0.1E 13.4 ± 0.2F
16.8 ± 0.1A 16.1 ± 0.2B 13.1 ± 0.2C 8.2 ± 0.2D 5.4 ± 0.2E 1.6 ± 0.1F 1.0 ± 0.2G
HMF 0.1 0.1 0.3 0.4 0.3 1.3 0.9
± ± ± ± ± ± ±
0.01A 0.01A 0.01A 0.01B 0.02A 0.04B 0.03C
glucose rich residue
furfural
glucose equivalentc (g)
glucose releasedd (%)
± ± ± ± ± ± ±
3.1 ± 0.1A 2.7 ± 0.2B 4.9 ± 0.3C 8.9 ± 0.2D 10.2 ± 0.3E 14.6 ± 0.4F 13.1 ± 0.2F
5.9 ± 0.2A 5.2 ± 0.2A 9.4 ± 0.1B 17.0 ± 0.5C 19.4 ± 0.4D 28.0 ± 0.3E 25.0 ± 0.3F
1.0 1.4 2.6 4.4 4.2 5.2 3.1
0.1A 0.1B 0.1C 0.2D 0.1D 0.1E 0.1F
residual glucosee (%) 94.1 94.8 90.6 83.0 80.6 72.0 75.0
± ± ± ± ± ± ±
0.2A 0.2A 0.3B 0.2C 0.3D 0.3E 0.5F
hydrolysis yieldf (%) 31.5 45.8 50.1 60.3 52.9 56.2 58.9
± ± ± ± ± ± ±
0.2A 0.2B 0.3C 0.3D 0.2E 0.2F 0.2G
overall conversion efficiency to glucose (%) 29.6 43.4 45.4 50.0 42.6 40.5 44.2
± ± ± ± ± ± ±
0.1A 0.4B 0.2C 0.3D 0.1E 0.2F 0.2G
a
T, t and C refers to pretreatment temperature (°C), residence time (min) and acid concentration (% w/w), respectively. bAmount of components determined by HPLC is presented in 900 mL of xylose rich hydrolsate. cGlucose equivalent = (glucose (g) + 1.29 × HMF (g)) × 0.9. dGlucose released (%) = glucose equivalent/glucose present in 100 g native biomass. eResidual glucose = 100 − glucose released. fHydrolysis yield = Glucose obtained in enzymatic hydrolysis × 100. gOverall conversion efficiency to glucose = (% residual glucose) × (% hydrolysis yield) . Glucose present in pretreated biomass
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catalyst added before and the other after the reactor temperature reached 160 °C (selected value). The reaction was allowed to proceed for 30 min, after which cooling commenced. The slurry thus obtained was filtered to yield xylose rich hydrolyzate and cellulose rich residue. The native and pretreated solid residues were abbreviated as AM (native A. mangium), W (water treated), DA (H2SO4 treated AM), SS (Na2SO3 treated AM), SS→DA (addition of Na2SO3 with biomass followed by H2SO4), DA→SS (addition of H2SO4 with biomass followed by Na2SO3), DA+SS (addition of H2SO4 and Na2SO3 together with the native AM). Statistical Analysis. The compositional analysis and enzymatic hydrolysis data have been reported as the average of three replicates. Standard deviation of each data point has been calculated by one way ANOVA using the post hoc Tuckey test available at Statistica.mooo.com. p < 0.05 was set as the level of statistical significance. FT-IR and SEM. Diffuse reflectance spectra of biomass samples were recorded using Shimadzu (Model IRPrestige21), FTIR spectrometer. All spectra were recorded in the absorbance mode from an accumulation of 200 scans at a 4 cm−1 resolution over the range 4000−400 cm−1. Microcrystalline cellulose (Avicel PH 101) was used as the reference materials. The zero baseline correction with 10 points smoothing using KubelkaMunk correction was used. The FT-IR spectra were normalized with respect to 1504 cm−1. SEM images were conducted at 2000× magnifications using HITACHI S-3400 SEM. The specimens to be coated were mounted on a conductive tape and coated with gold using a fine coater (2 nm) and observed at an accelerating voltage of 3.0 kV. Adsorption of Enzyme on Biomass. Protein adsorption experiments were performed at 4 °C in 0.05 M sodium citrate buffer (pH 4.8) in 15 mL glass vials. The substrate (native or pretreated residues) loading of 1.25% (w/w) with enzyme loadings of 0 to 60 mg protein/g substrate was maintained. The vials containing substrate, buffer and protein were mounted on a variable rotator and incubated for overnight at 100−150 rpm at 4 °C. Thereafter, solid−liquid separation was performed by centrifugation at 5000 rpm for 10 min. The solid residues were dried overnight at 105 °C. The adsorbed protein amount was directly determined by the nitrogen factor method.26,27 The nitrogen content of the dried and homogenized biomass solids was measured using a CHNS/O analyzer (Vario EL III) with sulfanilic acid as a standard. The Langmuir isotherm model was used to describe the equilibrium adsorption behavior according to the following equation.28
