Assisted Single-step Acid Pretreatment (ASAP) process for enhanced

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Assisted Single-step Acid Pretreatment (ASAP) process for enhanced delignification of rice straw for bioethanol production Thulluri Chiranjeevi, Anu Jose Mattam, Kusum K Vishwakarma, Uma Addepally, VC Rao Peddy, Sriganesh Gandham, and Harshad Ravindra Velankar ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.8b01113 • Publication Date (Web): 05 Jun 2018 Downloaded from http://pubs.acs.org on June 5, 2018

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ACS Sustainable Chemistry & Engineering

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Assisted Single-step Acid Pretreatment (ASAP) process for enhanced

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delignification of rice straw for bioethanol production

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Thulluri Chiranjeevi , Anu Jose Mattam , Kusum K Vishwakarma , Addepally Uma , Peddy

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V.C. Rao , Gandham Sriganesh and Harshad Ravindra Velankar

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†‡

†

†

†

†‡

†

†*

Bioprocess Group, Hindustan Petroleum Corporation Limited, HP Green R&D Centre, KIADB

Industrial Area, Tarabahalli, Devanagundi, Hoskote, Bengaluru 560067, India ‡

Centre for Biotechnology, IST, Jawaharlal Nehru Technological University Hyderabad

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(JNTUH), Kukatpally-500 085, Hyderabad, Telangana, India.

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*Correspondence: [email protected]

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‡

Abstract

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Dilute acid pretreatment of lignocellulosic biomass at higher temperatures (>160°C)

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solubilizes/removes hemicelluloses (xylan, arabinan, mannan, galactan) and acid-soluble lignin

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(ASL), but, it does not remove acid-insoluble lignin (AIL). During acid pretreatment, the

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condensation and re-deposition of coalesced lignin over cellulose fibers reduces the access of

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cellulose to cellulases. For higher delignification, various multi-stage pretreatments are available,

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however, all these are energy/chemical intensive methods. Therefore, an effective pretreatment

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which provides increased cellulose accessibility by enhanced removal of hemicelluloses and

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lignin in a single-step would be preferred. Our investigation reports a novel ASAP process for

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enhanced delignification of biomass under acidic conditions. Pretreatment of rice straw (particle

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size of 20 mm) with H2SO4 (0.75 % v/v) + boric acid (1% w/v) + glycerol (0.5 % v/v) (S/L, 1:5)

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at 150 °C for 20 min removed hemicelluloses completely, 44% of the lignin and ~48.5% of the

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silica leaving a solid consisting of 69 ± 1.5 % glucan, 0.7 ± 0.06 % ASL, 20 ± 2.0 % AIL and 12 1 ACS Paragon Plus Environment

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± 1.5% silica. The C/L (cellulose/lignin) ratio of solids resulted from ASAP was found to be

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>3.00, while it was 90%) and

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delignification (>40 %) with the assistance of boric acid and glycerol, a polyalcohol. Our studies

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revealed that the solid fraction (rich in cellulose) generated via this process is highly amenable to

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enzymatic hydrolysis to produce sugars which can be readily fermented to ethanol. To the best of

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our knowledge, this is the first report that demonstrates a 2.5-fold enhancement in lignin removal

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under acidic conditions that are less severe.

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Experimental

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Materials and reagents

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Rice straw was procured from local sources around Bangalore, Karnataka, India. D-

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xylose, D-glucose, Di-nitro salicylic acid (DNSA), sodium potassium tartrate, citric acid, xylan,

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carboxymethyl cellulose (CMC), microcrystalline cellulose(MCC), NaOH, CaCO3, and H2SO4 5 ACS Paragon Plus Environment


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(98 % w/v) were of analytical grade and obtained from SRL, Mumbai or Hi-Media, Mumbai,

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India. Cellulase enzyme (SachariSEB C6 Plus) used in this study was generously supplied by

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Advanced Enzymes, Mumbai, India.

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Compositional analysis of biomass

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To begin with, the rice straw was chopped into a size of ~7-9 cm and then dried overnight

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in a hot air oven (ED53, Binder, Germany) at 85 ºC to attain a constant weight. Afterwards, the

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dried material was shredded using a laboratory mixer and then particles of approximate size of

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about 0.5 mm were separated using a laboratory sieve. The processed biomass was stored at

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room temperature in a Ziploc bag until further usage. The biomass compositional analysis was

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carried out by following the NREL standard procedures.69 In brief, 300±1.0 mg of dried

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extractive free biomass sample was hydrolysed in presence of 3 mL of 72 % (w/v) sulphuric acid

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for 1 h at 30 °C in a Teflon screw-capped pressure tubes and after pre-hydrolysis, the acid

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concentration was brought down to 4 % (w/v) by diluting with 84 mL of distilled water and

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subsequently autoclaved at 121 °C for 60 min. Soon after, the hydrolysate was suitably diluted

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with distilled water and neutralized using solid CaCO3 and analyzed for sugar components and

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acid-soluble lignin. The solid residue obtained after acid digestion was used for the analysis of

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acid-insoluble lignin and silica through gravimetric analysis.69

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Pretreatment studies

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All the pretreatment studies were carried out in a 1 L batch reactor (Series. 4520, Parr

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Instruments, USA) at 100 g scale. The size of the biomass used in this study was about 2-3 cm.

