Soluble Lignin Research Articles

Effect of Microwave Pretreatment on Some Properties of Bamboo (Gigantochloa apus) for Bioethanol Production

Pretreatment of lignocellulosic biomass plays an essential role in bioethanol production as an alternative biofuel. This process reduces biomass recalcitrance in order to improve cellulose digestibility for saccharification and further fermentation reactions. In this study, microwave (MW) pretreatment was done on bamboo (Gigantochloa apus) to investigate the resulting physicochemical properties and bioethanol produced. Bamboo was irradiated in the microwave at different power (100-600 Watt) and irradiation time (5-20 minutes) followed by Water Soluble content (WSC) and lignin analysis. Simultaneous saccharification and fermentation (SSF) using cellulase enzyme was also done in five different treatment combinations (C1-C5) to investigate the effect of produced reducing sugar and bioethanol. The result shows that increasing MW power and irradiation time could decrease WSC gradually. The lowest WSC of 0.3% was obtained at 600-Watt MW power and 20 minutes irradiation time. Lignin content decreased from 18.9% to 16.0% concerning increasing irradiation time from 5 to 20 minutes under 300-Watt MW power. SEM images show that partial disruption and micro-scale pores existed in pretreated samples. The highest amount of ethanol was obtained at 24 hours fermentation for pretreated bamboo at 300-Watt MW power for 15 minutes followed by cellulase enzyme addition. The overall results showed that microwave pretreatment is a prospective method for future bioethanol production from bamboo due to effective WSC reduction and lignin degradation in a relatively short period of time.

Polystyrene sulfonate is effective for enhancing biomass enzymatic saccharification under green liquor pretreatment in bioenergy poplar

BackgroundWater-soluble lignin (particularly lignosulfonate, LS) has been well documented for its significance on enzymatic saccharification of lignocellulose, though the promotion mechanism has not been fully understood. Much attention has been paid to natural lignin or its derivatives. The disadvantage of using natural lignin-based polymers as promoting agents lies in the difficulty in tailor-incorporating functional groups due to their complex 3D structures. To further improve our understanding on the promotion mechanism of water-soluble lignin in the bioconversion of lignocellulose and to pursue better alternatives with different skeleton structures other than natural lignin, herein we reported a synthetic soluble linear aromatic polymer, sodium polystyrene sulfonate (PSS), to mimic LS for enhancing the efficiency of enzymatic saccharification.ResultsThe role of PSS in enzymatic saccharification of pure cellulose and green liquor-pretreated poplar (GL-P) was explored by analyzing substrate enzymatic digestibility (SED) under different addition dosages and various pH media, along with LS for comparison. At the cellulase loading of 13.3 FPU/g-glucan, the glucose yield of GL-P increased from 53% for the control to 81.5% with PSS addition of 0.1 g/g-substrate. It outperformed LS with the addition of 0.2 g/g-substrate by 6.3%. In the pH range from 4.5 to 6, PSS showed a positive effect on lignocellulose saccharification with the optimum pH at 4.8, where the most pronounced SED of GL-P was achieved. The underlying mechanism was unveiled by measuring zeta potential and using Quartz Crystal Microbalance (QCM) and Multi-parametric Surface Plasmon Resonance (MP-SPR). The results confirmed that the complexes of cellulase and PSS were conjugated and the negatively supercharged complexes reduced non-productive binding effectively along with the improved saccharification efficiency. The thickness of PSS required to block the binding sites of cellulase film was less than half of that of LS, and the PSS adlayer on cellulase film is also more hydrated and with a much lower shear modulus than LS adlayer.ConclusionsPSS as LS analogue is effective for enhancing the biomass enzymatic saccharification of GL-pretreated poplar. PSS exhibited a severer inhibition on the enzymatic saccharification of pure cellulose, while a more positive effect on bioconversion of lignocellulose (GL-P) than LS. In addition, a much lower dosage is required by PSS. The dynamic enzymatic hydrolysis indicated PSS could prolong the processive activity of cellulase. The valid data stemmed from QCM and SPR expressed that PSS bound to cellulases and the as-formed complexes reduced the non-productive adsorption of cellulase onto substrate lignin more efficiently than LS due to its flexible skeleton and highly hydrated structure. Therefore, PSS is a promising alternative promoting agent for lignocellulose saccharification. From another perspective, the synthetic lignin mimics with controllable structures enable us to reach an in-depth understanding of the promotion mechanism of soluble lignins on enzymatic saccharification.