[CE] =
σmax[St][Ef ] Kd + [Ef ]
100
RESULTS AND DISCUSSION Screening of Pretreatment Conditions. The pretreatment parameters: temperature, acid concentration and residence time were varied between 150 and 180 °C, 1.0− 2.0% and 20−40 min, respectively. Table 1 shows the components of the xylose rich hydrolyzate, viz. pentoses (xylose and arabinose), hexoses, HMF and furfural released across a different set of pretreatment experiments. Combined Severity Factor (CSF) which is a function of temperature, acid, concentration and residence time was calculated as {log10 (acid conc.× residence time × ((Temp-100)/14.5))}. CSF increased from 2.8 to 4.1 resulting in a reduction in xylose from 16.8 to 1.0 g/L attributed to its degradation to furfural (1.0 to 5.2 g/L). At CSF = 4.1, xylose is lowest in concentration while furfural is also less due to its further degradation to formic acid. At CSF = 4.1, formic acid formation was highest (4.9 g/L) among all the conditions viz. CSF 2.8−3.8 releasing 0.0−2.2 g/L formic acid. The decrease in xylose was also concomitant with the breakdown of cellulosic material reflected by an increase in glucose from 3.3 to 13.4 g/L in the hydrolysate with its subsequent degradation to HMF (0.1−1.3 g/L). Screening and selection of an optimum pretreatment condition was based on the overall glucose recovery during the pretreatment and enzymatic hydrolysis rather than only enzymatic hydrolysis of the resulting biomass residues. Table 1 shows that an increase in severity from 2.8 to 3.5 resulted in an increase in overall glucan conversion efficiency from 29.6 to 50.0% including glucose released in pretreatment and recovered after enzymatic hydrolysis. CSF = 3.5 was observed to be the optimum severity above which the glucan conversion did not increase. This may be attributed to higher glucan solubilization at CSF > 3.5 in the pretreatment itself and the formation of high amount of degradation products, i.e., HMF, furfural and formic acid. Thus, the condition at CSF = 3.5 corresponding to temperature = 160 °C, acid concentration = 2.0% and residence time = 30 min resulting into highest conversion efficiency (50.0%) was selected as the basal pretreatment condition. This efficiency, however, corresponded to the glucan conversion of 60.3% based on the residual cellulose content in pretreated biomass. It is evident from Table 1 that the higher severities resulted in relatively higher enzymatic hydrolysis. The optimum pretreatment condition was chosen on the basis of overall glucose recovery in the pretreatment hydrolysate and enzymatic hydrolysis. In order to further improve the sugar recovery with scale-up point of view, residence time and temperature were
(1)
Where [CE] is the amount of adsorbed enzyme in mg/mL, [Ef] is the free enzyme concentration in mg/mL, σmax is the maximum adsorption capacity of enzyme in mg/mg substrate, [St] is the substrate concentration in mg/mL and Kd is the equilibrium constant in mg/ mL equal to [C][E]/[CE]. C
DOI: 10.1021/acssuschemeng.6b00758 ACS Sustainable Chem. Eng. XXXX, XXX, XXX−XXX
Research Article
ACS Sustainable Chemistry & Engineering Table 2. Pretreatment Process Parameters and Compositional Analysis of Various Biomass Samples removal during pretreatment (%)
composition of pretreated biomass (%)
sample ID AM W DA SS SS→DA DA→SS DA+SS SS→DA
a
pretreatment conditions [T/t/C1/ C2]b N/A 160/30/0/0 160/30/2/0 160/30/0/0.5 160/30/2/0.5 160/30/2/0.5 160/30/2/0.5 160/30/2/1
solid recoveryc (%) N/A 85.0 ± 68.5 ± 89.8 ± 65.1 ± 65.7 ± 66.7 ± 69.6 ±
0.3A 0.2B 0.2C 0.1D 0.4D 0.2E 0.3B
glucan 47.6 49.3 47.9 45.6 52.7 51.3 53.3 58.7
± ± ± ± ± ± ±
0.2A 0.2B 0.3C 0.2D 0.3E 0.3D 0.2C
xyland
lignine
17.4 12.0 ± 0.1A 0.1 ± 0.1B 17.7 ± 0.2C 0.1 ± 0.1B 1.8 ± 0.1D 0.1 ± 0.1B 0.1 ± 0.1D
27 33.7 49.8 31.9 43.6 44.7 44.2 39.8
± ± ± ± ± ± ±
