Cellulosic Ethanol Production Research Articles

Spent yeast reuse as inoculum: a strategy to boost cellulosic ethanol productivity

AbstractFor the implementation of an integrated biorefinery into a pulping mill, the valorization of bark through cellulosic ethanol production and simultaneously the valorization of the residual yeast from each fermentation run should be considered to increase sustainability and pulp and paper profitability. Spent yeast recycling during cellulosic ethanol production is an opportunity to reduce waste according to environmental issues for disposal at the industrial level, boosting the circular economy. This work evaluated the reuse of spent yeast in successive fermentation experiments at Erlenmeyer and bioreactor scales for cellulosic ethanol from Eucalyptus globulus bark. The spent yeast recovered through centrifugation at the end of each experiment was used as inoculum for subsequent batch fermentation. Across five sequential fermentations, wherein only a fraction of spent yeast was recycled, a discernible decline in ethanol volumetric productivity was observed throughout successive fermentation cycles. Conversely, employing the complete spent yeast for recycling along 11 consecutive trials resulted in a notable enhancement of ethanol productivity, as evidenced by a reduction in fermentation duration to approximately one-third remaining the conversion efficiency above 70%. Four successive experimental trials were conducted at the bioreactor scale, resulting in volumetric productivities ranging from 2.00 to 5.83 g L−1 h−1. The reuse of spent yeast in consecutive fermentation cycles demonstrably augmented productivity, thereby validating a promising strategy for the industrial-scale production of cellulosic ethanol. This significantly contributes to advances in the circular economy principles within the overarching framework of biorefinery operations. Graphical Abstract

Novel class of aldehyde reductases identified from Scheffersomyces stipitis for detoxification processes in cellulosic ethanol production industry

The increase in industrialization has led to a significant energy crisis, sparking interest in lignocellulosic biomass for fuel ethanol production because of its renewable characteristics. The complex composition of this biomass requires pretreatment to reduce inhibitors like furfural and hydroxymethylfurfural (HMF), which hinder enzymatic hydrolysis and fermentation, ultimately decreasing ethanol yields. This study investigates the detoxification mechanisms of furan aldehydes in Scheffersomyces stipitis, particularly through the upregulation of genes SsOYE2.2, SsOYE2.7, and SsOYE3.1 under furfural and HMF stress. Enzyme characterization determined that SsOye3.3p is the most active enzyme for reducing both compounds using NADPH. Notably, SsOye2.6p showed the highest catalytic efficiency towards furfural, while SsOye2.8p was optimal for HMF. The study also established the optimal temperature and pH for these enzymatic reactions. Importantly, SsOye2.5p displayed broad substrate specificity, indicating its potential in detoxifying various aldehydes in microbial cells. The findings suggest that genes linked to enhanced enzymatic properties were not significantly induced, indicating that S. stipitis has more substantial potential for furan aldehyde detoxification and can be developed as a chassis organism exhibiting furan aldehyde tolerance. These insights facilitate the development of novel enzymes to counter furan aldehyde inhibitors and the creation of furan aldehyde-tolerant strains via genetic engineering.

Cellulosic Ethanol Production from High-Solids Corncob Residues by Simultaneous Saccharification and Fermentation on a Pilot Scale

Cellulosic Ethanol Production from High-Solids Corncob Residues by Simultaneous Saccharification and Fermentation on a Pilot Scale

Fungal Enzymes for Saccharification of Gamma-Valerolactone-Pretreated White Birch Wood: Optimization of the Production of Talaromyces amestolkiae Cellulolytic Cocktail.

Lignocellulosic biomass, the most abundant natural resource on earth, can be used for cellulosic ethanol production but requires a pretreatment to improve enzyme access to the polymeric sugars while obtaining value from the other components. γ-Valerolactone (GVL) is a promising candidate for biomass pretreatment since it is renewable and bio-based. In the present work, the effect of a pretreatment based on GVL on the enzymatic saccharification of white birch was evaluated at a laboratory scale and the importance of the washing procedure for the subsequent saccharification was demonstrated. Both the saccharification yield and the production of cellulosic ethanol were higher using a noncommercial enzyme crude from Talaromyces amestolkiae than with the commercial cocktail Cellic CTec2 from Novozymes. Furthermore, the production of extracellular cellulases by T.amestolkiae has been optimized in 2L bioreactors, with improvements ranging from 40% to 75%. Finally, it was corroborated by isoelectric focus that optimization of cellulase secretion by T.amestolkiae did not affect the pattern production of the mainβ-glucosidases and endoglucanases secreted by this fungus.

