Fractionation and characterization of lignin from waste rice straw: Biomass surface chemical composition analysis

Journal Pre-proofs Fractionation and characterization of lignin from waste rice straw: Biomass surface chemical composition analysis Shubhangi De, Shubham Mishra, Elangovan Poonguzhali, Mathur Rajesh, Krishnamurthi Tamilarasan PII: DOI: Reference:

S0141-8130(19)37225-3 https://doi.org/10.1016/j.ijbiomac.2019.10.068 BIOMAC 13562

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International Journal of Biological Macromolecules

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6 September 2019 29 September 2019 7 October 2019

Please cite this article as: S. De, S. Mishra, E. Poonguzhali, M. Rajesh, K. Tamilarasan, Fractionation and characterization of lignin from waste rice straw: Biomass surface chemical composition analysis, International Journal of Biological Macromolecules (2019), doi: https://doi.org/10.1016/j.ijbiomac.2019.10.068

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Fractionation and characterization of lignin from waste rice straw: Biomass surface chemical composition analysis Shubhangi De, Shubham Mishra, Elangovan Poonguzhali, Mathur Rajesh, Krishnamurthi Tamilarasan1 Department of Chemical Engineering, SRM Institute of Science and Technology, Chennai-603203, Tamilnadu, India.

Abstract Waste rice straw (RS) was fractionated to extract the lignin using alkaline (sodium hydroxide) treatment (SHT) and organic acid (Formic acid/Acetic acid) treatment (OAT) process. Rice straw fractionation by the acetic OAT and alkaline SHT methods resulted in the recovery of OAT-lignin and SHT-lignin respectively. The structural characterization of the extracted lignin fractions was done by UV-vis spectroscopy, FTIR and 1H NMR technique. Total phenolic content (TPC) present in SHT-lignin and OAT-lignin were determined to be 28.87 mg GAE/g and 24.75 mg GAE/g respectively. The DPPH radical scavenging activity 59.50 % was observed in SHTlignin and 45.74 % in OAT-lignin using the DPPH test for 30 min of incubation. Surface characterization of untreated rice straw (UT-RS) and treated rice straw (SHT-RS and OAT-RS) were carried out by XRD, FTIR and SEM. X-ray photoelectron spectroscopy (XPS) survey, C1s, and O1s spectra were used to determined the surface carbon and oxygen composition changes in RS after SHT and OAT. These structural characterizations of lignin and biomass are beneficial for further processing in bio-refinery industry. Keywords: Alkaline treatment, lignin fractions, antioxidant activity, chemical composition. 1. Introduction Lignin is the second largest organic plant polymer on earth. Generally, lignocellulosic biomass consists of ~15-35 % lignin. The low cost of this feedstock can be used to replace fossil source for synthesis of fine chemicals and energy production [1,2]. Globally, more than 1,000 million tons per year of rice straw are produced. Waste rice straw generated from agro-process has generated significant interest considering their availability and also their cellulose, hemicelluloses 1

Corresponding Author: Dr. Krishnamurthi Tamilarasan, Department of Chemical Engineering, SRM Institute of Science and Technology, Chennai-603203, Tamilnadu, India. E-mail:[email protected]

and lignin content. Lignin is a phenolic polymer with amorphous structure, wide range of molecular weight, and poor solubility in solvents [3]. Lignin primarily consist of three monomers units, namely guaiacyl propane (G), syringyl propane (S), and 4-hydroxyphenylpropane (H), which are chemically connected by alkyl-ether, carbon-carbon and aryl-ether bonds [4,5]. It exhibits various functional groups including carbonyls, carboxyls, phenolic and aliphatic hydroxyls etc, which finds various applications as phenolic resins, emulsifiers, dyes, additives, nutraceuticals and fuels synthesis [6-8]. Recent research primarily focused on the fractionation of lignocellulosic biomass to synthesis renewable chemicals [9]. Several kind of lignin derived from a specific biomass resource using different extraction method could be potentially valorize the lignin biomass. The extraction or fractionation method will have an influence on the composition and properties of lignin [10]. Several physical, chemical and solvent fractionation processes have been developed for lignin extraction from lignocelluloses