0.1A 0.2B 0.2C 0.3D 0.3D 0.2D 0.1E
ash 1.8 1.1 1.0 1.0 0.5 0.5 0.6 1.3
± ± ± ± ± ± ±
0.1A 0.2A 0.2A 0.1B 0.1B 0.1B 0.2A
xylanf N/A 41.2 ± 99.7 ± 8.40 ± 99.7 ± 93.3 ± 99.8 ± 99.6 ±
0.2A 0.2B 0.2C 0.2B 0.3D 0.2B 0.2B
ligning
glucose yield, 72 h (%)
N/A −6 −26.3 −6.2 −5.2 −8.9 −9.3 −2.7
8 12.8 57.8 33.8 77.0 51.9 64.3 80.2
± ± ± ± ± ± ±
0.2A 0.4B 0.3C 0.2D 0.2E 0.2F 0.2F
a AM, native A. mangium; W, water treated AM; DA, H2SO4 treated AM; SS, Na2SO3 treated AM; SS→DA, pretreatment by sequential addition of Na2SO3 and H2SO4; DA→SS, pretreatment by sequential addition of H2SO4 followed by Na2SO3; DA+SS, addition of mixture of H2SO4 and Na2SO3 together with the native biomass. bT, t, C1 and C2 refer to pretreatment temperature (°C), residence time (min), H2SO4 concentration (% w/w) and Na2SO3concentration (% w/w), respectively. cSolid recovery: mass of total solid recovered after pretreatment/mass of total biomass taken for pretreatment (on dry weight basis) × 100. dXylan (%) in pretreated residue = ((0.88 × xylose) + (0.88 × arabinose) + (0.78 × acetic acid) + (1.32 × furfural))/W × 100, where xylose, arabinose, acetic acid and furfural are in mg; W is the oven dry weight of initial biomass in mg. eLignin (%) in pretreated residue = (W2 − W1)/W × 100, where W is the oven dry weight of initial biomass in mg; W1 is the acid insoluble biomass after overnight drying at 105 °C; W 2 is the weight of residue left after heating at 550 °C for 4−5 h. f Xylan removal =
100 −
(% xylan in pretreated residue × % solid recovery after pretreatment) g . Lignin % xylan in the native biomass
removal = 100 −
(% lignin in pretreated residue × % solid recovery after pretreatment) ; % Lignin in the native biomass
negative values
of lignin removal indicate increase in lignin content in the pretreated residues.
by SS→DA during the enzymatic hydrolysis as shown in Table 2. The impact of sodium sulfite alone on the hemicellulose removal was seen by comparing the conditions “W” and “SS”. Whereas the former removed 41.2% hemicelluloses, the latter, known to be a delignifying agent, did not attack hemicellulose, but reduced lignin content from 33.7 to 31.9%. Although the significance of using SS for pretreatment does not show up here due to a very small concentration of SS, its addition in complementation with H2SO4 (DA) is noticeable during the subsequent enzymatic hydrolysis (77.0%) as shown in Table 2. In SS→DA, the addition of SS prior to DA brings the pH of the medium to approximately 5.0−5.2 until DA is added. During this period, lignin becomes vulnerable resulting in the formation of free phenyl hydroxyl groups. This may facilitate its sulphonation as well as depolymerization (Rio et al., 2013). Consequently, a reduction in residual lignin content from 49.8 in DA to 43.6% in SS→DA was observed (Table 2). Partial solubilization of sulfonated lignin renders the insoluble residual sulfonated/modified lignin more hydrophilic in nature. Thus, the nonproductive binding of lignin originating from hydrophobic interaction with the cellulase is reduced.30 As a result, the availability of cellulase for cellulose increased thereby, increasing the propensity of cellulose hydrolysis. However, this kind of sulfonation does not occur during DA pretreatment due to the formation of condensed lignin.12 Although removal of hemicellulose is the same in both the conditions (99.7%), the biomass in SS→DA condition became enriched in cellulose (47.9 versus 52.7%), which might be responsible in improving subsequent enzymatic cellulose conversion as discussed later. When acid was added along with the biomass followed by SS (DA→SS), by the time the reactor temperature reached 160 °C, it might have already solubilized a lot of hemicellulose leaving 1.8% in the solid. However, this residual hemicellulose content was higher than the other acid treatment conditions (DA, SS→DA and DA+SS) and may be attributed to the reduction in acidity of the medium brought about by SS addition.
