Lignocellulosic Bioethanol Production Research Articles

Current progress on lignocellulosic bioethanol including a technological and economical perspective

AbstractGrowing interest in lignocellulosic bioethanol stems from the European Union's renewable energy directive, targeting a global bioethanol output of 130 billion L to achieve a minimum threshold of 42.5%. Despite industrialization challenges, recent advancements, especially in crucial stages like pretreatment, hydrolysis, and fermentation, are transforming the landscape. This review delves into the progress of bioethanol production, addressing technological, environmental, and economic hurdles. Innovations such as deep eutectic solvent pretreatment and mechanocatalysis, offering advantages like 30% and 100% solid loading, respectively, surpass traditional techniques and enzymatic hydrolysis in yielding better results. Improved pretreatment methods, enzyme exploration, saccharification techniques, genetic engineering, and integrated biorefineries contribute to overall economic viability. Ongoing research involves techno-economic analysis for cost-effective strategies, aiming to enhance the competitiveness of lignocellulosic bioethanol production. Scrutinizing the feasibility of these innovative approaches not only highlights their potential to overcome existing shortcomings but also envisions a more attractive future for industrial bioethanol production. Embracing these advancements could pave the way for a vibrant and sustainable bioethanol industry.

Techno-enviro-economic analysis of second-generation bioethanol at plant-scale by different pre-treatments of biomass from palm oil waste

This study evaluates three pathways: supercritical water treatment (SCW), CO2-water (scCO2-H2O), and catalytic hydrothermal hydrolysis (CHH) to produce second-generation bioethanol using palm tree fronds (PTF) as feedstock. The analysis focuses on technical performances using process simulation with Aspen Plus™, environmental evaluation by applying a Life Cycle Assessment (LCA) framework and an economic analysis using the levelized cost. The results of the technical performances reveal CHH as the best pathway for bioethanol production, with the highest ethanol yield at 273 L/tonne dry PTF and a substantial NER of 2.2, outperforming SCW and scCO2-H2O. From an environmental evaluation, CHH reveals lower carbon footprints of 61 g CO2 per MJ. Economically, CHH is competitive, with an ethanol production cost of1.11 $/L, comparable to the enzymatic hydrolysis process. These findings provide the technical, environmental, and economic aspects of a promising route for lignocellulosic bioethanol production while suggesting pathways for further optimisation for future research.

Şanlıurfa Ekolojik Koşullarında Farklı Tatlı Sorgum (Sorghum bicolor var. saccharatum (L.) Mohlenbr.) Genotiplerinin Lignoselülozik Biyoetanol Verimleri

The use of products with high biomass yield per unit area as a renewable energy source, which are not consumed primarily for food purposes or can be used for multi-purposes, is of great importance in terms of food safety. For this purpose, sweet sorghum plant, which can grow in salty-alkaline soils, tolerant to drought and short-term floods, and has wide adaptability, has gained importance as a lignocellulosic bioethanol raw material in recent years. The aim of this study is to theoretically determine the lignocellulosic bioethanol yield of 21 different sweet sorghum (Sorghum bicolor var. saccharatum (L.) Mohlenbr.) genotypes grown under second crop conditions in the ecology of Şanlıurfa province, obtained from domestic and foreign sources. In the study, sweet sorghum pulp from plant sap was used as lignocellulosic bioethanol raw material. The cellulose and hemicellulose contents of the pulp were determined and the amount of lignocellulosic bioethanol that could be obtained per decare was calculated by using the bioethanol conversion rates. According to the data obtained, the highest yield was obtained from UNL-Hybrid-3 (805.5 L/da), Theis (766.5 L/da), Smith (698.1 L/da) genotypes. In order to solve problems such as increasing the octane of gasoline, reducing greenhouse gas emissions and gasoline imports, it is recommended that these three genotypes be cultivated and disseminated for lignocellulosic bioethanol production in Şanlıurfa ecological conditions.

Novel cyclic shifting of temperature strategy for simultaneous saccharification and fermentation for lignocellulosic bioethanol production

In the current study, a novel strategy using cyclic shifting of temperature was developed for simultaneous saccharification and fermentation (SSF) for bioethanol production from rice straw. The in-situ cellulase production, saccharification and fermentation was carried out using P. janthinellum and S. cerevisiae. Bioethanol titer of 14.98 g/l was obtained using base followed by acid pretreated rice straw by employing the cyclic shifting of temperature strategy “30 °C for 2 h to 40 °C for 2 h”. The holding time was further tuned to increase the productivity and the tuned condition 30 °C(1.7 h) – 40 °C(2 h) improved the bioethanol titer to 15.9 g/l. Using this strategy, resulted 5.1-fold and 2.8-fold increment of bioethanol production compared to known approaches, SSF at mutual optimum temperature and prolong prehydrolysis followed by fermentation respectively. The application of cyclic shifting of temperature strategy can unleash a great potential in enhancing the yield and efficiency for a sustainable lignocellulosic bioethanol production.