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The size reduced rice straw biomass was subjected to four different types pretreatments, A1)

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0.75 % w/v H2SO4; A2) 0.75 % w/v H2SO4 + 1 % w/v Boric acid; A3) 0.75 % w/v H2SO4 + 0.5 %

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w/v glycerol and A4) 0.75 % w/v H2SO4 + 1% w/v Boric acid + 0.5 % w/v glycerol. The 6 ACS Paragon Plus Environment

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conditions used for control pretreatment i.e., 'acid-only' (A1) and the boric acid concentrations

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used for test pretreatments were optimized in our laboratory (Table. S1 and Fig. S1). In all the

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cases, the biomass was manually pre-mixed with respective catalyst solution maintaining solid to

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liquid ratio at 1: 5 (20 % w/v solids) and catalyst impregnated solids were charged to the reactor

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vessel (Reactor loading was 10 %). Afterwards, the vessel was heated with the help of an

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electrical heater with ramping rate of 5 °C/min. The outer temperature was maintained at 160 °C

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while the process temperature was at 150 °C. Under these conditions, the autogenously generated

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pressure was detected to be in the range of 68-75 psi. After pretreatment, the whole slurry was

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filtered using a vacuum filtration system. The resulting hydrolysates were analyzed for soluble

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sugars and other inhibitors generated after pretreatments, while the solids were analyzed for its

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chemical components by NREL-LAP as described above. Before subjecting the pretreated solids

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for enzymatic hydrolysis, the solids were washed with tap water (~1 L) to wash off the residual

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catalyst and degraded lignin (after washing, the solids pH was around 4.5).

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Enzymatic hydrolysis of pretreated rice straw sample

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Enzymatic hydrolysis of pretreated biomass samples was carried out at 50 °C in 3 L

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reactor for about 72-96 h. The solid loading was maintained at 10 and 15 % (w/v) while the

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glucan concentrations of these reactions were 6.9 % (w/v) and 10.35 % (w/v), respectively.

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Sodium citrate buffer (0.05 M) was used to maintain the optimum pH of 4.5. The enzyme

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(SachariSEB C6 Plus) load was 20 FP units g-1 of dry solids. Around 100 g (for 10 % w/v solids

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concentration) and 150 g (15 % w/v solids concentration) of pretreated biomass was subjected to

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enzymatic hydrolysis in a volume of 1 L. The mixing of reactants was done by an anchor type

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impeller and the mixing speed was maintained at 250 RPM. One unit of enzyme activity is

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defined as the amount of enzyme that produces 1 µmol reducing sugar per minute in the reaction 7 ACS Paragon Plus Environment


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mixture under the specified conditions. The reducing sugars were estimated by the DNSA

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assay70 and also analyzed by HPLC. The conversion of cellulose to glucose (% Saccharification)

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was calculated as follows, Enzymatic hydrolysis (%) =

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[Total reducing sugar (Glucose) x 0.9/size of sample (g) x Cellulose content] X 100 ….(Equation no. 1)

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Analytical and microscopic methods

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HPLC analysis

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Analysis of sulfuric acid hydrolyzed (NREL-LAP) sugar components of plant biomass

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structural carbohydrates was carried out in UHPLC (Ultra High Performance/Pressure Liquid

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Chromatography) (Agilent 1290 series) equipped with a Hiplex H anion exchange column

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(Agilent) connected to RID (Refractive Index Detector). The sugar recovery standards of

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glucose, xylose, arabinose, mannose and galactose were analyzed using their respective sugar

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calibration curves. The conditions employed were, 5mM H2SO4, mobile phase (Filtered and

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degassed); 0.6 mLmin-1, flow rate; 45 °C, column temperature (external temperature); 55 °C,

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detector temperature (internal temperature).