increased, however, that resulted in degradation of glucose to HMF. This necessitated an improvement methodology in order to save the sugar loss and while getting better enzymatic hydrolysis. Supplementation of Sodium Sulfite. For pretreatment of woody biomass, standalone SPORL (sulfite pretreatment to overcome recalcitrance of lignocellulose) has been reported to be an efficient technology at a very high dosage.11,12 Hence, further intensification of process conditions was carried out by supplementing the basal pretreatment conditions (160 °C/30 min/2%) with 0.5 and 1.0% sodium sulfite (SS), which are very low concentrations compared to the ones used in comparison to SPORL. The addition of SS and H2SO4 was conducted either together or in sequence one after the other, and the potential of SS was investigated as an agent in improving the cellulase accessibility of A. mangium. Compositional Analysis of Cellulose Rich Pretreated Solid. Table 2 shows the pretreatment conditions and effect of sodium sulfite on the composition of resultant biomass. Solid recovery across the different conditions varied between 65.1 and 89.80%, mainly due to the solubilization of hemicelluloses. A direct relationship between the solid recovery and residual hemicellulose content in the solid was observed. The native biomass, AM consisted (%, w/w): glucan: 47.6, xylan: 17.4, lignin: 27.0, ash: 1.8 and extractives: 7.18. Interestingly, the hemicellulose content in the pretreated residues of DA, SS→ DA, DA→SS and DA+SS showed that almost all hemicellulose broke down and solubilized during the process, leaving the residue somewhat enriched in glucan with 47.9, 52.7, 51.3 and 53.3% (w/w) respectively. Apart from glucan, a surge in residual lignin content was also observed which may be attributed to the formation of pseudolignin. Pseudolignin formation from carbohydrates has also been reported by Hu et al. (2012)29 under DA pretreatment conditions. The synergistic effect of SS and DA was emphasized by conducting individual experiments exclusively with SS and DA resulting in a lower glucose yield of 33.8 and 57.8% in comparison to 77.0% for that D
DOI: 10.1021/acssuschemeng.6b00758 ACS Sustainable Chem. Eng. XXXX, XXX, XXX−XXX
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ACS Sustainable Chemistry & Engineering
improvement in cellulose conversion. However, this presumption of low lignin content being a cause for high cellulose accessibility failed when conditions with similar lignin content resulted in significantly different glucan conversion. DA→SS and DA+SS residues despite containing similar (44.7 and 44.2%) lignin content led to significantly different glucan conversion, i.e., 51.9 and 64.3% respectively. This difference may be attributed to the lower hemicellulose content, i.e., 0.1 and 1.8% in the DA+SS and DA→SS residues. These results show that the removal of hemicellulose was more critical than lignin content for high glucan conversion. Zhang et al. (2013)30 have also focused on crucial role of hemicellulose removal over lignin removal during SPORL pretreatment of switchgrass and attained better digestibility than DA pretreatment.31 To establish the positive impact of sodium sulfite, we have conducted experiment using 1% of sodium sulfite followed by DA which resulted in an increase of 3% glucose yield. This further confirms the beneficial impact of sodium sulfite supplementation. Cellulase Adsorption and Glucan Conversion. The first step in cellulose digestion is the cellulase adsorption onto it.14,32 This phenomenon is affected by enzyme/substrate features and various physicochemical parameters.33,34 To get an insight into the basis for difference in cellulase mediated glucan conversion across the pretreated biomasses, in the current study, their cellulase adsorption capacities were compared. The adsorption parameters were determined by nonlinear regression of maximum adsorption capacity (σmax) for untreated and pretreated biomass using Langmuir isotherm (eq 1). Table 3 gives the summary of kinetic parameters, viz.
The reagents when added together (DA+SS), again led to high efficiency of hemicellulose removal (residual 0.1%). Therefore, supplementation of SS either along with the acid and biomass or following the addition of DA gave comparable results as far as the hemicellulose removal is concerned with little gain in lignin removal during the former case. However, the implication of this effect may be perceived by evaluating the performance during enzymatic saccharification of the corresponding pretreated residues. Enzymatic Saccharification. The glucan conversion yield of pretreated A. mangium residue obtained after each pretreatment condition is represented in Figure 1. Clearly,
Figure 1. Enzymatic saccharification of A. mangium pretreated under various conditions.