Performance and robustness analysis reveals phenotypic trade-offs in yeast.

To design strains that can function efficiently in complex industrial settings, it is crucial to consider their robustness, that is, the stability of their performance when faced with perturbations. In the present study, we cultivated 24 Saccharomyces cerevisiae strains under conditions that simulated perturbations encountered during lignocellulosic bioethanol production, and assessed the performance and robustness of multiple phenotypes simultaneously. The observed negative correlations confirmed a trade-off between performance and robustness of ethanol yield, biomass yield, and cell dry weight. Conversely, the specific growth rate performance positively correlated with the robustness, presumably because of evolutionary selection for robust, fast-growing cells. The Ethanol Red strain exhibited both high performance and robustness, making it a good candidate for bioproduction in the tested perturbation space. Our results experimentally map the robustness-performance trade-offs, previously demonstrated mainly by single-phenotype and computational studies.

Life cycle environmental benefits of recycling waste liquor and chemicals in production of lignocellulosic bioethanol

In recent decades, numerous bioconversion processes and techniques have been developed to utilize lignocellulosic biomass as feedstock in the production of bio-based fuels and materials. However, waste treatment, an important sub-system, is seldom considered in the life cycle assessment of lignocellulose derived products. This study comprehensively investigated the environmental impacts of bioethanol and electricity cogeneration from sugarcane bagasse, with a focus on recycling techniques adopted in waste treatment. A life cycle assessment indicated that high recycle rate of black liquor, acid and waste washing water can substantially reduce the consumption of fresh water, related chemicals and energy by 70–80%. Environmental impacts relating to global warming, acidification potential and primary energy demand can be decreased by 5–10 times or even entirely eliminated. These study outcomes demonstrate significant environmental benefits of integrating waste recycling techniques into lignocellulose biorefinery process, providing a solid foundation for future industrial development.

Improvement of Large-Scale Production of Lignocellulosic Bioethanol through Synthetic Biology Approaches: A Comprehensive Review

This review article explores the use of synthetic biology approaches like gene editing and metabolic engineering to create genetically-modified organisms that can produce commercially-valuable products on large-scale, clean environmental pollutants, and serve as efficient biosensors. The Biobrick system includes a registry of available genetic information and a set of standard protocols for assembling and testing them, which finds various significant uses in synthetic biology, such as in the production of "Fourth generation” biofuel. Genetic engineering techniques may be applied to manipulate crops or microbes for more efficient biofuel production. Such synthetic biology techniques also include modification of lignin structure and content, use of bacterial enzymes abundantly expressed in plants, and manipulation of hemicellulose biosynthesis. However, challenges such as high temperature, efficient conversion of all substrate molecules for maximum product yield, efficient pentose fermentation, and tolerance to acetic acid and bioethanol concentration can all impact the yield of the product. This article explores innovative genetic modification approaches designed to decrease lignin levels and enhance crop digestibility in plants, with a specific focus on the model plant Arabidopsis thaliana and herbaceous plants such as Alfalfa. The aim is to manipulate the Monolignol pathway and modify the CSE gene, known for its ability to decrease lignin content. These modifications have been shown to significantly improve saccharification efficiency and increase the yield of lignocellulosic bioethanol. Besides, the role of xylulose-5-phosphate as an intermediate in the non-oxidative pentose phosphate pathway and the oxidative pentose phosphate pathway, and the use of heterologous xylose transporters and modified sugar transporters as potential solutions to improve the biofuel production efficiency is also discussed in this review article.