Lignin properties and cell wall response to deconstruction by alkaline pretreatment and enzymatic hydrolysis in brown midrib sorghums

Lignin has an adverse impact on the deconstruction of plant cell wall biopolymers in biorefining processes and its reduction and/or alteration during biosynthesis is one target for decreasing plant cell wall recalcitrance. In this work, the impact of two brown midrib mutations (bmr6 and bmr12) in two sorghum background lines (the commercial hybrid Atlas and near-isogenic BTx623) on lignin properties and the plants’ response to cell wall deconstruction to monomeric sugars via alkaline pretreatment and enzymatic hydrolysis is investigated with the goal of assessing how differences in lignin content and properties impact the plant’s response to pretreatment. We identify that both bmr sorghum lines show significantly lower abundance of water-extractable sugars (glucose, sucrose, and fructose) and alkali-saponifiable p-coumarate. Furthermore, both these properties exhibited identical trends across both background lines. Next, both untreated and mild alkali-pretreated bmr sorghums were shown to exhibit higher glucose hydrolysis yields following enzymatic hydrolysis than the control lines. Following pretreatment, the Atlas bmr sorghums exhibited more lignin solubilization and the solubilized lignin was of lower molar mass than the background control line suggesting that differences in the lignin response to pretreatment resulted these differences. Finally, significant differences were observed in the lignin content, lignin monomer distribution, and inter-unit linkages in the Atlas bmr line relative to the control line with key differences including lower syringyl monomer content in both bmr lines, higher relative abundance of β-O-4 linkages in the bmr6 line, and the presence of 5-hydroxy guaiacyl monomers and benzodioxane (α-O-5/β-O-4) linkages in the bmr12 line.

The effects of methyl jasmonate on growth, gene expression and metabolite accumulation in Isatis indigotica Fort.

Methyl jasmonate (MeJA) plays important roles on growth, gene expression and metabolite accumulation in plants. In this study, the physiological and biochemical characteristics, transcriptome and metabolome profiles of Isatis indigotica Fort., responded to MeJA were explored. Under the treatment, the contents of dry matter, chlorophyll and carotenoids increased significantly, while the water content did not. And the contents of soluble protein, flavonoid and lignin were also raised significantly, but the soluble sugar decreased. Besides, the enzyme activities of phenylalanine ammonia lyase (PAL), lipoxygenase (LOX), polyphenol oxidase (PPO), peroxidase (POD), catalase (CAT) and superoxide dismutase (SOD) increased, showing certain resistance characteristics of this plant. Based on RNA-seq, 31,769 unigenes were functionally annotated, and 2725 differential expressed genes (DEGs) identified. Among them, 57 DEGs covered most of the catalytic steps in general biosynthesis pathways of jasmonic acid, glucosinolates and phenylpropanoid. Referring to the metabolomics analysis, 175 significant differential metabolites were detected by GC-MS, combined with LC-MS method. Moreover, 53 differential metabolites were identified by the multivariate statistical analysis, mainly enriched in 37 different metabolic pathways, with most differential metabolites found in phenylalanine metabolism pathway. The results provided the comprehensive analysis on growth, transcriptome and metabolite profiles responded to MeJA, and gave new insights into revealing and enriching the functions of MeJA, contributing to explore the key genes and their functions on growth and metabolite accumulation and regulation process in I. indigotica.

Application of ligninolytic bacteria to the enhancement of lignocellulose breakdown and methane production from oil palm empty fruit bunches (OPEFB)

Efficient pretreatment of lignocellulosic biomass is needed to reduce associated energy costs. Lignin-degrading bacteria (Comamonas testosteroni, Agrobacterium sp., Lysinibacillus sphaericus, and Paenibacillus sp.) were tested as potential pre-treatment methods for oil palm empty fruit bunches (OPEFB) to enhance its methane yield during anaerobic digestion (AD). The lignocellulose fractions were analysed for total reducing sugar (TRS), total soluble phenols (TSP), lignin content, weight loss, pH and microscopic analysis carried out both before and after treatment. The methane production was tested using the biochemical methane potential (BMP) test. Bacteria mediated depolymerisation of OPEFB was confirmed through the observation of increased release of TSP and associated lignin breakdown and weight loss of the cultured OPEFB biomass. The highest percentage of lignin breakdown (25.84%) was obtained from OPEFB treated by Lysinibacillus sphaericus, while the highest average specific methane potential (0.042 m3/kg VS) was obtained from treatment with Comamonas testosteroni.