Table 3. BET Surface Area and Cellulase Adsorption Parameters
water (W) removed up to 41% hemicellulose (Table 2) at 160 °C during a residence time of 30 min, however, did not result into noticeable glucan conversion (12%). DA, which resulted in >99% hemicellulose removal was also observed to be limited by its ability to adequately target the recalcitrant factors of A. mangium leading to only 57.8% glucan conversion. Thus, hemicellulose removal was considered a desirable but not sufficient for exposing cellulose completely. Table 2 shows, although, DA, DA+SS and SS→DA treated biomasses retained equal amounts of xylan (0.1%), their glucan conversion yields were significantly different as 57.8 and 64.3 for DA and SS+DA respectively, while, 77% for SS→DA. In SS→DA, the addition of SS prior to DA brings the pH of the medium to approximately 5.0−5.2 until DA is added. During this period, lignin becomes vulnerable to the formation of free phenyl hydroxyl groups. This may facilitate its sulphonation as well as depolymerization.15 This reduces the probability of nonproductive binding of lignin with cellulase due to reduced hydrophobic interaction.30 Sodium sulfite, when added before the acid, might have resulted in partial deploymerization/dissolution of lignin and increase its hydrophilicity by sulphonation facilitating its detachment from hemicelluloses.30 Eventual addition of H2SO4 targeted hemicellulose, efficiently enriching the biomass in cellulose (52.7%) and exposing it to enzymatic attack. Thus, the lowest lignin (43.6%) present in the SS→DA residue in addition to high hemicellulose removal may be ascribed for its highest glucan conversion (77%). Here, W and SS were not included as these conditions did not lead to appreciable hemicelluloses removal, which has direct impact on solid recovery. Thus, the idea of targeting lignin using sodium sulfite was presumed as a potential alternative to achieve further
substrate
BET surface area (m2/g)
max. adsorption capacity, σmax (mg/g substrate)
equilibrium constant, Kd (mg/mL)
affinity constant, Ka (mL/mg)
AM W DA SS SS→DA DA→SS DA+SS Avicel
1.0 1.4 2.3 2.3 3.7 2.8 3.2 1.0
44.2 48.7 35.4 34.3 29.2 38.7 34.0 39.9
19.5 16.6 8.4 9.2 4.0 10.1 6.7 4.5
0.05 0.06 0.12 0.11 0.25 0.10 0.15 0.22
maximum adsorption capacity (σmax): defined as the amount of adsorbate per unit gram of adsorbent; equilibrium constant (Kd), which represents the dissociation of an enzyme−substrate complex; and affinity constant (Ka), which describes the binding affinity of two molecules at equilibrium. The results showed that the addition of sodium sulfite followed by sulfuric acid (SS→DA) had the lowest σmax (29.2 mg/g) contrary to the expectation based on its highest glucan conversion among all pretreated residues. This sample also showed maximum BET surface area of 3.7 m2/g and this may be prominent factor for enhanced glucan conversion. This may be the result of relatively higher lignin removal/alteration as evidenced by the FTIR analysis of the residues (Figure 5). The DA, SS and DA+SS had almost same or moderate σmax (34.0 to 35.4 mg/g substrate) values, whereas the maximum σmax was shown by DA→SS as 38.7 mg/g substrate. Higher σmax values may be a result of nonspecific binding of cellulase to the E
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Figure 2. Correlation of maximum adsorption capacity of substrate (σmax) and enzymatic hydrolysis (%). Glucose yield was determined at 4 and 72 h of hydrolysis of native and pretreated A. mangium using 20 FPU/g Advanced enzyme.
Figure 3. Correlation of affinity constant (Ka) and enzymatic hydrolysis (%). Glucose yield was determined at 4 and 72 h of hydrolysis of native and pretreated A. mangium using 20 FPU/g Advanced enzyme.
residual lignin in the pretreated biomass.32,14 DA residue has higher lignin content than DA→SS, yet lower maximum adsorption capacity. In DA pretreatment, lignin content increases due to hemicellulose removal and likely pseudolignin formation. During this process, lignin undergoes condensation into droplets and migrate to the surface of cellulose, possibly leading to reduced cellulose adsorption. This may be due to lignin condensation into droplets that may migrate to the surface of cellulose during DA pretreatment, thereby leading to reduced cellulose adsorption.35 However, the nature of lignin is anticipated to change when sulfite is being used. Therefore, it may not be a good idea to evaluate saccharification efficiency based upon σmax values alone as it is total adsorption efficiency including nonspecific irreversible binding. Partial fragmentation and sulphonation of lignin following sodium sulfite addition might result in the formation of lignosulfonates (LS).14 As a result of SS addition, LS fragments bind to the cellulose due to electrostatic interaction forming LSC complex. Whereas, binding of lignin with cellulase is completely nonproductive, LSC complex is reported to facilitate glucan conversion depending upon the pH and molecular weight of the LS fragments. Lou et al. (2014)36 referred this mechanism to “LS− cellulase aggregate stabilization and enhanced cellulose binding.” Wang et al. (2013)14 reported that LS−cellulase aggregate binds to a hydrophobic cellulose site, the hydrophilic group of LS faces toward water to produce a stable binding between cellulase and cellulose resulting in enhanced glucan conversion. Following this, in our study, SS→DA may have resulted in better sulfonated/fragmented lignin for LSC complex formation and improved enzymatic saccharification. A correlation was drawn to account for the relationship between the maximum adsorption capacity (σmax) and glucan conversion at 4 and 72 h of hydrolysis across the native and pretreated samples. A significant negative correlation was