A critical review of machine learning for lignocellulosic ethanol production via fermentation route

In this work, machine learning (ML) applications in lignocellulosic bioethanol production were reviewed. First, the pretreatment-hydrolysis-fermentation route, the most commonly studied alternative, was summarized. Next, a bibliometric analysis was performed to identify the current trends in the field; it was found that ML applications in the field are not only increasing but also expanding their relative share in publications, with bioethanol seeming to be the most frequently researched topic while biochar and biogas are also receiving increased attention in recent years. Then, the implementation of ML for lignocellulosic bioethanol production via this route was reviewed in depth. It was observed that artificial neural network (ANN) is the most commonly used algorithm (appeared in almost 90% of articles), followed by response surface methodology (RSM) (in about 25% of articles) and random forest (RF) (in about 10% of articles). Bioethanol concentration is the most common output variable in the fermentation step, while fermentable sugar and glucose concentration are studied most in hydrolysis. The datasets are usually small, while the fitnesses of the models (R2) are usually high in the papers reviewed. Finally, a perspective for future studies, mostly considering improving data availability, was provided.

Co-production of ethanol and polyhydroxybutyrate from lignocellulosic biomass using an engineered Saccharomyces cerevisiae

The development of glucose/xylose co-fermenting Saccharomyces cerevisiae has improved bioethanol yield from lignocellulosic biomass, the most abundant and sustainable resource for net-zero production of fuels and chemicals. The co-production of value-added chemicals would further improve the economic feasibility of lignocellulosic bioethanol production. Here, we developed a glucose/xylose co-fermenting S. cerevisiae strain capable of co-producing polyhydroxybutyrate, a prominent biodegradable polymer, as an intracellularly accumulated co-product by introducing a polycistronic polyhydroxybutyrate biosynthetic pathway. The engineered strain accumulated polyhydroxybutyrate with a content of 64 mg/g DCW while maintaining extracellular production of ethanol with a high yield (0.43 g ethanol/g sugar). The co-production of ethanol and polyhydroxybutyrate was then evaluated using various types of biomass, including sugarcane bagasse, silver grass, and even cardboard boxes. This study demonstrates the feasibility of co-production of bioethanol and value-added chemicals to maximize the values derivable from lignocellulosic biomass.

Effect of Laccase Detoxification on Bioethanol Production from Liquid Fraction of Steam-Pretreated Olive Tree Pruning

During lignocellulosic bioethanol production, the whole slurry obtained by steam explosion is filtered, generating a water-insoluble fraction rich in cellulose which is used for saccharification and ethanol fermentation, as well as a liquid fraction containing solubilised glucose and xylose but also some inhibitory by-products (furan derivatives, weak acids and phenols), which limits its use for this purpose. Since utilization of this liquid fraction to ethanol is essential for an economically feasible cellulosic ethanol process, this work studied a laccase from Myceliophthora thermophila to detoxify the liquid fraction obtained from steam-pretreated olive tree pruning (OTP) and to overcome the effects of these inhibitors. Then, the fermentation of laccase-treated liquid fraction was evaluated on ethanol production by different Saccharomyces cerevisiae strains, including the Ethanol Red, with the capacity to ferment glucose but not xylose, and the xylose-fermenting recombinant strain F12. Laccase treatment reduced total phenols content by 87% from OTP liquid fraction, not affecting furan derivatives and weak acids concentration. Consequently, the fermentative behavior of both Ethanol Red and F12 strains was improved, and ethanol production and yields were increased. Moreover, F12 strain was capable of utilizing some xylose, which increased ethanol production (10.1 g/L) compared to Ethanol Red strain (8.6 g/L).

Study of washing method of oil palm empty fruit bunch-treated with alkali explosion in lignocellulosic bioethanol production

The conventional process of lignocellulosic bioethanol production includes four steps: pretreatment, hydrolysis, fermentation, and purification. Pretreatment is the crucial way to enhance cellulose purity and remove lignin as well as other impurities compounds in biomass. Alkaline explosion is one of the most used pretreatment methods but pulp-produced is still in base condition. This pulp is not suitable as a substrate for enzymatic hydrolysis that needed a pH of 4.5-5.5. Therefore, pulp washing is required for separating pulp with black liquor and obtaining neutral pH pulp before going to hydrolysis. This study explored the washing method of oil palm empty fruit bunch (EFB)-treated with alkaline explosion in bioethanol production. The washing method using freshwater, acid solution, and a combination of freshwater and acid solution was investigated. As a result, the washing used freshwater needed the highest of water and wash cycles. The washing method using acid solution could obtain neutral pulp in two cycles wash step or more, thus, requiring higher acid consumption. The washing with combination method provided neutral pulp with fewer wash cycles, water, and acid. This method also provided the highest glucose and ethanol concentration. Therefore, the combination washing method is considered more efficient, economical, and environmentally for EFB pulp washing in bioethanol production.