Valorization of lignocellulosic biomass: Progress in the production of second-generation bioethanol

Cellulosic ethanol, a biofuel product derived from microbial fermentation of cellulose-rich agricultural waste biomass, holds the potential to significantly contribute to sustainable development, energy security, and environmental friendliness as a green and renewable alternative energy source. Cellulosic ethanol offers remarkable energy benefits and carbon emission reductions. However, the biorefining process of cellulosic ethanol faces challenges and complexities. This article revolves around five aspects: feedstock, composition, pretreatment, cellulase hydrolysis, and ethanol fermentation processes in the biorefining of cellulosic ethanol. It provides an overview of the process flow and characteristics of cellulosic ethanol biorefining, emphasizing its significance. The main technological bottlenecks in cellulosic ethanol production are analyzed. Recent research advancements in cellulosic ethanol biorefining are summarized, followed by an outlook on the research focus and future prospects in the field of cellulosic ethanol.

EVALUATION OF SILAGE OF SORGHUM AND SUDAN GRASS HYBRID PULP FOR BIOENERGY PURPOSES

This research was carried out to determine the potential for using the pulp of 10 different sorghum x sudan grass hybrid varieties (Aneto, Greengo, Jumbo, Master BMR, Nutri Honey, Nutrima, Sugar Graze II, Supergraze 1000, Süper Su 22, Tonka) used to obtain ethanol in Bingöl, Eastern Anatolian ecological conditions as silage. Field trials were carried out in 3 replications in 2020 according to the randomized block design. The plants were harvested during the pulping period of the grains. The stems from which the sap was extracted were silaged and left for fermentation at room temperature for 45 days. Feed quality characteristics and organic acid contents were determined in silage materials. Depending on the varieties; silages have a physical score of 7-19, dry matter rate 37.58-50.94%, pH value 3.36-4.30, ADF rate 39.33-50.76%, NDF rate 53.40-66.86%, ADL rate 10.90-13.96%, CP rate 2.76-4.46%, DDM ratio 49.33-50.26%, DMI ratio 1.80-2.36%, RFV ratio 68.66-107.00, net energy value 1.176-1.337, AA ratio 0.11-0.66%, BA ratio 0.76-1.16%, LA ratio 0.07-1.05% and PA ratio was found to vary between 0.0013-0.0026%. As a result, it was concluded that silages made with pulp in cultivars of sorghum x sudan grass hybrid plant can be used more appropriately in cellulosic ethanol production than forage.

Production of bioethanol from Opuntia ficus-indica using Saccharomyces cerevisiae and Water Kefir following an autohydrolysis pretreatment

This study investigates the conversion of Opuntia ficus-indica waste (pears, peels, and seeds) into bioethanol. The research was done in two steps. The first objective of this work was to evaluate the optimal conditions of autohydrolysis pretreatment (AP) on these lignocellulosic materials. Experimentation involved assessing various temperatures (121 °C, 150 °C, and 180 °C) against a fixed solid-to-liquid ratio of 1:10. The Response Surface Methodology was employed for this optimization process. The highest glucose and saccharose concentrations, 0.251 mol/l and 2.81 mol/l, respectively, were observed at 150 °C over 15 min. The second part of this study focused on fermenting the pretreated biomass to maximize ethanol concentration after autohydrolysis. Both water kefir and Saccharomyces cerevisiae were employed separately and combined to assess their effectiveness in fermenting the pretreated biomass to maximize ethanol concentration. This fermentation was conducted at 30 °C with a solution-yeast ferment ratio of 70:30. The most efficacious fermentation condition was observed with a pretreatment at 121 °C for 15 min using water kefir, which resulted in an ethanol concentration of 16%. This process highlights the potential of cactus pear waste in cellulosic ethanol production, offering a sustainable energy source and a solution for agricultural waste management.