observed with R2 = 0.62 at 4 h and R2 = 0.77 at 72 h (Figure 2). It is apparent that the W residue had the highest adsorption of cellulases followed by acid and sodium sulfite (DA, DA→SS, DA+SS) treated residues (34.0−38.7 mg/g substrate) with SS→DA being exceptionally low (29.2 mg/g substrate). The equilibrium constant, Kd of the untreated and pretreated biomass for cellulase was in accordance with the σmax values (Table 3) having inverse relationship with glucose (Figure S1) (R2 = 0.76 and 0.91 for 4 and 72 h, respectively). However, a positive correlation was observed when affinity constant “Ka”, which reflects the firmness of enzyme−substrate binding versus glucose yield, was plotted (R2 = 0.73 and 0.77 for 4 and 72 h, respectively). Among the kinetic parameters, the affinity constant of cellulose to cellulase was found to be the only one to account for the difference between the six pretreated substrates in adsorbing enzymes leading to higher glucan conversion (Figure 3). High affinity constant signifies tighter hold of enzyme on to the substrate attributed to the formation of more adsorption sites on cellulose post pretreatment/ sulphonation, finally leading to higher enzymatic hydrolysis. Highest affinity constant value for SS→DA (0.25 mL/mg) is also supported by its highest surface area (3.7 m2/g) in comparison to other pretreatment conditions (1.0−3.2 m2/g) as shown in Table 3. The results signified that it is not necessary that a faster and better cellulose hydrolysis is always the consequence of higher enzyme adsorption capacity (σmax) of the biomass. Apart from cellulose, even the nonproductive binding of enzymes to lignin may result in inflated values of adsorption capacity and impeded saccharification. Impact of Surface Area on Glucose Yield. The specific surface areas of native/pretreated biomasses and Avicel as measured by BET equation are presented in Table 3. The values ranged from 1.0 to 3.7 m2/g with the surface area of F
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Figure 4. Correlation of BET surface area and enzymatic hydrolysis (%). Glucose yield was determined at 4 and 72 h of hydrolysis of native and pretreated A. mangium using 20 FPU/g Advanced enzyme.
SS→DA residue being the highest (3.7 m2/g) among other pretreated residues and followed the order SS→DA > DA+SS > DA→SS > DA=SS > W>AM. This is because pretreatment results in reduction in size of biomass, removal of xylan and removal/redistribution of lignin consequently, increasing the surface area. Thus, water treated and native A. mangium showed lowest surface areas, viz. 1.4 and 1.0 m2/g, respectively. Several studies have also indicated that the breakdown and loosening of the compact biomass architecture by various pretreatment methods led to increased specific surface area.34 The glucan conversion of pretreated residues was evaluated at 4 and 72 h of hydrolysis. It appears that specific surface area is one of the most crucial physicochemical characteristics that facilitates strong initial binding of enzyme to substrate and bears a positive correlation (R2 = 0.80) with glucan conversion (Figure 4). Similar trend continues until 72 h of hydrolysis as shown by the R2 value (0.92). It suggests that higher surface area leads to increased cellulose accessibility to enzymes reflected by glucose yield. Kapoor et al. (2015)5 have reported increased glucan conversion due to increase in surface area of mustard stalk pretreated by DA, alkali and steam explosion. FT-IR spectroscopy. FT-IR spectroscopic analysis was employed to investigate the alteration of chemical composition and molecular conformation in A. mangium. The analyses of the spectra was based upon the assignments given by previous investigations.37 Table 4 summarizes the peak assignments for different functional groups. Figure 5 shows that all the samples showed broad bands at 3350−3460 and 2897−2905 cm−1, attributed to the stretching of −OH groups and to CH stretching, respectively, corresponding to the aliphatic moieties in cellulose, and residual lignin. The band at 1735 cm−1