sources [11,12]. Among the process organosolv and alkali treatment process are efficient methods to obtained high yield and high quality of lignin [4,13]. The organosolv treatment is sustainable, simple and nonsulfur treatment process for lignin extraction [10]. Alkali treatment involves selective cleavage of β-aryl ether bonds in lignocelluloses to significantly enhance the solubility of lignin [4]. Lignin and cellulose compounds are more useful to bio-refinery applications when the structures and characteristics are analyzed using various techniques [14]. The aim of current research was to investigate the extraction of lignin from rice straw by organosolv and alkali treatment method. The total phenol content and antioxidant activity of extracted lignin has been investigated. Structural characterization of fractionated lignin was studied using UV-vis, FTIR, and 1H NMR spectroscopy. In addition, the surface chemical structure changes of biomass during extraction were investigated by SEM, XRD, FTIR and XPS analysis.

2. Materials and methods 2.1 Materials and reagents Formic acid (98.99%), Ethylene glycol (extra pure AR), acetic acid glacial, Folin– Ciocalteu reagent, sulfuric acid (99%), DPPH (2,2-diphenyl-1-picryhydrazyl) were bought from Sisco research laboratories pvt ltd, India. Gallic acid (99.5%) was purchased from Loba chemie pvt ltd, India. Dimethyl sulfoxide-d6 (99.9 atom % D) was purchased from Sigma-Aldrich. Other chemicals were of highest purity available commercially.

2.2 Lignocellulosic biomass preparation Rice straw was collected from fields near Chennai, India. Raw rice straw was washed with distilled water to remove unwanted materials and impurities. It was kept in hot air oven at 105 °C for 12 h of drying. Dry biomass was ground into powder using kitchen blender machine, and then sieved in a 18 mesh size sieve. The powder form of rice straw was kept for drying in a hot air oven for 2 h and then directly used as feedstock for lignin extraction [15].

2.3 Lignin extraction by formic acid/acetic acid treatment The lignin extraction processes was carried out in a 250 ml conical flask. The dried rice straw (solid-to-liquor ratio of 1:8) was mixed with 85 % organic acid (70:30 v/v ratio of formic acid/acetic acid) mixture heated at 90 °C for 180 min of extraction. At the end of extraction, solid fractions were separated from mixture by centrifugation (10000 rpm for 10 min). Subsequently, the lignin precipitated from liquid fraction by adjusting the distilled water to pH 2. The lignin residue was dried in the vacuum oven at 80 °C and stored for further characterization [16].

2.4 Lignin extraction by sodium hydroxide treatment The dried rice straw were treated with 5 % sodium hydroxide solution (1:8 (w/v) solid to liquor ratio) heated at 90 °C for 180 min. After treatment, reaction mixture was allowed to centrifugation to removed residual biomass. The supernatant liquor was adjusted to pH 2 by adding H2SO4 drop wise to precipitate lignin. The resulting solution was centrifuged to remove the supernatant liquor and the residual lignin was kept in a separate petri dish. The recovered lignin sample was dried in the vacuum oven at 80 °C and stored for further analysis [17].

2.5 Characterization of lignin 2.5.1. UV-vis and FTIR spectroscopy analysis Phenolic content of lignin fractions were analyzed using a UV-visible spectrophotometer (Cary 60 UV-Vis, Agilent Technologies). 0.01g lignin sample was dissolved in 10 ml 0.1 % NaOH solution then the sample absorbance was recorded between 250 to 400 nm. The FTIR spectra of biomass and the lignin samples were recorded using FTIR spectrophotometer (Cary 600 series FTIR Spectrometer, Agilent technologies). The sample and KBr powder (1:100) were uniformly ground and pressed to form a pellet and the spectra recorded in the range of 400 to 4000 cm−1 with a resolution of 4 cm−1. Structural characterizations of lignin samples were done using nuclear

magnetic resonance (NMR) spectrometry (Bruker-Avance-500 spectrometer). Lignin samples were dissolved in DMSO-d6 under gentle heating with micro-stirring conditions.