Characterization of sugarcane bagasse solubilization and utilization by thermophilic cellulolytic and saccharolytic bacteria at increasing solid loadings

In Brazil the main feedstock used for ethanol production is sugarcane juice, resulting in large amounts of bagasse. Bagasse has high potential for cellulosic ethanol production, and consolidated bioprocessing (CBP) has potential for lowering costs. However, economic feasibility requires bioprocessing at high solids loadings, entailing engineering and biological challenges. This study aims to document and characterize carbohydrate solubilization and utilization by defined cocultures of Clostridium thermocellum and Thermoanaerobacterium thermosaccharolyticum at increasing loadings of sugarcane bagasse. Results show that fractional carbohydrate solubilization decreases as solids loading increases from 10 g/L to 80 g/L. Cocultures enhance solubilization and carbohydrate utilization compared to monocultures, irrespective of initial solids loading. Rinsing bagasse before fermentation slightly decreases solubilization. Experiments studying inhibitory effects using spent media and dilution of broth show that negative effects are temporary or reversible. These findings highlight the potential of converting sugarcane bagasse via CBP, pointing out performance limitations that must be addressed.

Influence of feedstock selection on cellulosic ethanol production based on densified biomass with calcium hydroxide and regular steam pretreatment

Feedstock selection is a key factor affecting the process of cellulosic ethanol production. Densifying lignocellulosic biomass with chemicals (calcium hydroxide) followed by regular steam autoclave (DLCA(ch)) is an emerging and efficient pretreatment technology. In this study, different crop straws were selected as feedstock in a DLCA(ch) based process for cellulosic ethanol production, and their experimental and economic performances were evaluated. Both single crop straws and mixed crop straws were used and compared as feedstock for DLCA(ch) based biorefinery. Among single crop straws of corn stover, rice straw and wheat straw, wheat straw had the highest ethanol yield and the lowest feedstock cost, with a minimum ethanol selling price of 2.03 $/gal ethanol considering a 10 % internal rate of return and a 2000 tons crop straw per day biorefinery scale. When mixed crop straws were used as the feedstock, both feedstock cost and ethanol production cost were further reduced, and the minimum ethanol selling price was reduced to 2.02 $/gal ethanol. In addition, the use of mixed crop straws has great potential for large scale DLCA(ch) based process for cellulosic ethanol production.

A cellulosomal yeast reaction system of lignin-degrading enzymes for cellulosic ethanol fermentation.

Biofuel production from lignocellulose feedstocks is sustainable and environmentally friendly. However, the lignocellulosic pretreatment could produce fermentation inhibitors causing multiple stresses and low yield. Therefore, the engineering construction of highly resistant microorganisms is greatly significant. In this study, a composite functional chimeric cellulosome equipped with laccase, versatile peroxidase, and lytic polysaccharide monooxygenase was riveted on the surface of Saccharomyces cerevisiae to construct a novel yeast strain YI/LVP for synergistic lignin degradation and cellulosic ethanol production. The assembly of cellulosome was assayed by immunofluorescence microscopy and flow cytometry. During the whole process of fermentation, the maximum ethanol concentration and cellulose conversion of engineering strain YI/LVP reached 8.68g/L and 83.41%, respectively. The results proved the availability of artificial chimeric cellulosome containing lignin-degradation enzymes for cellulosic ethanol production. The purpose of the study was to improve the inhibitor tolerance and fermentation performance of S. cerevisiae through the construction and optimization of a synergistic lignin-degrading enzyme system based on cellulosome.

Engineering Compositionally Uniform Yeast Whole-Cell Biocatalysts with Maximized Surface Enzyme Density for Cellulosic Biofuel Production.

In recent decades, whole-cell biocatalysis has played an increasingly important role in the food, pharmaceutical, and energy sector. One promising application is the use of ethanologenic yeast displaying minicellulosomes on the cell surface to combine cellulose hydrolysis and fermentation into a single step for consolidated bioprocessing. However, cellulosic ethanol production using existing yeast whole-cell biocatalysts (yWCBs) has not reached industrial feasibility due to their inefficient cellulose hydrolysis. As prior studies have demonstrated enzyme density on the yWCB surface to be one of the most important parameters for enhancing cellulose hydrolysis, we sought to maximize this parameter at both the population and single-cell levels in yWCBs displaying tetrafunctional minicellulosomes. At the population level, enzyme density is limited by the presence of a nondisplay population constituting 25-50% of all cells. In this study, we identified the cause to be plasmid loss and successfully eliminated the nondisplay population to generate compositionally uniform yWCBs. At the single-cell level, we demonstrate that enzyme density is limited by molecular crowding, which hinders minicellulosome assembly. By adjusting the integrated gene copy number, we obtained yWCBs of tunable enzyme display levels. This tunability allowed us to avoid the crowding-limited regime and achieve a maximum enzyme density per cell. As a result, the best strain showed a cellulose-to-ethanol yield of 4.92 g/g, corresponding to 96% of the theoretical maximum and near-complete conversion (∼96%) of the starting cellulose (1% PASC). Our holistic engineering strategy that combines a population and single-cell level approach is broadly applicable to enhance the WCB performance in other biocatalytic cascade schemes.