Integrated Method of Pretreatment, Saccharification and Electro-Fermentation of Saccharum Spontaneum to Produce Bioethanol Using Microbial Consortium

Production of lignocellulosic ethanol using microbial consortium can be an economical strategy. Consortium of ligninolytic and cellulolytic microbe(s) that can depolymerize lignin and hydrolyze cellulose along with fermenting yeast (CDBT-2) can replace the separate techniques used for biomass pretreatment and hydrolysis prior to fermentation. So, the aim of this study was to screen fungi capable of producing laccase (lignin depolymerizing enzyme) and cellulase (converts cellulose to fermentable sugars), and use the strains along with Saccharomyces cerevisiae (CDBT-2) to develop an integrated method of simultaneous pretreatment saccharification and electro-fermentation of Saccharum spontaneum powder to produce ethanol. 18-s rRNA sequencing and BLAST analysis of screened isolates confirmed, cellulolytic isolate (F1) resembled as Aspergillus niger and, ligninocellulolytic isolate (F2) resembled to Ganoderma sessile. These fungi were used along with CDBT2 for ethanol production. Different consortiums sets of these isolates were tested for fermentation efficiency. All the analysis were done using crude untreated as well as hot water pretreated S. spontaneum powder to compare the ethanol production efficiency. In controlled environment of 28oC, shaking at 80 rpm and 5.0 pH, culture with consortium of G. sessile and CDBT2 produced higher ethanol with yield 57 mg/g biomass powder followed by consortium of G. sessile, A. niger and CDBT2 with yield of 44±0.18 mg/g biomass. Monoculture of G. sessile produced higher amount of phenol ie 292.48±17.74 µg/mL. Similarly, G. sessile, A. niger and CDBT2 consortium produced higher furfural of 58.48±3.86 µg/mL. However, the amount of phenol and furfural generated were very low in both cases. The ethanol yield was higher in crude biomass compared to hot water pretreated S. spontaneum powder. In addition, electro-fermentation of crude S. spontaneum enhanced ethanol production by 12% with G. sessile and CDBT2 consortium while 18% with the set of G. sessile, A. niger and CDBT2 consortium. This monophasic method of integrated pretreatment saccharification and electro-fermentation can be an alternative approach for lignocellulosic bioethanol production as it does not require separate pretreatment and fermentation approach.Keywords: Bioethanol, Saccharum spontaneum, microbial consortium, simultaneous saccharification and fermentation, and electro-fermentation Figure 1

Effects of harvest time on bioethanol production and bagasse characteristics for combustion and forage in sweet sorghum

Determining the optimal harvest time is very crucial to achieve sustainable production of first-generation bioethanol as well as efficient use of its bagasse for combustion or feed in sweet sorghum. The aim of the current research was to understand the effects of different harvest times, which comprised flowering (FLW), soft dough (SD), hard dough (HD), and physiological maturity (PM) on the bioethanol production and the combustion and forage characteristics of its bagasse in sweet sorghum. In one study, averaged over a period of 2 years, the mean theoretical total ethanol yield (TEY) was between 4802 and 5316 L ha–1. The PM harvest produced the highest TEY, which was followed by the HD and SD harvests, with sligh differences. These results suggested that sweet sorghum should be harvested at any phenological stage after FLW to achieve sustainable total (juice + lignocellulosic) bioethanol production in a semi-arid Mediterranean environment. Averaged over a period of 2 years, the mean dry bagasse yield (DBY) achieved from the harvest times herein ranged between 12.0 and 14.1 t ha–1. As with TEY, the PM harvest exhibited the highest DBY and desirable bagasse combustion caharacteristics due to exhibiting the lowest moisture and ash contents, which indicated that it seemed be the most suitable harvest time if obtaining solid biofuel production from the bagasse is the main target after the production of juice ethanol. On the other hand, the HD harvest provided the highest bagasse forage quality among the harvest times due to exhibiting the significantly lowest neutral detergent fiber and acid detergent fiber contents, but the significantly highest relative feed value. These results suggested that the HD harvest time was the most suitable for sweet sorghum cultivation when producing juice ethanol that is complemented by the production of forage utilizing the bagasse.

Quantification of Microbial Robustness in Yeast.