2.5.2. Total phenol and antioxidant activity of lignin The total phenolic content present in the fractionated lignin sample was analyzed by FolinCiocalteu method with some modifications [18]. The analysis sample mixture consists of 0.5 mL of lignin sample (1 mg/mL in ethylene glycol), 2.5 mL of Folin’s reagent (10% v/v) and 5 mL sodium carbonate (20 % w/v). After 30 min incubation, the intensity of blue color was measured at 750 nm in a UV-vis spectrophotometer. The total phenolic content was expressed as microgram gallic acid equivalent per dry weight of material (µg GAE/mg sample). The antioxidant activity of lignin samples were analyzed by evaluating free radical scavenging effect. The DPPH radical scavenging capacities of lignin samples were determined according to the slightly modified method of Aadil et al., 2014 [6]. The DPPH solution was prepared by dissolving 11.8 mg DPPH in 50 mL methanol water solution (30:20 v/v).The lignin stock solution (1 mg/mL) was prepared by dissolving lignin in ethylene glycol. The reaction mixture consists of 2.5 mL DPPH (dissolved in methanol) and 0.5 mL lignin stock solution. Then the mixtures were incubated at 30 min in the dark room and then absorbance was measured at 517 nm using gallic acid as standard. The antioxidant activity was calculated as follows: DPPH radical scavenging activity (%) = (ODc − ODs)/ ODc X 100

(1)

where ODc was the absorbance of the control, and ODs was the absorbance of the sample. 2.6 Analytical characterization of biomass 2.6.1 SEM, XRD, FTIR and XPS analysis The SEM (FEI Quenta 200 F) was used to observe the surface microstructure changes of the raw rice straw after treatments. The samples were placed on the specimen stub, coated with gold and subjected to image analysis. Raw and treated biomass crystallinity indexes were determined by X-ray diffractometer (X’Pert PRO diffractometer, PANalytical (Netherlands)). The X-ray unit was operated at 25 °C using Cu Kα radiation (λ=1.542 Å) generated at 45 kV and 40 mA. The diffraction profile was analyzed in the 2θ range from 10 to 50°. The crystallinity index (CrI) of the samples is calculated according to CrI = (Icry – Iamp)/ Icry X 100 Where Icry and Iamp is the CrI of crystalline region and amorphous region, respectively [19].

(2)

The surface elemental compositions of untreated and treated RS were analysis by XPS (PHI 5000 Versaprobe III scanning XPS microscope analytical instrument). Monochromatic Al Kα sources operated under 15 kV and 25W were used to record the spectra. The chemical composition of the biomass sample is analyzed in the range from 1100 eV to 0 eV binding energy. The pass energy of 280 eV with a step size of 1 eV was used to record the Cls and O1s spectra. Origin version and XPS peak fit software were utilized in processing the data.

3. Results and discussion 3.1. Rice straw fractionation process In this study, RS was used as feedstock for lignin extraction using OAT and SHT process, as shown in Fig 1. Rice straw fractionation process observed 45.9 % biomass dissolution in OAT and 40.7 % in SHT process at 90 °C for 180 min of treatment. Under this condition 107.5 mg/(g of RS) of lignin obtained in OAT method and 141.2 mg/(g of RS) of lignin in SHT method. Delignification of RS results suggest that, OAT is more efficient than SHT under the process conditions, which is consistent with the results in a previous work by Florian et al, 2019. The study is important from understanding that SHT selectively hydrolyzes the covalent lignin-carbohydrates linkages compared to the OAT pulping process [20]. On the other hand, the biomass dissolution rate was more for OAT method than the SHT method. This phenomenon could be due to effective penetration of organic acid causing the solubilization of celluloses and hemicelluloses fractions during the delignification process. This pattern has been consistent to those obtained during lignin extraction by Florian et al., 2019 [21].

3.2. Lignin characterization UV-vis absorption spectra of the OAT-lignin and SHT-lignin samples were carried out from 250-400 nm. As shown in Fig 2A, non-conjugated phenolic groups in the lignin sample exhibits at 280-285 nm, and conjugated phenolic groups (p-coumaric and ferulic acids) absorption at 345-350 nm. In OAT-lignin, additional small peaks at 300-310 nm were observed due to the carbonyl groups present in the sample. FTIR spectra of OAT-lignin and SHT-lignin samples were characterized based upon the assignments given in previous literature [11]. FTIR spectra of both lignin samples show (Fig 2B) that variations of band intensities observed due to functional groups were affected by the chemicals used in the extraction process. The broad vibration at 3400-3450 cm−1, was associated to