Life cycle assessment and techno-economic analysis of wood-based biorefineries for cellulosic ethanol production

Poplar is widely used in the paper industry and accompanied by abundant branches waste, which is potential feedstock for bioethanol production. Acid-chlorite pretreatment can selectively remove lignin, thereby significantly increasing enzymatic efficiency. Moreover, lignin residues valorization via gasification-syngas fermentation can achieve higher fuel yield. Herein, environmental and economic aspects were conducted to assess technological routes, which guides further process optimization. Life cycle assessment results show that wood-based biorefineries especially coupling scenarios have significant advantages in reducing global warming potential in contrast to fossil-based automotive fuels. Normalization results indicate that acidification potential surpasses other indicators as the primary impact category. In terms of economic feasibility, coupling scenarios present better investment prospects. Bioethanol yield is the most critical factor affecting market competitiveness. Minimum ethanol selling price below ethanol international market price is promising with higher-levels technology. Further work should be focused on technological breakthrough, consumable reduction or replacement.

Study of acid hydrolysis and submerged fermentation parameters in second generation ethanol production using cassava waste

Biofuels are an alternative to reduce the environmental impacts caused by the use of petroleum-based fuels. Second generation ethanol comes from lignocellulosic materials that are little used or discarded in the environment, including agro-industrial waste. These residues are rich in sugars that can be converted by ethanol-producing microorganisms. In this context, the present work uses cassava waste (Manihot esculenta Crantz) obtained during the processing of starch as a raw material for the production of cellulosic ethanol, as well as evaluating different parameters in acid hydrolysis and during submerged fermentation using the strain Saccharomyces cerevisiae ATCC 26602. Experiments of acid hydrolysis of these residues with sulfuric acid in concentrations of 0.5% to 5.0% (v/v) were carried out. The results demonstrated that 2.0% H2SO4 for 10 min. reaction at 121 ºC/1.1 atm in an autoclave reached the highest release of reducing sugars present in the residue (131.09 g.L-1). The ideal conditions for alcoholic fermentation using yeast were pH 6.5, temperature of 35 ºC, without rotation and initial reducing sugar concentration of 50 g.L-1, resulting in 21.23 g.L-1 of ethanol with productivity of 1.86 g.L-¹.h-¹ and theoretical yield of 96.5% after 12 hours of fermentation. The results indicated that the cassava waste serve as a potential substrate for the production of second generation ethanol. Thus, this study provides data on the most suitable conditions for the use of these industrial wastes aiming at the generation of a renewable fuel, an increasingly attractive feature for the world, where the economic and environmental concern is growing.

Characteristics and evolution of bioethanol from Manila Tamarind (Pithecellobium dulce) leaf through fermentation

<p>This study's two primary goals are to synthesize bioethanol from biowaste and examine its thermal characteristics. Bioethanol must first undergo a comprehensive assessment of its thermal characteristics in order to be approved for usage in spark-ignition engines. A multi-step procedure comprising extraction, pretreatment, enzymatic hydrolysis, and fermentation was used to manufacture the bioethanol. The raw material was put through a preliminary screening using thermogravimetric analysis to find the mass loss rate as a function of temperature before to starting this process. Remarkably, the Manila tamarind leaves exhibited the highest mass loss, up to 34%. Enzymatic cellulose conversion to fermentable sugars was a critical step in the production of cellulosic ethanol. Following hydrolysis, Saccharomyces cerevisiae was employed in the fermentation process, leading to the bioethanol synthesis phase. The Manila Tamarind leaves yielded the most, around 29% by weight, which is amazing. In order to assess the thermal properties of the extracted ethanol, a variety of parameters were carefully examined, including the viscosity, density, cetane number, and calorific value. In each of these areas, mixtures of tire oil, gasoline, and bioethanol consistently outperformed the others. Furthermore, Fourier Transform Infrared Spectrometry spectra were utilized to validate the concentrations of potential groups, including alcohol, aromatic, alkyne, amide, and carbonyl groups. Clear objectives guide the fermentation process, aiming for consistent quality, safety, and functionality in the end product.</p>