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Estimation of total reducing sugars (TRS) and phenol (TPC) contents

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Both, total reducing sugars (TRS) and the total phenolic content (TPC) present in the

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pretreatment generated liquid hydrolysates were estimated spectrophotometrically in a multi-well

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plate reader (Epoch, Biotek Instruments, USA). TRS content was determined by the DNSA

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method70 at 540 nm using glucose as a reference standard while TPC was determined using

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Gallic acid (GA) as a reference standard.71 8 ACS Paragon Plus Environment

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Powder-X-Ray Diffraction

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The pretreated biomass samples were also characterized by P-XRD measurements using

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X-ray diffractometer (X’pert3PANalytical, Netherlands) to determine the crystallinity of

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cellulose before and after treatments. The X-ray source used was Cu K-alpha, 0.15418 nm

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(Bragg-Brentano geometry) at a wavelength of 1.5406 nm. The samples were scanned over the

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angular range2-50°, 2θ with a step size of 0.030°, smooth operation (0.302) and step time of 0.5s.

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Crystallinity was calculated according to Segal empirical method.72 Crystallinity index (%) = (I002-I 18 °]/ I002 X 100

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Where, I002= Maximum intensity of the diffraction

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I 18 °= Intensity diffraction at 18 ° on 2 theta scale

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.... (Equation no. 2)

Fourier Transform Infra-Red (FTIR)

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The biomass samples were analyzed by FTIR (Perkin Elmer, USA) in ATR (Attenuated

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Total Internal Reflection) mode having MCT detector. The samples were analyzed over a wave

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number range of 400-4000 cm-1 region to characterize the cellulose in untreated and treated

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materials.

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FE-SEM (Field Emission Scanning Electron Microscope)

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The changes in the morphology of both untreated and pretreated rice straw solids were

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observed by scanning electron microscope (JSM-7610F, JEOL, Japan). Prior to acquiring

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images, the samples were mounted on pre-cut brass stubs using carbon paste and then sputter

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coated with Pd-Pt prior to imaging.

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Lignin staining.

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The staining of lignin in the biomass samples was carried by the phloroglucinol-HCl stain. 73

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Solid state CP/MAS

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samples

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C NMR characterization of pretreated rice straw solids and lignin

The solid-state CP/MAS

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C NMR experiments were performed on a Bruker Avance

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III400MHz spectrometer operating at frequencies of 100.59 MHz for 13C using a Bruker double-

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resonance 4-mm MAS probe head at ambient temperature. The samples were packed in a 4mm

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ZrO2 rotor fitted with a Kel-F cap and spun at 8,000Hz. CP/MAS 13Cdata were acquired with a

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Bruker CP pulse sequence under the following acquisitions: pulse delay 4s, contact pulse

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2000ms, and 2k to 4k numbers of scans. Bruker TopSpin (version 2.1) software and

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MestReNova software were used for data processing.

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Results and discussion

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Effect of acid-only and assisted-acid pretreatments on the chemical components of rice

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straw biomass:

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The compositional analysis of untreated and pretreated biomass has been summarized in

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Table 1. In general, the dilute acid pretreatment at higher temperatures promotes dissolution of

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hemicelluloses from the lignocellulose complex.39,

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biomass pretreated with ‘only-acid’ (A1) and assisted-acid pretreatments (A2-A4) caused near

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complete removal of arabinan and partial removal of ASL (acid-soluble lignin) resulting in

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biomass with higher glucan content (on dry weight basis) in the range of 33.8±1.4 g (49±1.8 %)

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to 36.9±1.2 g (69 ± 1.5 %) (Table 1). In the present study, the main objective was to enhance

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delignification of rice straw via a single-step acid pretreatment assisted by glycerol, so that the

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accessibility of cellulose could be improved. Contrary to our expectations, glycerol addition

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during acid pretreatment did not result in considerable delignification (Table 1) and instead

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caused increased xylan removal (A2) (83.2 %) as compared to only-acid pretreatment (A1) (78

74-76

The results obtained revealed that


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%). Further, the addition of boric acid to only-acid (A3) and to acid-glycerol (A4) processes

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resulted in almost complete removal of xylan which also contributed higher residual glucan

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content of about 36.9 ± 1.2 g (66 ± 2.2 %) and 36.5 ± 0.8 g (69 ± 1.5 %) in A3 and A4

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pretreatments, respectively (Table 1).

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Another interesting observation made during the present study was the increased removal

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of klason-lignin and silica during A3 (44 %) and A4 (48 %) pretreatments (Table 1 & Fig. 1).