the hydroxyl groups in phenolic and aliphatic compounds. The vibrations at 2930 and 2840 cm−1 were attributed to the C-H vibrations in aromatic methoxyl and methylene groups of the side chains. The absorbance band at 1710 cm−1 corresponding to unconjugated carbonyl/carboxyl stretching in OAT-lignin and SHT-lignin. The different peak intensity observed in both samples at 1600, 1514 and 1424 cm−1 due to varying quantity of phenyl-propane skeleton present in the samples [10]. In addition the absorbance signal at 1330 and 1125 cm−1 represent syringyl unit (S), while bond at 1259 cm−1 associate to guaiacyl unit (G), confirming that both lignin composed of S and G basic units [9]. The bond between1300 and 1000 cm−1 represented that C-C, C=O and C-O groups present in the lignin samples at different quantities. The 1H NMR was used to investigate structural features of SHT-lignin and OAT-lignin and H chemical shifts were shown in Fig 3. Table 1 summarizes the results of chemical shifts assignment of 1H NMR signals. The chemical shifts in the range of 6.00–8.00 ppm represented aromatic protons. The signal at 7.60–7.40 ppm could be assigned to p-hydroxyphenyl, while signals at 7.30–7.00 and 6.9–6.30 ppm originated from the G and S unit, respectively [2,15,22]. The two weak peaks at 6.0–4.0 ppm were associated to aliphatic protons with the β-O-4′, β-1 and β-β structures [23]. In addition, both lignins showed strong signal at 4.0–3.6 ppm attributed to methoxyl protons (–OCH3).The solvent peak for DMSO was assigned at 2.5 ppm. Sharp acetyl signal was observed between 2.5–1.5 ppm indicating the presence of the protons of aromatic and aliphatic acetates. These protons are obtained from the acetylation of phenolic and alcoholic hydroxyl groups. The signal obtained between 1.4 and 0.6 ppm may be corresponded to the aliphatic moiety of the lignin [24-26].

3.3. Analysis of phenolic and antioxidant power of lignin samples The total phenolic content and antioxidant activity of lignin samples was summarized in Table 2. The maximum TPC was observed in SHT-lignin (28.87 mg of GAE/g) and OAT-lignin (24.75 mg of GAE/g). The results suggest that basic condition of SHT process solubilize the phenolic hydroxyl groups better compared to the acidic condition of OAT process. Michelin et al. reported TPC values of 25.6 mg GAE/g obtained from hydrothermally pretreated ethanol organosolv lignin. On the other, hand Qazi et al. reported TPC value of 50 mg GAE/g observed from kraft lignin [27,28]. The antioxidant activity was quantified in terms of percentage inhibition of lignin. The highest inhibition of 55.91 % was observed in the SHT-lignin at 15 min and 59.50 % at 30 min,

while OAT-lignin observed 42.25 % incubation at 15 min and 45.74 % at 30 min at the same concentration. FTIR results also support our contention that the higher antioxidant activity of SHTlignin. The overall results suggest higher TPC and antioxidant activity in SHT-lignin compared to OAT-lignin. Aadil et al described that more than 90 % inhibition (DPPH assay) and more than 350 µg GAE of TPC were observed in acetone and methanol fractionated lignin [6].

3.4. Biomass characterization 3.4.1. SEM analysis of pulps and raw rice straw The SEM observation revealed that surface morphological of raw rice straw was clean and smooth due to its wax and lignin substance covering the fiber surface (Fig. 4A). In Fig 4B, the fibre surface is less damage and some gaps between nearby cellulosic fibres due to the dilute alkali SHT process removed part of the lignin in the fiber surface. The smooth surface appeared on the treated OAT biomass sample in Fig. 4C, indicates the complete removal of wax and lignin in the biomass by OAT process [20, 29, 30].