Further insights into the solubilization and surface modification of lignin on enzymatic hydrolysis and ethanol production

In this study, the effect of residual lignin and its structural modification of pretreated sugarcane bagasse (SCB) on enzymatic hydrolysis and fermentation was comprehensively evaluated by alkali and alkaline hydrogen peroxide (AHP) pretreatment. It was found that the AHP pretreatment had a negative influence on lignin dissolution compared with alkali pretreatment accompanied by a relatively low NaOH concentration (≤1%, w/v). However, there was less significance of enzymatic hydrolysis efficiency between NaOH and AHP pretreatment with >1% NaOH loading due to a high lignin removal above 78%. In addition, PEG 6000-assisted pretreatment was also performed to stabilize the enzymatic activity and further promote enzymatic digestibility and fermentability. The highest ethanol concentration of 59.96 g/L (61.22%) was obtained with PEG 6000-assisted 1% NaOH pretreated SCB at 30% solid loading. The structural features of the residual lignin indicated that most of the side chain structures were oxidized and that some aryl-ether bonds were dissociated after the pretreatments, especially in alkali pretreatment. The reduction of molecular weight and the breaking of lignin substructures help to achieve a more effective yield of enzymatic hydrolysis and fermentation.

Technological characterisation of wood from Australian red cedar after first thinning of a plantation at five years

Australian red cedar (Toona ciliata M. Roem.) is a forest species planted in Brazil especially for lumber production. This study aimed to evaluate technological properties of the wood after selective first thinning of a plantation 5 years after planting. The technological properties evaluated were: (i) physical – apparent specific mass at 12% moisture content and shrinkage; (ii) mechanical – modulus of elasticity (E), modulus of rupture (σ), and brittleness; (iii) weathering; (iv) surface macrostructure – colour and roughness; (v) chemical – soluble and insoluble lignins, and holocellulose and ash contents; and vi) thermal – thermogravimetric analysis and calorific value. Apparent specific mass was 380 g m−3; the calculated shrinkage anisotropy of 1.04 and swelling anisotropy of 1.14 fit the classification ‘excellent quality’; the elasticity modulus was calculated as 4 700 MPa, and strength to flexion was 45 MPa. The mechanical properties other than brittleness were not compromised with weathering exposure. However, the accelerated weathering testing as a simulation of natural weathering showed modified colour of the wood and reduction of the mechanical properties. The calorific value of the wood (4 634.03 Kcal g−1) was similar to that reported for other fast-growing woods at even more advanced planting ages. Thermal stability of the wood was comparable to other fast-growing species used for energy purposes, owing especially to high holocellulose content.

Amino Acid-Based Deep Eutectic Solvents in Biomass Processing - Recent Advances

The use of non-conventional solvent systems, such as deep eutectic solvents (DES), for biomass processing is a growing interest. DES are formed by two or more components, usually solids at room temperature, which can interact with each other via hydrogen bonding, from a hydrogen bond acceptor (HBA) and a hydrogen bond donor (HBD), resulting in a liquid phase. The most studied HBA in the literature is choline chloride with several HBD and their use have been extensively reviewed. However, other abundant and natural HBA can be successfully applied on the preparation of different DES, e.g., amino acids. These amino acid-based DES have been used in biomass pretreatment, providing the fractionation of the main macromolecular components by lignin solubilization. In addition, amino acid-based DES can be applied in biomass chemical conversion to obtaining platform chemicals such as furanic derivatives. Bearing this in mind, this review focuses on exploring the use of amino acid-based DES on biomass processing, from pretreatment to chemical conversion.

Production of kraft pulp from Ochroma pyramidale wood

ABSTRACT Brazil stands out in the international scenario in the production of short-fiber pulp. Despite the great Brazilian biodiversity, that production is based on exotic Eucalyptus clones. In this sense, there may be great potential in the assessment of new sources of fibers from the Brazilian flora, including the Amazon. The present study aimed to assess the technical potential of the wood of Ochroma pyramidale (Malvaceae) for the production of kraft pulp. Four-year-old trees were harvested from a commercial forest for lumber production in Mato Grosso state (Brazil). We determined the wood’s chemical compositions (holocellulose, Klason lignin, soluble lignin, extractives and ash contents), physical properties (density and porosity), and fiber morphology (fiber length, width and thickness, lumen diameter, wall fraction, coefficient of flexibility, and slenderness and Runkel ratios). The wood was subjected to pulping with an effective alkali charge ranging from 10 to 24%, with intervals of 2%. Ochroma pyramidale wood presented characteristics favorable to the production of cellulosic pulp, such as appropriate fiber dimensions and low lignin, extractives, and ash content. The amount of residual active alkali and pH of the black liquor were positively related to the increase of the alkali charge employed in the pulping process. The increase of alkali charge decreased the pulp yield, kappa number and waste content, and increased the hexenuronic acid concentration.