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The separated lignin was obtained as insoluble material in the C5 rich hydrolysate and could be

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easily recovered by filtration (to be used as boiler fuel). It is now known that the removal of

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lignin and hemicellulose from biomass is an energy intensive process due to the extensive cross-

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linking among these components by atleast 14 types of bonds77-80, most of which comprise of

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thermo-labile ether linkages (β-O-4).80 Biomass pretreatment using acids usually results in the

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cleavage of β-O-4 ether bonds in lignin.16 Despite the extensive cleavage of β-O-4 ether bonds

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under severe conditions, the condensation and re-deposition of coalesced/transformed lignin over

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the cellulose fibers causes ‘masking’ of cellulose fibers and a reduction in their accessibility to

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cellulolytic enzymes during saccharification. In a separate study, it was shown that the re-

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polymerization of lignin could be avoided by the capping of reactive phenolic groups in lignin

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with boric acid. 60 The same study also reported that boric acid also acted as a catalyst for acidic

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driven α-ether hydrolysis of lignin.60 In line with earlier reports60, the higher delignification

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observed during our studies could be attributed to the capping of lignin moieties by boric acid

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resulting in lower condensation /re-deposition of lignin on the pretreated biomass. This

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eventually resulted in about 15 % higher ASL removal during A3 and A4 processes in

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comparison with A1 and A2 pretreatments (Table 1). Further, the higher delignification during



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A3 and A4 pretreatments also caused increased removal of silica since in rice straw, the lignin

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layer is overlaid by silica (Table 1).

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Mass balance calculation of all pretreatments (A1, A2, A3, A4) indicated that around

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88.9 % of cellulose was recovered in A1, which slightly increased to 91 % in case of A2. The

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addition of boric acid (A3 and A4) to reaction mixtures further increased the cellulose recoveries

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to 97 and 96 % respectively (Fig. 1). In addition, the desilication obtained during A3 and A4

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pretreatments were significantly higher (~48.5%) than during A1 and A2 pretreatments (~4%)

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(Fig. 1).

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Liquid hydrolysate analysis:

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Detailed analysis of pretreated hydrolysate generated during A1, A2, A3 and A4

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pretreatments of rice straw mainly revealed the presence of reducing sugars, inhibitors (HMF,

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furfural and phenolics), ASL and xylooligosaccharides (XOS). Our studies indicated that during

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all the 4 pretreatments (A1, A2, A3 and A4), the xylan component was either completely

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converted to xylose or partially hydrolyzed to XOS (Table 2). HPLC analysis of hydrolysates

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revealed that the reducing sugars comprised mainly of xylose (~60 % of TRS), glucose (10.5 %

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of TRS) and arabinose (9 % of TRS) (Table 2). Overall, the TRS of A3 (26.4 ± 1.1g) and A4

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(27.5 ± 1.5 g) pretreatments was higher than A1 (18 ± 1.8g) and A2 (19 ± 1.5g) hydrolysates

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(Table 2). Consequently, the XOS contents of A1 (11.5±1.5 mgmL-1) and A2 (9.5±1.1 mgmL-1)

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hydrolysates were higher than A3 (2.7±0.2 mgmL-1) and A4 (1.8±0.12 mgmL-1) (Table 2). It is

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generally acknowledged that severe acid pretreatments of biomass may also lead to the

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conversion of hemicellulosic sugars into inhibitors along with monomeric sugars

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our study, we observed that the concentrations of inhibitors obtained in hydrolysates generated


81, 57

. During

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from all the four pretreatments was minimal (Table 2). The higher total phenolic contents of the

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hydrolysates could be attributed to the increased removal of ASL from the biomass (Table 2) 82,

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83

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subsequent unit operations such as fermentation.

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during ASAP that uses dilute sulfuric acid as the catalyst is even lesser than the levels of

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inhibitors (HMF and Furfural) generated during earlier pretreatment studies with dilute acid

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albeit on a different type of biomass (corn stover).86 Another advantage of the ASAP process is

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the lower formation of pseudolignins that correlates with higher reducing sugar recoveries (Table

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2). The formation of pseudolignins causes sugar losses since these acid-insoluble (klason-

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positive) aromatic compounds are a result of condensation of released lignocellulosic sugars and

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the dislocated lignin under severe conditions.49,87,88 Moreover, pseudolignins tend to re-deposit

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on the cellulose fibers thereby impeding the accessibility of cellulolytic enzymes during

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enzymatic hydrolysis.