corresponding to CO and CO bonds of the acetyl ester units originating from residual hemicelluloses (in AM, W and SS residues) either reduced in intensity or shifted to lower wavelength (1703 cm−1) in the case of pretreated residues showing removal of hemicellulose. This resulted in increased enzymatic hydrolysis from 12.8 to 77.0%. Consequent enrichment in pretreated residues was evident by the increased strength of bands around 1072 and 1123 cm−1 associated with cellulose CO bond and ring stretching frequencies, respectively. The peak intensities in the range of 1327−1508 cm−1 were ascribed for lignin. Band intensity at 1508 cm−1 was attributed to the skeletal and stretching vibration of aromatic moieties in lignin. The CO in the alkyl groups of the lignin side chains has been suggested to conjugate with the aromatic structure and resulting in an absorption peak at 1650 cm−1.38 In comparison to the AM, W and DA residues, SS→DA, DA→SS and DA+SS samples showed a flattening of the shoulder at 1650 cm−1 indicating delignification, further supported by the weakening of relative intensity at 1425 and 1456 cm−1 (attributed to bending vibration of the methoxyl on benzene rings) implying the removal of methoxyl in the lignin. This observation was confirmed by the variation of the bands at 1270 and 1230 cm−1, attributed to the aromatic core of guaiacyl and syringyl, respectively. The absorption peak due to aromatic CH is observed at 1370 cm−1 generated by cleavage of ether bonds within the lignin. The intensity of this band decreased after sulfite pretreatement due to removal of lignin. This effect was most prominent for SS→DA sample showing a flattening at band position 1370 cm−1 (CH bending 1370) and 1329 cm−1 (CC, CO vibration absorption). Lignin removal may be ascribed for highest enzymatic hydrolysis (77.0%) achieved for SS→DA residue. Critical analysis of FT-IR spectra reflected a sulfonated lignin peak appearing in all the cases wherein SS was used irrespective of any order. Therefore, the formation of sulfonated lignin could be one of the important factors which improved enzymatic hydrolysis. Moreover, this peak was absent in water, native A. mangium and Avicel. The alterations in cellulose and lignin related peaks in sulfite and acid pretreated samples suggest a beneficial effect during enzymatic hydrolysis. The effect being the most important for SS→DA residue that shows highest alteration in structure with respect to lignin supports the lowest cellulase adsorption capacity (29.2 mg/g) of SS→DA sample. Scanning Electron Microscopy (SEM). Surface morphological information on the substrate before and after the pretreatment was studied using SEM microscopy (Figure 6). The untreated sample exhibits the regular, compact surface structure and a highly fibrillated, intact and smooth
Table 4. Assignment of FT-IR Adsorption Bands (cm−1) adsorption bands (cm−1) 3360 2913 1735 1650 1603, 1508 1456 1329 1242 1123 1043 899
assignment OH stretching vibration in cellulose CH stretching vibration CO stretching vibration in acetyl group of hemicellulose CO stretching in alkyl groups of lignin side chains CC stretching vibration in aromatic ring of lignin bending vibration of methoxyl on benzene ring syringyl ring breathing with CO streching CO stretching vibration in lignin and hemicellulose aromatic CH in-plane deformation for syringyl type CO stretching vibration in cellulose and hemicellulose COC stretching at the β-(1→4)-glycosidic bond G
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Figure 5. FT-IR spectrum of native and pretreated A. mangium. The pretreatment conditions were 160 °C and residence time of 30 min. Spectra of Avicel is shown as a reference.
Figure 6. Scanning electron microscope (3.0 kV, 2000× magnification) of native and pretreated A. mangium with pretreated condition of 160 °C for 30 min.
bioconversion as is evident by the highest glucose yield (77%) obtained for the same pretreatment.
morphology. DA and DA+SS pretreatments caused defibration of biomass surface resulting in structural breakdown and cracks development.39 Finally, the structure of A. mangium was further degraded after pretreatment resulting into an irregular and broken structure (SS→DA). Increase in porosity results in enhanced exposure of cellulosic material for effective
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CONCLUSION
In the current study, pretreatment conditions have been optimized for a woody biomass Acacia mangium. On the basis H
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(4) Sharma, S.; Kumar, R.; Gaur, R.; Agrawal, R.; Gupta, R. P.; Tuli, D. K. Pilot scale study on steam explosion and mass balance for higher sugar recovery from rice straw. Bioresour. Technol. 2014, 98, 696−699. (5) Kapoor, M.; Raj, T.; Vijayaraj, M.; Chopra, A.; Gupta, R. P.; Tuli, D. K.; Kumar, R. Structural features of dilute acid, steam exploded, and alkali pretreated mustard stalk and their impact on enzymatic hydrolysis. Carbohydr. Polym. 2015, 124, 265−73. (6) Raj, T.; Kapoor, M.; Semwal, S.; Sadula, S.; Pandey, V.; Gupta, R. P.; Kumar, R.; Tuli, D. K.; Das, B. P. The cellulose structural transformation for higher enzymatic hydrolysis by ionic liquids and predicting their solvating capabilities. J. Cleaner Prod. 2016, 113, 1005−14. (7) Gaur, R.; Soam, S.; Sharma, S.; Gupta, R. P.; Bansal, V. R.; Kumar, R.; Tuli, D. K. Bench scale dilute acid pretreatment optimization for producing fermentable sugars from cotton stalk and physicochemical characterization. Ind. Crops Prod. 2016, 83, 104−12. (8) Arora, A.; Carrier, D. J. Understanding the Pine Dilute Acid Pretreatment System for Enhanced Enzymatic Hydrolysis. ACS Sustainable Chem. Eng. 2015, 3 (10), 2423−8. (9) Kshirsagar, S. D.; Waghmare, P. R.; Chandrakant Loni, P.; Patil, S. A.; Govindwar, S. P. Dilute acid pretreatment of rice straw, structural characterization and optimization of enzymatic hydrolysis conditions by response surface methodology. RSC Adv. 2015, 5 (58), 46525−33. (10) Gu, F.; Wang, W.; Jing, L.; Jin, Y. Sulfite-formaldehyde pretreatment on rice straw for the improvement of enzymatic saccharification. Bioresour. Technol. 