Changes in chemical composition of cover crops residue during decomposition

ABSTRACT: Crop residues decomposition are controlled by chemical tissue components. This study evaluated changes on plant tissue components, separated by the Van Soest partitioning method, during cover crop decomposition. The Van Soest soluble fraction was the first to be released from the crop residues, followed by cellulose and hemicellulose. Lignin was the crop residue component that suffered the least degradation, and for certain crop residue types, lignin degradation was not detected. The degradation of the main components of crop residues (soluble fraction, cellulose, hemicellulose and lignin) is determined by the chemical and structural composition of each fraction.

Biocatalytic pretreatment of rice straw by ligninolytic enzymes produced by newly isolated Micrococcus unnanensis strain B4 for downstream cellulolytic saccharification.

Rice (Oryza sativa L.) straw is an agricultural byproduct of high yield, and its disposal by burning has detrimental effect on ecosystem. It has potential as source of fermentable sugars for industrial use; however, it requires effective pretreatment to remove lignin. Bacterial enzymes based pretreatment is advantageous due to their extracellular nature, and tolerance to higher temperature, pH and oxygen limitation. We herein report screening of lignocellulose degradation environment of vermicompost for ligninolytic bacteria, and studying role of Micrococcus unnanensis strain B4 in delignification of rice straw. The bacterium was capable to degrade acid soluble and insoluble lignin; and produced lignin degrading laccase and peroxidase having maximum activity at pH 6.5 and 72 h incubation. Both enzymes exhibited alkaline pH stability, and thermal stability with retention of 100 % activity on pre-incubation at 60 ℃ for 1 h. The enzymes were used for pretreatment of rice straw using chemicals (acetic acid:hydrogen peroxide) pretreatment as reference. Scanning electron microscopy of pretreated rice straw samples showed alteration in morphology with exposure of cellulosic components. Enzymatically pretreated rice straw on saccharification by a commercial cellulase yielded about 400 mg of reducing sugar per gram, comparable to that released on chemical pretreatment. Hence, pretreatment based on M.unnanensis strain B4 and its ligninolytic enzymes can be an alternative to chemical pretreatment for saccharification of rice straw to fermentable sugars.

Transcriptome analysis reveals the main metabolic pathway of c-GMP induced by salt stress in tomato (Solanum lycopersicum) seedlings.

Tomato (Solanum lycopersicum L.) is a model crop as well as an important food worldwide. In arid areas, increasing soil salinity has limited higher yields in tomato production. As a second messenger molecule, cyclic guanosine monophosphate (c-GMP) plays an indispensable role in plant response to salt stress by regulating cell processes to promote plant growth and development. However, this mechanism has not been fully explored in tomato seedlings. In this experiment, tomato seeds were cultured in four treatments: (1) distilled water (CK); (2) 20μM c-GMP (T1); (3) 50mM NaCl (T2); and (4) 20μM c-GMP+50mM NaCl (T3). The results show that 20μM c-GMP effectively alleviated the inhibitory effect of 50mM NaCl on growth and development, and induced the expression of 1580 differentially expressed genes (DEGs). Seedlings in the CK vs T1 shared 95 upregulated and 442 downregulated DEGs, whereas T2 vs T3 shared 271 upregulated and 772 downregulated DEGs. Based on KEGG (Kyoto Encyclopaedia of Genes and Genomes) analysis, the majority of DEGs were involved in metabolism; exogenous c-GMP induced significant enrichment of pathways associated with carbohydrates, phenylpropanoids and fatty acid metabolism. Most PMEs , acCoA , PAL , PODs , FADs , and AD were upregulated, and GAPDHs , PL , PG , BXL4 , and β-G were downregulated, which reduced susceptibility of tomato seedlings to salt and promoted their salt tolerance. The application of c-GMP increased soluble sugar, flavonoid and lignin contents, reduced accumulation of malondialdehyde (MDA), and enhanced the activity of peroxidase (POD). Thus, our results provide insights into the molecular mechanisms associated with salt tolerance of tomato seedlings.