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Structural characterization of pretreated solids

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FTIR analysis:

Based on earlier reports, the low inhibitor generation during ASAP process may not affect the 84,85

The inhibitor concentrations obtained

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The functional group alterations that occur in the biomass due to A1, A2, A3 and A4

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pretreatments were analyzed by FTIR and compared with the untreated biomass. Overall, the

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FTIR results indicated considerable breakage of bonds that provided structural integrity to the

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biomass. In comparison with untreated biomass, the changes in the stretching and bending

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vibrations were observed for all the pretreated solids. The results (Fig. 2) indicate two

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characteristic bands near 1155 cm-1 and 905 cm -1for A3 and A4 pretreated biomass and not in

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A1 and A2 pretreated biomass. Earlier reports indicate that these bands arise from C-O stretching



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at β-(1-4)-glycosidic linkages of cellulose due to the exposure of cellulose.89 Further, for A3 and

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A4 pretreated biomass, the intensities at 1160 cm-1 and 1318 cm-1 had marginally decreased

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representing C-O anti-symmetric stretching and CH wagging in cellulose, C-O-C, C-C groups as

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well as increase in bending vibrations in C-H and OH groups. In all the solid samples (untreated

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and treated), a broad peak between 3561 cm-1 and 3412 cm-1 indicated the stretching of H-

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bonded OH. However, when compared to untreated biomass, the intensity of two characteristic

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peaks at 2800 cm-1, 2900 cm-1decreased in A1 and A2 indicating sp3 C-H stretching due to which

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peak broadening happened and may be attributed to the removal of hemicelluloses. In case of A3

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and A4 treated biomass, the peaks 2800 cm-1, 2900 cm-1were not observed probably due to the

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absence of xylan component in the solids. Similarly, the ester linkage of C=O between

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hemicelluloses and lignin at 1720 cm-1 and C-O-C stretch of the acetyl group in hemicelluloses

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at 1245 cm-1 disappeared in the case of A3 and A4. In contrast to acid-only pretreatment (A1),

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only the absorption bands related to hemicellulose slightly decreased in A2, while the bands

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related to lignin were not changed. Further, two strong peaks at 1049 cm-1 and 1022 cm-1 are

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indicative of C-O stretching at C-3, C-C stretching and C-O stretching at C-6, while the small

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peaks near to 580 cm-1and 615cm-1ranges correspond to the out of plane bending of C-O-H. In

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the untreated rice straw, the intensity of the peaks around 1500-1620 cm-1 and 1380 cm-1,

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characteristic of C=C stretching of the aromatic ring (guaicyl unit) and C-H stretching vibrations

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of the syringyl units, respectively decreased for solids of A3 and A4 pretreatments as compared

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to A1 and A2 pretreated solids.

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X-ray diffraction studies

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X-ray diffraction studies of treated (A1, A2, A3 and A4) and untreated rice straw showed

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different diffraction patterns (Fig. 3). The divergence in the position of the diffracting peaks 14 ACS Paragon Plus Environment

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designate the distance between the hydrogen bonded sheets of cellulose. As can be observed

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from Figure 3, two characteristic peaks present at 2θ of 22° and 18° were seen in both, untreated

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and pretreated biomass which correspond to cellulose-I and II respectively.90 The major peak at

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2θ of 22° is the main peak signifying the presence of a highly organized crystalline region (Iα

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and Iβ), whereas the minor peak at 2θ of 18owas the less organized region (Cellulose-II).

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Earlier reports indicate that crystallinity of pretreated biomass alter post the pretreatment.

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Similar observations were made during our studies and the crystallinity of A3 pretreated samples

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was found to be higher (98.4 %) than the untreated sample (41 %). The higher intensity of the

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crystalline region (2θ of 22°) could be attributed to the dissolution of amorphous cellulose during

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A3 pretreatment. The removal of amorphous cellulose may occur due to the delignification

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during pretreatment thereby exposing the cellulose component and its heat-labile amorphous

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regions. Hydrolysis of these amorphous regions would lead to an increase in total reducing sugar

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concentrations as observed during A3 and A4 pretreatments (Table 2). Earlier investigations

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have indicated that the crystallinity of cellulose in biomass is mainly contributed by the inter-

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hydrogen bonding which can get altered during acid-thermal treatments leading to the expansion

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of lattice spacing in cellulose

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biomass after A4 pretreatment (in presence of glycerol) got lowered probably due to the inter-

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crystalline swelling of cellulose in presence of aqueous glycerol91,

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observed that delignification of biomass due to A3 and A4 pretreatments was significantly higher

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than only-acid pretreatments (A1 and A2) although the treatment temperature was same in all

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cases. Microscopic observations of the pretreated biomass also revealed higher exposure of

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cellulose in the case of A3 and A4 pretreatments (Fig. S2).

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Lignin staining and microscopy for understanding the cellulose accessibility:

39

. Based on previous reports, the crystallinity of cellulose in


92.

During our studies, we


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The histochemical staining and scanning electron microscopy of the solids before and

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after pretreatments distinctly showed the microstructure of biomass consisting of lignin,

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cellulosic fibrils and hemicellulose. A lignin specific stain- phloroglucinol-HCl was used for the

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staining work. Microscopic images of untreated biomass showed stained (brown) lignin sheath

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covering the compactly arranged cellulose fibrils surrounded by the hemicellulosic regions (Fig.