3.4.2. Structure analysis of rice straw by XRD and FTIR The XRD patterns (Fig 5A) obtained from UT-RS, OAT-RS and SHT-RS exhibited two sharp peaks appearing at 22.3° and 15.6°, which represent the intensity of crystalline and amorphous regions of biomass. Table 3 shows that, CrI of UT-RS was determined to be 32.66 %, which subsequently increased to 38.18 % after SHT process due to removal of lignin and extractives. Similar study was reported in previous literature, such as higher crystalline size (73.2 %) sugarcane tops obtained after alkali pretreatment compared to raw sample crystalline size (61.63 %) [29]. The CrI reached high value of 45.10 % in OAT-RS biomass. The higher crystallinity index obtained in OAT-RS compared to SHT-RS (45.10 % vs 38.18 %), suggests the complete removal of amorphous non-cellulosic compounds (lignin, extractives and hemicelluloses) in the strong acidic treatment. Previous reports also support that crystallinity index of native rice straw (49.03 %), which increased to 58.59 % after organosolv pretreatment process [31]. FTIR spectrum evaluates the functional groups changes in the surface of rice straw samples as shown in Fig 5B. Clear absorption spectra differences can be detected at 3416 cm-1 in UT-RS, which is shifted to 3435 cm-1 after SHT and 3441 cm-1 after OAT due to the dissolution of amorphous lignin and hemicelluloses components [31]. The SHT-RS and OAT-RS biomass show that lignin weak peaks observed at 1630, 1529 and 1427 cm-1. In addition to that, very weak

intensity of syringyl ring and guaiacyl vibrations at 1325 and 1222 cm-1 observed in treated RS due to the significant removal lignin. Similarly, weak aromatic ring vibrations (1215 and 1270 cm−1) appeared in SHT-RS and OAT-RS compared to untreated RS [20,32].

3.4.3. Surface composition of biomass analysis by XPS The surface element compositions of untreated-RS and treated-RS were analyzed using an XPS survey method, which were performed with a snapshot model. The XPS spectra reveal that carbon and oxygen elements peaks appeared in the surface of rice straw at 284 and 534 eV respectively, which is in excellent agreement with previous literature [33].

3.4.4. XPS survey spectra analysis An XPS survey spectrum was used to measure the biomass surface O/C atomic ratio. The O/C ratio of UT-RS (0.41), SHT-RS (0.44), and OAT-RS (0.78) were calculated from XPS spectra (Fig 6) and presented in Table 4. According to the literature, the O/C ratio of lignin and cellulose are calculated as 0.33 and 0.8 respectively [34]. From the UT-RS survey, spectra O/C ratio value was near to the theoretical value of lignin, which confirms that more lignin component is present in the surface of untreated biomass. The O/C ratio value of biomass slightly increased after SHT process due to partial removal of lignin from surface of biomass. The maximum O/C ratio of 0.78 was obtained after OAT process, which confirms complete removal of lignin and only cellulosic fiber is present in biomass surface. This finding is good agreement with previous literature [34].

3.4.5. Analysis of C1s and O1s spectra The high-resolution XPS C1s spectra of rice straw were obtained in a scanned model, which indicated the presence of three components (C1-C3) peak between 291 to 281 eV as shown in Fig 7. The C1 peak is related to carbon linked to carbon (C-C) or hydrogen (C-H) groups, from the aliphatic and aromatic carbons of lignin and extractives in rice straw biomass [33,35]. The C1 peak decreased from 72.9 to 52.9 % and 43.1 % in SHT-RS and OAT-RS respectively,which is probably due to the removal of lignin in delignification process. On the other hand, C-O assigned C2 peak and C═O assigned C3 peak are increased in SHT-RS and OAT-RS. The C2 peak increased from 20 % to 36.2 % and 40.1 % after SHT and OAT process respectvely. It can be concluded that SHT-RS surface consists of both lignin and cellulose components, where as OATRS surface contine only cellulose component. The results confirmed that the OAT process

significantly changes the biomass surface compared to the SHT process, as supported by the SEM (Fig 4), XRD and FTIR (Fig 5) analyses [34,36]. Rice straw O1s spectra observed two subunit peaks in between 528 and 537 eV shown in Fig 8. The O1peak corresponded to (O–C=O), which is mainly originates from lignin. O2 peak corresponded to (C–O–), which is associated with hemicelluloses and cellulose in biomass sample. The results of the O1s spectra analysis shows that the O1 components decreased, whereas the O2 component increased after OAT process. On the other hand, in the SHT-RS, O1 peak increased and O2 peak decreased compared to untreated RS, due to the significant amount of lignin and cellulose present in SHT-RS biomass.