Profitable disperser coupled surfactant pretreatment of aquatic phytomass for energy efficient solubilization and biomethanation: a study on lignin inhibition and its possible solutions

The effect of cost-effective combined pretreatment on rice straw for lignin solubilization and the recovery of biomethane.

Study on the removal of lignin from pre-hydrolysis liquor by laccase-induced polymerization and the conversion of xylose to furfural

High-value use of PHL to transform the platform compound furfural. Laccase polymerization through the combination of 5-5′ and 4-O-5′ bonds removes soluble lignin in PHL while protecting the sugar content from loss.

Controlling lignin solubility and hydrogenolysis selectivity by acetal-mediated functionalization

Aldehyde assisted fractionation of biomass produces an acetal-stabilised lignin that is fully upgradeable. We exploit the aldehyde to control the lignin's physical properties, producing lignins that can be soluble in both polar and apolar solvents.

Valorization of waste bark for biorefineries: chemical characterization of Eucalyptus camaldulensis inner and outer barks

Abstract Bark wastes today are viewed as a high-value resource for biorefinery due to their chemical richness and diversity. This work presents a comprehensive chemical characterization of the inner bark and the outer bark of Eucalyptus camaldulensis cultivated in Algeria. The extractives were first isolated with an Accelerated Solvent Extractor (ASE) and then analyzed by Gas Chromatography-Mass Spectrometry (GC-MS). The content of pre-extracted bark in cellulosic polysaccharide and free sugar monomers was determined by Gas Chromatography (GC). The hemicellulose composition and amount was determined after the acid methanolysis and GC. The amount of lignin was determined gravimetrically by the Klason lignin method and the acid soluble lignin was determined by the UV method. Formic and acetic acids in the bark were determined by HPLC after alkaline hydrolysis. It was found that the extractives content were similar in the outer bark (0.85%) and the inner bark (0.88%). The cellulose content was higher in the outer bark (33.4%) than in the inner bark (28.7%). Lignin and the total hemicellulose contents were more abundant in the outer bark (31.7, 26.2%) than in the inner bark (28.6, 19.3%) whereas, sugar monomers were more abundant in the inner bark (4.4%) than in the outer bark (3.8%). The variation in acetic and formic acids and ash contents between the outer bark (1.5, 0.006 and 2.5%) and the inner bark (1.3, 0.005 and 2.4%) was small. The obtained results showed that the bark can be considered a suitable feedstock for lignocellulosic biorefinery and also for the extraction of bioactive compounds that can be used in different sectors.

HETEROGENEOUS CATALYTIC FRACTIONATION OF BIRCH-WOOD BIOMASS INTO MICROCRYSTALLINE CELLULOSE, XYLOSE AND ENTEROSORBENTS

For the first time, it was proposed to fractionate the main components of birch wood into microcrystalline cellulose, xylose and enterosorbents by integrating heterogeneous catalytic processes of acid hydrolysis and peroxide delignification of wood biomass. The hydrolysis of wood hemicelluloses into xylose is carried out at a temperature of 150 °C in the presence of a solid acid catalyst Amberlyst® 15. Then the lignocellulosic product undergoes peroxide delignification in a "formic acid – water" medium in the presence of a solid TiO2 catalyst to obtain microcrystalline cellulose (MCC) and soluble lignin. Under the determined optimal conditions (100 °С, Н2О2 – 7.2 wt.%, НСООН – 37.8 wt.%, LWR 15, time 4 h), the yield of MCC reaches 64.5 wt.% and of organosolvent lignin 11.5 wt% from the weight of prehydrolyzed wood. By the treatment of organosolvent lignin with a solution of 0.4% NaHCO3 or hot water the enterosorbents were obtained, whose sorption capacity for methylene blue (97.7 mg/g) and gelatin (236.7 mg/g) is significantly higher than that of the commercial enterosorbent Polyphepan (44 mg/g and 115 mg/g, respectively). The products of catalytic fractionation of birch wood are characterized by physicochemical (FTIR, XRD, SEM, GC) and chemical methods.