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4a). The A1 and A2 pretreatments caused the overall reduction in lignin content (brown

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coloration) associated with the cellulose and the hemicellulose regions (Fig 4b and 4c). Earlier

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studies that have used only acids for biomass pretreatment have reported re-deposition of lignin

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onto cellulose fibrils thereby affecting subsequent enzymatichydrolysis.48,

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pretreatment of rice straw, (Fig 4d), although there was an overall reduction in the lignin content

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(brown coloration), lignin agglomerates were observed around cellulose fibrils. It was interesting

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to observe the differing surfaces morphologies of the boric acid (A3) and boric acid + glycerol

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assisted-acid (A4) pretreated solids (Fig. 4d and 4e). The lignin agglomerates observed in A3

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were significantly reduced in A4 which would naturally increase cellulose accessibility. The

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SEM analysis (Fig. S2) of the untreated biomass clearly shows the presence of silica knots which

357

are reduced after ASAP treatment (Fig. S2e) indicating desilication.

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13

93-96

With A3

C-solid-state NMR analysis of pretreated solid-residues and respective lignin fractions

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The lignin recovered from the C5 hydrolysates of all pretreatments (A1, A2, A3 and A4)

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was analyzed by solid-state CP/MAS 13C NMR (Fig. S3) along with the cellulose rich pretreated

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solids to obtain insights about the nature of cellulose and lignin. In general, solid-state 13C-NMR

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of cellulose exhibits six singlets which correspond to 13C chemical shifts of cellulose carbons (in

363

glucose units). These are designated as C1 (105 ppm), C4 (79-92 ppm), C2/C3/C5 (70-80 ppm)

364

and C6 (60-69 ppm).99 Moreover, the changes in the C6 resonance region may indicate alteration 16 ACS Paragon Plus Environment

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365

of specific hydrogen bonding with other hydroxyl groups in the adjacent cellulose chains. Broad

366

shoulders in the C4 region at 83 ppm and in the C6 region at 63 ppm indicate the presence of

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amorphous cellulose.100 Our studies revealed that ASAP (A4) pretreated rice straw showed

368

strong signals at 88 and 65 ppm and broad signals at 83 and 63 ppm indicating that it contains

369

both, the crystalline and amorphous fractions (Fig. 5a & 5b). In comparison with A3, the NMR

370

spectra of A4 solids showed increased amorphous regions, probably due to glycerol induced

371

swelling of biomass and the possible disruption of hydrogen bonds. These observations can be

372

correlated with similar decrease in crystallinity as determined by X-ray diffractograms (Fig. 3).

373

Also, since the removal of klason-lignin during A3 and A4 treatments was higher, the NMR

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signals characteristic of aromatic/unsaturated carbon (usually observed between 109 and 167

375

ppm) were not detected in the pretreated (A3 & A4) solids (Fig 5a, 5b). As anticipated, the lignin

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recovered in C5 hydrolysate stream after A3 and A4 pretreatments showed a strong peak signal

377

in the range of 0-40 ppm, at 56 ppm, and in between 110-160 ppm in the CP/MAS

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spectrum (Fig 5c, 5d), which respectively corresponded to aliphatic carbon, aromatic methoxyl (-

379

OCH3) resonance, and the aromatic region (Fig 5c, 5d). 101,102

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Enzymatic hydrolysis of pretreated solids

13

C-NMR

381

The amenability of pretreated cellulosic material to cellulolytic enzymes is one of the

382

indicators of the effectiveness of the pretreatment process. In the present study, untreated and

383

A1, A2, A3 and A4 pretreated rice straw samples were subjected to enzymatic hydrolysis at 50

384

°C for 96 h by adding cellulase enzymes at concentrations of 20 FPUg-1 of dry solids. All the

385

hydrolysis reactions were carried out with two different solid concentrations viz., 10 % (w/v) and

386

15 % (w/v) and the conversions were expressed as the percentage of actual glucose released

387

versus theoretical glucose equivalent. The glucan-to-glucose conversion yields of rice straw 17 ACS Paragon Plus Environment


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before (untreated/UT) and after all pretreatments (A1, A2, A3 and A4) are shown in Figure 6.