Stable cell performance in a fluctuating environment is essential for sustainable bioproduction and synthetic cell functionality; however, microbial robustness is rarely quantified. Here, we describe a high-throughput strategy for quantifying robustness of multiple cellular functions and strains in a perturbation space. We evaluated quantification theory on experimental data and concluded that the mean-normalized Fano factor allowed accurate, reliable, and standardized quantification. Our methodology applied to perturbations related to lignocellulosic bioethanol production showed that the industrial bioethanol producing strain Saccharomyces cerevisiae Ethanol Red exhibited both higher and more robust growth rates than the laboratory strain CEN.PK and industrial strain PE-2, while a more robust product yield traded off for lower mean levels. The methodology validated that robustness is function-specific and characterized by positive and negative function-specific trade-offs. Systematic quantification of robustness to end-use perturbations will be important to analyze and construct robust strains with more predictable functions.

Partial energy integration between biofuels production processes: Effect on costs, CO2 emissions and process safety

Energy integration is a tool which allows reducing the heating and cooling requirements for production processes. This is particularly important in the processes for production of biofuels, since such processes are expected to have low environmental impact, which can be achieved by reducing the need for steam and cooling water. It is common to perform energy integration by making use of all the available streams. This approach may allow reducing as much as possible utilities’ requirements, but other indicators may be affected, such as capital costs, since the number of required equipment is increased. Thus, in this work the effect of performing partial integration is assessed, i.e., selecting only a few streams to perform the energy integration. The effect of increasing the number of integrated streams is assessed in terms of sustainability indicators based on the green chemistry principles. The studied indicators are utilities’ requirements, total annual cost, environmental impact (assessed through CO2 emissions) and safety (assessed through the HPSI index). The study is applied to the energy integration of a supercritical biodiesel production process and a lignocellulosic bioethanol production process.

Bioethanol from Napier grass employing different fermentation strategies to evaluate a suitable operation for batch bioethanol production

• Variations in substrate and enzyme additions in SSF of Napier grass were compared. • SHF and SSF resulted in 30.6 g/L and 28.5 g/L ethanol from 15% Napier grass. • Alleviate mixing problem by portioning addition did not improve ethanol production. • Pre-incubating the grass before SSF negatively impacts ethanol production. • Extra loading of substrate during SSF resulted in higher ethanol production. Napier grass has been used as animal feed especially in countries with tropical climates. As interest in alternative feedstocks for fuel bioethanol increases, Napier grass has been considered as a potential feedstock for lignocellulosic bioethanol production. Two main fermentation strategies, including separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF), were investigated with various substrate and enzyme feeding patterns in SSF. Total substrate loading of 15% and enzyme dosage of 40 FPU/g substrate were controlled to compare the performance of each fermentation variation. SHF and SSF resulted in similar ethanol production of 30.6 ± 0.4 g/L and 28.5 ± 2.3 g/L. Preincubation of grass and enzyme resulted in significant lower ethanol at 20.3 ± 1.6 g/L. Portioning the addition of grass and enzyme did not change the ethanol production significantly. Increasing the grass loading to 30% via portioning approach resulted in an increase in ethanol production in batch fermentation, while the addition of extra enzyme did not help with increasing ethanol production.

Selection of Superior Yeast Strains for the Fermentation of Lignocellulosic Steam-Exploded Residues.

The production of lignocellulosic ethanol calls for a robust fermentative yeast able to tolerate a wide range of toxic molecules that occur in the pre-treated lignocellulose. The concentration of inhibitors varies according to the composition of the lignocellulosic material and the harshness of the pre-treatment used. It follows that the versatility of the yeast should be considered when selecting a robust strain. This work aimed at the validation of seven natural Saccharomyces cerevisiae strains, previously selected for their industrial fitness, for their application in the production of lignocellulosic bioethanol. Their inhibitor resistance and fermentative performances were compared to those of the benchmark industrial yeast S. cerevisiae Ethanol Red, currently utilized in the second-generation ethanol plants. The yeast strains were characterized for their tolerance using a synthetic inhibitor mixture formulated with increasing concentrations of weak acids and furans, as well as steam-exploded lignocellulosic pre-hydrolysates, generally containing the same inhibitors. The eight non-diluted liquors have been adopted to assess yeast ability to withstand bioethanol industrial conditions. The most tolerant S. cerevisiae Fm17 strain, together with the reference Ethanol Red, was evaluated for fermentative performances in two pre-hydrolysates obtained from cardoon and common reed, chosen for their large inhibitor concentrations. S. cerevisiae Fm17 outperformed the industrial strain Ethanol Red, producing up to 18 and 39 g/L ethanol from cardoon and common reed, respectively, with ethanol yields always higher than those of the benchmark strain. This natural strain exhibits great potential to be used as superior yeast in the lignocellulosic ethanol plants.