Acidolysis mechanism of lignin from bagasse during p-toluenesulfonic acid treatment

Lignin was efficiently separated from lignocellulosic biomass using p-toluenesulfonic acid (p-TsOH) treatment. However, lignin depolymerization and acidolysis have not been studied to date. In this study, the mechanism of lignin acidolysis was investigated. The structure of lignin in bagasse before and after the p-TsOH treatment was analyzed. The efficiency and selectivity of lignin depolymerization were evaluated. The lignin removal rate reached 88.81%. Hemicellulose was completely removed from lignin, and cellulose destruction was inhibited. The structure of lignin in bagasse was analyzed using nuclear magnetic resonance (NMR). The results revealed that α-methoxylated β-O-4 intermediates were formed during lignin depolymerization. This indicated that the benzyl ether bonds were effectively catalyzed and broke, thereby promoting lignin dissolution. The lignin–carbohydrate complex structure was destroyed. The insoluble and soluble lignin in the hydrolysate were analyzed using NMR and gas chromatography–mass spectrometry. The results indicated that the catalyst promoted breaking of the Cβ–O bonds in the β-O-4 structures. Phenols and Hibbert ketones were formed. In addition, lignin sulfonation was confirmed by the presence of di-o-tolusulfone and di-p-tolusulfone. The low contents of β-β and β-5 bonds in the reaction products indicated that lignin condensation was inhibited. The results demonstrated that lignin acidolysis was synergistically catalyzed by H+ and p-TsOH. It provides important theoretical support for efficient and clean separation of lignin using the p-TsOH treatment.

γ-Valerolactone/water system for lignin fractionation to enhance antibacterial and antioxidant capacities

• A simple and green method for fractionating lignin by γ-valerolactone was proposed. • Lignin fractionation mechanism was investigated through solvatochromic parameters. • Mechanisms of improving antibacterial/antioxidant activities were proposed. • The low molecular weight lignin had excellent antibacterial/antioxidant properties. • The recovered γ-valerolactone had excellent lignin fractionation performance. In this study, γ-valerolactone, a green and recyclable organic solvent, was applied to fractionate organosolv lignin. The original lignin was dissolved in 60% γ-valerolactone/water solution, and sequentially sedimentated in 50%, 40%, 30%, and 1% γ-valerolactone aqueous solution with the addition of water afterwards, producing four fractions (L1, L2, L3, and L4). Multiple analytical methods were used to characterize the lignin fractions in detail. Results manifested that weight average molecular weight of lignin decreased gradually from 7900 (L1) to 1890 g/mol (L4), and the total phenolic hydroxyl content increased from 2.18 (L2) to 2.80 mmol/g (L4). Here, due to the increase of water content in the solvent system, the hydrogen bond capacity with lignin decreased and the solvent polarizability increased. Therefore, the solubility of high molecular weight lignin with low polar groups (hydroxyl and carboxyl) decreased, thus realizing the sequential fractionation of lignin. DPPH and agar diffusion test results indicated that low molecular weight lignin exhibited good antimicrobial/antioxidant activities, which was mainly due to the positive effects of the phenolic hydroxyl content and side chain structure of lignin. The mechanisms of improving antibacterial/antioxidant activities of lignin were proposed for potential practical applications.

Organosolv lignin aggregation behaviour of soluble lignin extract from Miscanthus x giganteus at different ethanol concentrations and its influence on the lignin esterification

BackgroundLignin is the second most abundant naturally occurring biopolymer from lignocellulosic biomass. While there are several lignin applications, attempts to add value to lignin are hampered by its inherent complex and heterogenous chemical structure. This work assesses the organosolv lignin aggregates behaviour of soluble lignin extract derived from Miscanthus × giganteus using different ethanol concentrations (50%, 40%, 30%, 20%, 10% and 1%). The effect of two different lignin concentrations using similar ethanol concentration on the efficacy of esterification was studied.ResultsOverall, particle size of lignin analysis showed that the particle size of lignin aggregates decreased with lower ethanol concentrations. 50% ethanol concentration of soluble lignin extract showed the highest particle size of lignin (3001.8 nm), while 331.7 nm of lignin particle size was recorded at 1% ethanol concentration. Such findings of particle size correlated well with the morphology of the lignin macromolecules. The lignin aggregates appeared to be disaggregated from population of large aggregates to sub-population of small aggregates when the ethanol concentration was reduced. Light microscopy images analysis by ImageJ shows that the average diameter and circularity of the corresponding lignin macromolecules differs according to different ethanol concentrations. The dispersion of lignin aggregates at low ethanol concentration resulted in high availability of hydroxyl group in the soluble lignin extract. The efficacy of the lignin modification via esterification was evidenced directly via FTIR using the similar ethanol concentration of soluble lignin extract at different lignin concentrations.ConclusionThis study provided the understanding of detail analysis on particle size determination, microscopic properties and structural insights of lignin aggregates at wider ethanol concentrations. The esterified lignin derived at 5 mg/mL is suggested to expand greater lignin functionality in the preparation of lignin bio-based materials.