4. Conclusion The extracted SHT-lignin and OAT-lignin were analyzed by UV-vis, FTIR and 1H NMR spectroscopic techniques to elucidate their structural characteristics. It was found that SHT-lignin had more purity with more content of aromatic compound compared to OAT-lignin. The SHT process facilitated the selective cleavage of the β-O-4 linkages in the lignocelluloses biomass resulting in a higher phenolic content in SHT-lignin. Furthermore, total phenolic content and antioxidant activity analysis also indicated SHT-lignin contains more phenolic OH groups compared to OAT-lignin. The morphological and structural changes of biomass after the treatment were confirmed using SEM, XRD and FTIR. The XPS spectroscopy survey spectra atomic O/C ratio value of untreated and treated biomass suggested that SHT process selectively remove lignin, where as OAT process completely fractionates the lignin and hemicelluloses. These findings will support future lignin valorization and cellulose bio-refineries process in fine chemicals and biofuels synthesis.

Acknowledgements: The authors are thankful to the Management of SRM Institute of Science and Technology and department of Chemical Engineering for their support to carry out this research work and kindly supported by Nanotechnology Research Centre for providing analytical facilities.

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List of figure Figure 1.Schematic representation of the rice straw fractionation process and corresponding analysis techniques Figure 2.UV-visible spectra of lignin samples (A) and FTIR spectra of lignin samples (B) Figure 3.1H NMR spectra of lignin samples Figure 4. FE-SEM images of UT-RS (A), SHT-RS (B), and OAT-RS (C) Figure 5. XRD pattern of biomass samples (A), and FT-IR spectra of biomass samples (B) Figure 6. XPS survey spectra of UT-RS (A), SHT-RS (B), and OAT-RS (C) Figure 7. The high-resolution XPS C1s spectra of UT-RS (A), SHT-RS (B), and OAT-RS (C) Figure 8. The high-resolution XPS O1s spectra of UT-RS (A), SHT-RS (B), and OAT-RS (C)

Table 1. Proton shift assignment for 1H NMR for lignin samples Signal (ppm)

Assignment

7.6-7.4

Aromatic H of p-hydroxyphenyl units

7.3-7.0

Aromatic H of guaiacyl units

6.9-6.3

Aromatic H of syringyl units

6.0-4.0

Aliphatic protons with β-O-4′, β-1 and β-β structures

4.0-3.6

H of methoxyl groups

2.5-1.5

H in aromatic acetates

1.4-0.6

H in aliphatic acetates

Table 2. Total phenolic and antioxidant capacity (DPPH assay) of lignin samples. Lignin

DPPH assay (%) 15 min 30 min

OAT-lignin SHT-lignin

42.25 55.91

45.74 59.50

TPC (mg GAE/g) 24.75 28.87

Table 3. XRD analysis the crystallinity index of untreated and treated rice straw Biomass

CrI of crystalline region at 22°

CrI of amorphous region at 15°

Crystallinity Index

UT-RS 27366 18427 32.66 SHT-RS 22910 14162 38.18 OAT-RS 23398 12844 45.10 Icr : CrI of crystalline region at 22.1° ; Iam : CrI of amorphous region at 15.3°

Table 4. Summary of XPS spectral parameters of untreated and treated rice straw Biomass

C/%

Component % (C1s survey)

O/% O/C

Component % (O1s survey)

C1

C2

C3

O1

O2

70.8

29.2 0.41

72.9

20.0

6.9

5.7

94.2

69.4

30.6 0.44

52.9

36.2

10.8

13.6

86.3

OAT-RS 55.9

44.1 0.78

43.1

40.1

16.6

0.9

99.1

UT-RS SHT-RS

Highlights

x

Rice straw was fractionated to extract the lignin by alkaline and acidic treatment process

x

Structural characterization of the SHT-lignin and OAT-lignin fractions were analyzed by UV, FTIR and 1H NMR technique

x

Surface characterization of UT-RS, SHT-RS and OAT-RS biomass were carried out by SEM, XRD and FTIR technique

x

Surface chemical composition changes of SHT-RS and OAT-RS analysis by XPS survey, Cls and O1s spectra