389

With 10% (w/v) solid concentration, enzymatic hydrolysis of untreated rice straw (UT) after 96 h

390

yielded only 25 % glucan-to-glucose conversion, while A1and A2 treated samples yielded 39 %

391

and 42 % glucan-to-glucose conversion, respectively. For A3 and A4 pretreatments, the glucose

392

yields were much higher i.e., 66 % and 77 %, respectively. Only a few earlier studies have

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reported similar glucan-to-glucose conversions (of ~70%) with ‘only-acid’ catalyzed

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pretreatment of rice straw; wherein the catalyst concentration used was higher (1 % w/v) and the

395

solid loading during enzymatic hydrolysis was very low (2 % w/v)34 compared to the present

396

study wherein 0.75 % w/v acid was used for pretreatment and the pretreated biomass could be

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loaded at higher concentrations (10-15 % w/v) for enzymatic hydrolysis. The higher glucose

398

yield obtained during hydrolysis of ASAP pretreated biomass could be attributed to enhanced

399

delignification (Fig. 1) and the subsequent structural changes (Fig. 2, 3 & 5). In the present

400

study, increased loading of untreated rice straw from 10 % (w/v) to 15% (w/v) during enzymatic

401

hydrolysis resulted in the lowering of glucan to glucose conversion by around 8 %, whereas for

402

A1, A2, A3 and A4 pretreated biomass, the corresponding glucan conversion decreased by 11%,

403

11%, 6% and 5% (Fig. 6). Similar observations about decreased hydrolysis on increasing the

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solids concentrations have been previously reported with steam-exploded sunflower stocks.103.

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Still, the hydrolysis of A3 and A4 was around 2 folds higher than A1 and A2 pretreated biomass

406

(Fig. 6). Some of the reasons for the differences in hydrolysis could be attributed to insufficient

407

mass and heat transfer due to heterogeneous nature of reaction mixture,104 feedback inhibition of

408

enzymes due to released sugars,105 changes in biomass composition104-106 and their

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concentrations,107 synergistic disparities in enzyme components108,109 etc. For a better

410

understanding of the present hydrolysis results, we analyzed the cellulose to lignin (C/L) ratio for


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untreated (UT) and pretreated solids (A1-A4) in order to directly correlate the sugar yields with

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the degree of delignification (Fig.6a). The C/L ratio of A1 and A2 pretreated solids at 10 % (w/v)

413

and 15 % (w/v) loading was < 2.00 while for A3 and A4 was >3.00 (Fig 6a); despite the decrease

414

on glucose yields due to higher loadings (Fig. 6). The impact of differing C/L ratios on the

415

efficiency of enzymatic hydrolysis is clearer when an approximation of the cellulose and lignin

416

quantities entering the enzymatic hydrolysis is done. For instance, for a scale of 100 mL

417

hydrolysis reaction at 10 % (w/v) and 15 % (w/v) solids loading, respectively 2 g and 3 g of

418

lignin was present (Fig. S4). Further, according to the same study, cellulases adsorption to

419

cellulose was found to decrease at higher solids concentrations which in turn leads to reduced

420

sugar conversion yields.112 In our study, the increase in relative amount of lignin concentration

421

due to increasing solids concentration could be one of the plausible reasons for decrease in

422

hydrolysis yield, however, this needs further investigation.

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An appraisal of ASAP in contrast to other single-step acid pretreatment processes

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In the present study, Table 3 compares previously reported single-step acid catalyzed

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pretreatments of different lignocellulosic biomass materials (wheat straw, sugarcane bagasse,

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rice straw, corn stover, wood chips, A. aspera and oat hulls) with their subsequent enzymatic

427

hydrolysis yields. Different parameters considered for comparison include the catalyst system,

428

type of biomass, size of biomass, solids concentration, reaction temperature, treatment duration,

429

washing of pretreated solids and the degree of delignification achieved. Likewise, for enzymatic

430

hydrolysis; the solids concentration, enzyme loading, hydrolysis duration and the obtained yields

431

have been compared (Table 3). Based on whether ‘only-acid’ or acid combined with an additive

432

were used, pretreatment processes could be broadly classified into two groups. The first group

433

includes studies that uses only-acid catalysts for biomass pretreatment (Table 3; No. 1, 2, 3, 4, 6, 19 ACS Paragon Plus Environment


434

8, 11 and 12); while the second group includes reports that have used acid along with another

435

catalyst/additive (Table 3; No. 5, 7, 9, 10) in comparison with ASAP process (Table 3; No. 13).