2013, 142, 218−24. (11) Zhu, W.; Houtman, C. J.; Zhu, J. Y.; Gleisner, R.; Chen, K. F. Quantitative predictions of bioconversion of aspen by dilute acid and SPORL pretreatments using a unified combined hydrolysis factor (CHF). Process Biochem. 2012, 47 (5), 785−91. (12) Shuai, L.; Yang, Q.; Zhu, J. Y.; Lu, F. C.; Weimer, P. J.; Ralph, J.; Pan, X. J. Comparative study of SPORL and dilute-acid pretreatments of spruce for cellulosic ethanol production. Bioresour. Technol. 2010, 101 (9), 3106−14. (13) Tian, S.; Luo, X. L.; Yang, X. S.; Zhu, J. Y. Robust cellulosic ethanol production from SPORL-pretreated lodgepole pine using an adapted strain Saccharomyces cerevisiae without detoxification. Bioresour. Technol. 2010, 101 (22), 8678−85. (14) Wang, Z.; Zhu, J.; Fu, Y.; Qin, M.; Shao, Z.; Jiang, J.; Yang, F. Lignosulfonate-mediated cellulase adsorption: enhanced enzymatic saccharification of lignocellulose through weakening nonproductive binding to lignin. Biotechnol. Biofuels 2013, 6, 156. (15) Del Rio, L. F.; Chandra, R. P.; Saddler, J. N. The effects of increasing swelling and anionic charges on the enzymatic hydrolysis of organosolv-pretreated softwoods at low enzyme loadings. Biotechnol. Bioeng. 2011, 108 (7), 1549−58. (16) Wang, Z. J.; Zhu, J. Y.; Zalesny, R. S., Jr.; Chen, K. F. Ethanol production from poplar wood through enzymatic saccharification and fermentation by dilute acid and SPORL pretreatments. Fuel 2012, 95, 606−14. (17) Patil, S.; Patil, H.; Mutanal, S.; Shahpurmath, G. Growth and productivity of Acacia mangium clones on shallow red soil. Karnataka J. Agric. Sci. 2012, 25 (1), 94−95. (18) Kaida, R.; Kaku, T.; Baba, K.; Oyadomari, M.; Watanabe, T.; Hartati, S.; Sudarmonowati, E.; Hayashi, T. Enzymatic saccharification and ethanol production of Acacia mangium and Paraserianthes falcataria wood, and Elaeis guineensis trunk. J. Wood Sci. 2009, 55 (5), 381−6. (19) Boondaeng, A.; Vaithanomsat, P.; Apiwatanapiwat, W.; Trakunjae, C.; Kongtud, W. Statistical approach for optimization of ethanol production from fast-growing trees: Acacia mangium and Acacia hybrid. BioResources 2015, 10 (2), 3154−3168. (20) Bradford, M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 1976, 72 (1), 248−54. (21) Jäger, S.; Brumbauer, A.; Fehér, E.; Réczey, K.; Kiss, L. Production and characterization of β-glucosidases from different Aspergillus strains. World J. Microbiol. Biotechnol. 2001, 17 (5), 455−61.
of the overall sugar recovery (50%), combining the pretreatment and enzymatic saccharification steps, the basal pretreatment conditions were selected by detailed optimization process; however, desirable glucan conversion could not be achieved. Further intensification of the pretreatment was accomplished by the supplemention of 0.5% sodium sulfite, which improved the glucan conversion to from 57.8 to 77%. Physico-chemical characterization of pretreated biomass revealed the favorable alterations in the lignocellulosic structure with respect to cellulose and lignin. The synergistic action for improved hydrolysis was imparted by the sequence in which the two reagents DA and SS were added during pretreatment. Increased surface area for SS→DA (3.7 m2/g) also supported its highest glucan conversion. Studies on cellulose adsorption indicated that the quality of binding between cellulase and cellulose, as evident by the highest affinity constant (Ka = 0.25 mL/mg) for SS→DA was the predominant kinetic parameter offsetting the lower value of σmax in improving saccharification yield. This study bears high potential to pave the way for improving overall glucose recovery during the scale-up process.
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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.6b00758. Correlation of equilibrium constant with 20 FPU advance enzyme at 4 h and 72 h of native and pretreated A. mangium (PDF).
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AUTHOR INFORMATION
Corresponding Author
*Ravindra Kumar. Tel.: +91 0129 2294463. Fax: +91 0129 2286221. E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We acknowledge the Department of Biotechnology, Government of India and DBT-IOC Centre for Advanced Bio Energy Research, Indian Oil Corporation Ltd., Research & Development Centre, Faridabad (India) (BT/PB/08/03/2007) for funding and support. We express gratitude to Analytical Division, R&D Centre, IOCL for nitrogen content determination (Protein analysis) by CHNS analyzer and FT-IR. We also thank Punjab Renewable Energy Systems Pvt. Ltd (PRESPL), Thane, Maharashtra, India for providing Acacia mangium biomass.
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J
DOI: 10.1021/acssuschemeng.6b00758 ACS Sustainable Chem. Eng. XXXX, XXX, XXX−XXX