Transcriptomic analysis of ncRNAs and mRNAs interactions during drought stress in switchgrass

Switchgrass (Panicum virgatum L.) plays a pivotal role as a bioenergy feedstock in the production of cellulosic ethanol and contributes significantly to enhancing ecological grasslands and soil quality. The utilization of non-coding RNAs (ncRNAs) has gained momentum in deciphering the intricate genetic responses to abiotic stress in various plant species. Nevertheless, the current research landscape lacks a comprehensive exploration of the responses of diverse ncRNAs, including long non-coding RNAs (lncRNAs), circular RNAs (circRNAs), and microRNAs (miRNAs), to drought stress in switchgrass. In this study, we employed whole transcriptome sequencing to comprehensively characterize the expression profiles of both mRNA and ncRNAs during episodes of drought stress in switchgrass. Our analysis identified a total of 12,511 mRNAs, 59 miRNAs, 38 circRNAs, and 368 lncRNAs that exhibited significant differential expression between normal and drought-treated switchgrass leaves. Notably, the majority of up-regulated mRNAs displayed pronounced enrichment within the starch and sucrose metabolism pathway, as validated through KEGG analysis. Co-expression analysis illuminated that differentially expressed (DE) lncRNAs conceivably regulated 1308 protein-coding genes in trans and 7110 protein-coding genes in cis. Furthermore, both cis- and trans-target mRNAs of DE lncRNAs exhibited enrichment in four common KEGG pathways. The intricate interplay between lncRNAs and circRNAs with miRNAs via miRNA response elements was explored within the competitive endogenous RNA (ceRNA) network framework. As a result, we constructed elaborate regulatory networks, including lncRNA-novel_miRNA480-mRNA, lncRNA-novel_miRNA304-mRNA, lncRNA/circRNA-novel_miRNA122-PvSS4, and lncRNA/circRNA-novel_miRNA14-PvSS4, and subsequently validated the functionality of the target gene, starch synthase 4 (PvSS4). Furthermore, through the overexpression of PvSS4, we ascertained its capacity to enhance drought tolerance in yeast. However, it is noteworthy that PvSS4 did not exhibit any discernible impact under salt stress conditions. These findings, as presented herein, not only contribute substantively to our understanding of ceRNA networks but also offer a basis for further investigations into their potential functions in response to drought stress in switchgrass.

Application of Aromatic Ring Quaternary Ammonium and Phosphonium Salts–Carboxylic Acids-Based Deep Eutectic Solvent for Enhanced Sugarcane Bagasse Pretreatment, Enzymatic Hydrolysis, and Cellulosic Ethanol Production

Deep eutectic solvents (DESs) with a hydrophobic aromatic ring structure offer a promising pretreatment method for the selective delignification of lignocellulosic biomass, thereby enhancing enzymatic hydrolysis. Further investigation is needed to determine whether the increased presence of aromatic rings in hydrogen bond receptors leads to a more pronounced enhancement of lignin removal. In this study, six DES systems were prepared using lactic acid (LA)/acetic acid (AA)/levulinic acid (LEA) as hydrogen bond donors (HBD), along with two independent hydrogen bond acceptors (HBA) (benzyl triethyl ammonium chloride (TEBAC)/benzyl triphenyl phosphonium chloride (BPP)) to evaluate their ability to break down sugarcane bagasse (SCB). The pretreatment of the SCB (raw material) was carried out with the above DESs at 120 °C for 90 min with a solid–liquid ratio of 1:15. The results indicated that an increase in the number of aromatic rings may result in steric hindrance during DES pretreatment, potentially diminishing the efficacy of delignification. Notably, the use of the TEBAC:LA-based DES under mild operating conditions proved highly efficient in lignin removal, achieving 85.33 ± 0.52% for lignin removal and 98.67 ± 2.84% for cellulose recovery, respectively. The maximum digestibilities of glucan (56.85 ± 0.73%) and xylan (66.41 ± 3.06%) were attained after TEBAC:LA pretreatment. Furthermore, the maximum ethanol concentration and productivity attained from TEBAC:LA-based DES-pretreated SCB were 24.50 g/L and 0.68 g/(L·h), respectively. Finally, the comprehensive structural analyses of SCB, employing X-rays, FT-IR, and SEM techniques, provided valuable insights into the deconstruction process facilitated by different combinations of HBDs and HBAs within the DES pretreatment.