Growth of thermotolerant Pichia kudriavzevii UniMAP 3-1 strain for ethanol production using xylose and glucose at different fermentation temperatures

The applications of thermotolerant microorganisms in the production of lignocellulosic bioethanol is the key factor for successful simultaneous saccharification and fermentation process. Thus, this study aimed to isolate a thermotolerant yeast strain that was able to convert both glucose and xylose into ethanol. An analysis based on D1/D2 region of the large-subunit ribosomal DNA identified the isolated strain namely as Pichia kudriavzevii UniMAP 3-1. The growth of this newly isolated yeast was tested with fermentation temperature at 30°C and 40°C on xylose and glucose. P. kudriavzevii UniMAP 3-1 was able to ferment xylose to ethanol at both 30°C and 40°C with a yield of 0.013 g/g and 0.019 g/g with concomitant xylitol yield of 0.24 g/g and 0.25 g/g, respectively. Fermentation of glucose to ethanol was also tested at 30°C and 40°C and the yields were 0.42 g/g and 0.41 g/g, respectively. The potential of this thermotolerant yeast to be used in high-temperature fermentation in both glucose and xylose are proven in this study.

Optimal fed-batch bioreactor operating strategies for the microbial production of lignocellulosic bioethanol and exploration of their economic implications: A step forward towards sustainability and commercialization

The enduring adoption of greener technologies from the biofuel manufacturing industries requires the balanced integration of multiple inter-disciplinary concerns (i.e., engineering, business, sustainability, societal) to make production processes more attractive to the industrial sector. From an engineering perspective, the commercial establishment of lignocellulosic bioethanol plants is still hindered by increased production cost. High product concentrations with improved yield and productivity at the fermentation stage seem to be inviolable condition to straightly compete with fossil-based and first-generation bioethanol facilities. This study proposes a model-based multi-objective dynamic optimization approach to systematically derive optimal operating policies for the fermentation bioreactor as a step forward towards enhancing the economic performance of lignocellulosic bioethanol plants. The economic benefit of the derived optimal bioreactor strategies is evaluated via a scalable economic index, linking for the first time the performance of the fermentation bioreactor to the economics of the entire production process. It is demonstrated that high values in productivity and substantial compromises in yield and final bioethanol concentration are conditions which favor economic feasibility with the achieved fermentation performance (3.32 g L−1 h−1, 0.399 g g−1, 106.0 g L−1) being comparable to first-generation bioethanol production. Finally, a global system analysis framework is employed to investigate the robustness of the optimal value of the economic index to the variability of key model parameters, illustrating the impact of model uncertainty on the process economic performance.

Variations in structure and saccharification efficiency of biomass of different sorghum varieties subjected to aqueous ammonia and glycerol pretreatments

Sorghum biomass is a potential feedstock for lignocellulosic bioethanol production. The selection of suitable sorghum variety is essential to obtain high ethanol yield. In this paper we screened sorghum varieties belonging to sweet sorghum, post rainy sorghum, and hybrid sorghum. These varieties were screened based on their agronomic traits, amenability to pretreatment methods, and enzymatic digestibility. The sorghum biomass was pretreated using glycerol (60 %) at 190 ̊C for 60 min and aqueous ammonia (15 %) at 120 ̊C for 60 min. The digestibility of the pretreated biomass was determined using commercial cellulase (Cellic CTec2) at 10U/g loading, and the structural changes in the pretreated biomass were analyzed by spectroscopy and scanning electron microscopy. Sweet sorghum varieties showed significant variations in phenotypic traits such as fresh stalk yield, dry fodder yield, and juice yield. The cellulose digestibility among the sorghum varieties after the pretreatment also differed significantly. The cellulose digestibility levels of glycerol range from 64 % to 89 % and ammonia pretreated sorghum from 63 % to 81 %. The total sugar yields varied from 227 mg/g to 356 mg/g and 209 mg/g to 313 mg/g for sorghum pretreated with ammonia and glycerol, respectively. Although the delignification of sorghum varieties was higher (31%–65%) after ammonia pretreatment than glycerol pretreatment, the cellulose digestibility was higher for the glycerol pretreated biomass. These results indicated that effect of delignification on cellulose digestibility is trivial. This study explores factors affecting pretreatment and cellulose digestibility of sorghum varieties for maximum sugar yield in the cellulosic ethanol process.