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Comparing all the pretreatments listed in Table 3 with ASAP process, high delignification of

437

around 42% was achieved at 20 % (w/v) solids loading by the addition of 0.75 % H2SO4(w/v)

438

assisted with 1 % (w/v) boric acid+ 0.5 % (w/v) glycerol. All other processes were carried out at

439

either higher catalyst concentrations (Table 3; No. 1, 2, 3, 5, 7, 11 & 12), at lower solids loading

440

(Table 3; No. 1-12), require extensive water washing (Table 3; No. 4, 7, 11 & 12) or utilize

441

solvents as the reaction medium (Table 3; No. 7, 9 & 10). During ASAP pretreatment of

442

biomass, the catalyst concentration was the lowest, the solid loading was the highest, water

443

requirement was significantly lower during biomass washing and the concentration of glycerol

444

(organic solvent) required was lesser. The ASAP-pretreated biomass was washed with water

445

(1:20 ratio) till insoluble material stopped coming out in the wash. The pH of the biomass after

446

washing was found to be around 4.5. As mentioned earlier, the selection of a pretreatment

447

method does not solely depend upon its potential to delignify or hydrolyze biomass but also upon

448

several factors such as low chemical consumption, high biomass loading, generation of highly

449

amenable cellulose etc. 53,54 which was obtained for ASAP process. Despite appreciable yields of

450

hydrolysis as could be seen for entry no. 1, 7, 11 and 12, lesser solids loading (5 % w/v or 20 FPUg-1), very high quantities of chemical usage (eg. 4:1

452

Ethylene carbonate-Ethylene glycol, 80% w/v H3PO4, 4% w/v HNO3, etc.) for pretreatment as

453

well as extensive water washings to remove the catalysts are some of the drawbacks of these

454

processes. The ASAP process developed in the present study used higher solids loading (20%

455

w/v) with larger sized biomass (20 mm) to achieve enhanced delignification and considerable


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enzymatic hydrolysis at higher solids loading of even 15% (w/v) with standard enzyme

457

concentration of 20 FPUg-1.

458

Mass balance study

459

The mass balance for ASAP process using 1 kg dry rice straw biomass followed by

460

enzymatic hydrolysis along with the possible ethanol yield has been depicted (Fig. 7). The

461

conditions for ASAP pretreatment in the reactor were standardized at 150°C, 20 min, 72 psi of

462

autogenous pressure at solid to liquid ratio of 1:5. The catalyst composition was fixed at 0.75%

463

(v/v) H2SO4, 1% (w/v) boric acid and 0.5 % (v/v) glycerol. Following the ASAP, 0.275 kg of

464

soluble reducing sugars containing mainly the hemicellulosic fraction was separated into the

465

liquid hydrolysate, wherein 0.162 kg xylose equivalent, 0.025 kg arabinose equivalent and 0.025

466

kg of glucose equivalent was detected. The inhibitors detected were 0.014 kg of furfural and

467

0.006 kg of HMF. On the other hand, around 53 to 55 % solids (0.53-0.55 kg) recovery was

468

obtained. The major components of these solids was cellulose which accounted for 0.365 kg and

469

the remaining included AIL (0.106 kg), ash (silica) (0.063 kg), ASL (0.0042 kg). Further, the

470

pretreated cellulose-rich solid material was subjected to cellulase hydrolysis at 20 FPUg-1

471

and15% (w/v) solid loading to release 0.289 kg of glucose equivalent. The overall hydrolysis

472

under these conditions was observed to be 72%. Considering around 85 % hydrolysis, the value

473

of glucose equivalent would come to 0.340 kg. Considering the present C6 sugar (glucose) yield

474

(72 %) obtained during our study, around 0.144 kg of ethanol could be produced from 1kg of

475

raw rice straw biomass. Correspondingly, 0.170 kg of ethanol can be produced by considering 85

476

% of glucose yields.

477

CONCLUSIONS



478

Biomass pretreatment is a critical unit operation in the process of producing

479

lignocellulosic biofuels. The newly developed ASAP process achieved higher delignification of

480

about 42 % and near-complete removal of hemicellulose which resulted in the generation of

481

pretreated solids that showed high amenability to cellulolytic enzymes. This was also further

482

supported by morphological (Lignin staining & SEM) and functional group analysis (FTIR).

483

After pretreatment, there was a change in cellulose crystallinity as could be seen from the XRD

484

diffractograms and CP/MAS

485

liquid hydrolysate nor redeposited on cellulose fibers, instead it came out into the C5 hydrolysate

486

and the wash liquid in the form of agglomerates which can be easily separated by filtration and

487

the wash water can be recycled. Moreover, this separated lignin was also confirmed by CP/MAS

488

13

489

w/v (81.9 gL-1), which was 2.6 and 4.2 times higher than from the solids of acid-only

490

pretreatment and untreated rice straw, respectively. The increased enzymatic hydrolysis was

491

further supported by analyzing the cellulose to lignin ratio, where it was found to be higher

492

(>3.00) for the solids from ASAP than untreated and acid-only treated solids (

Assisted Single-step Acid Pretreatment (ASAP) process for enhanced

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