High titer (>100 g/L) ethanol production from pretreated corn stover hydrolysate by modified yeast strains

Developing a reliable lignocellulose pretreatment method to extract mixed sugars and engineering efficient strains capable of utilizing xylose are crucial for advancing cellulosic ethanol production. In this study, chemical and characterization analyses revealed that alkali cooking can significantly remove lignin from lignocellulose crops. The highest amount of mixed sugar was obtained from corn stover hydrolysates with a 15 % solid loading. Our genetically engineered yeast strain ΔsnR4, derived from a well-staged WXY70, demonstrated excellent performance in low 10 % solids loading corn stover hydrolysate, producing a high ethanol yield of 0.485 g/g total sugars. When a combined NaOH-ball milling pretreatment strategy was applied at high solids loading, ΔsnR4 exhibited the maximum ethanol titer of 110.9 g/L within 36 h, achieving an ethanol yield of 92.9 % theoretical maximum. Therefore, ΔsnR4 is highly compatible with high solid loading NaOH-ball milling pretreatment, making it a potential candidate for industrial cellulosic ethanol.

Cellulosic ethanol production: Assessment of the impacts of learning and plant capacity

Cellulosic ethanol has great potential to displace fossil fuels and reduce greenhouse gas emissions, but supply is low due to high cost. Learning-by-doing is a strategy to reduce cost with increases in capacity. This study attempts to understand how learning-by-doing affects the growth of cellulosic ethanol in synergy with plant capacity. We modeled a three-phase learning effect using a hybrid general equilibrium model to project the U.S cellulosic ethanol production. The reference case projects an annual production of 5.49 million gallons (MG) from 2030 to 2040. With strong policy support of a high mandate, the U.S. is projected to produce 171 MG of cellulosic ethanol in 2040. However, the learning-by-doing effect cannot be initiated because only 4 large-scale centralized plants will be built. When plant size reduces, learning-by-doing is enabled, and 2040 cellulosic ethanol production can reach 3.27 billion gallons. Similar result is predicted for the cases representing an actual biorefinery. Fast and widespread learning is estimated to greatly increase production when plants have lower capital cost. The findings show that policy incentives are critical to technology development when cost is a significant barrier. Additionally, building small-size plants is a strategy that will enable and facilitate the learning-by-doing effect.

The impact of lignocellulosic ethanol production on the environment from a life cycle perspective

In the production process of cellulosic ethanol, all sections are closely related. It is difficult to study its producing law and key factors that affect the process only by experimental means. Computer simulation has a powerful data mining ability, which can analyze, design, and evaluate the production process of cellulosic ethanol. Specifically, it can use data calculated by Aspen Plus before, and analyze the impact of cellulose ethanol production on the environment by life cycle assessment (LCA). Results show that the total environmental impact value of the whole process is 0.20∼0.37 person·a/t. And in the whole process, the growth stage and the production stage have the biggest impact.

Changes in various lignocellulose biomasses structure after microwave-assisted hydrotropic pretreatment

Effective pretreatment of lignocellulosic biomass combined with delignification is a condition for effective enzymatic hydrolysis of cellulose, which is of key significance for the efficiency of the process of production of cellulosic ethanol. Becoming aware of the full range of changes in the structure of lignocellulose as a result of biomass pretreatment is important in view of the necessity to optimise the process of technological production of bioethanol. Our research was aimed at a comprehensive analysis of changes in structure of pine chip (softwood), beech chip (hardwood) and wheat straw (non-wood) biomasses resulting from a newly developed pretreatment method combining the use of hydrotrope in the form of sodium cumene sulfonate and microwave radiation. The performed analyses of the biomass composition indicate a high effectiveness of the proposed pretreatment method in the area of biomass delignification and hemicellulose removal. An effective removal of amorphous substances, i.e. lignin and hemicellulose, led to an increase in the crystallinity of pine chip biomass to 55%, and beech chips and wheat straw biomasses to 61%.