Hydrolysis Of Biomass Research Articles

Characterization of a novel thermostable α-l-arabinofuranosidase for improved synergistic effect with xylanase on lignocellulosic biomass hydrolysis without prior pretreatment

Utilizing thermostable enzymes in biomass conversion processes presents a promising approach to bypass pretreatment, garnering significant attention from the biorefinery industry. A novel discovered α-l-arabinofuranosidase, Abf4980, exhibits exceptional thermostability by maintaining full activity after 24 h of incubation at 70 °C. It effectively acts on polyarabinosides, cleaving α-1,2- and α-1,3-linked arabinofuranose side chains from water-soluble wheat arabinoxylan while releasing xylose. When synergistically combined with the thermostable bifunctional xylanase/β-glucanase CbXyn10C from Caldicellulosiruptor bescii at an enzyme-activity ratio of 6:1, Abf4980 achieves the highest degradation efficiency for wheat arabinoxylan. Furthermore, Abf4980 and CbXyn10C demonstrated remarkable efficacy in hydrolyzing unmodified wheat bran and corn cob to generate arabinose and xylooligosaccharides. This discovery holds promising opportunities for improving the efficiency of lignocellulosic biomass conversion into fermentable sugars.

Efficient utilization of monosaccharides from agri-food byproducts supports Chlorella vulgaris biomass production under mixotrophic conditions

Microalgae are promising resources for the sustainable production of biofuels, feed, and high-value chemicals. Several strains can grow heterotrophically or mixotrophically on multiple organic substrates even if the high cost associated to their use can hinder scalability and economical sustainability of the overall process. The use of agri-food waste biomass hydrolysates might make the cultivation procedure more sustainable, while at the same time valorising underutilized by-products. In this study, Chlorella vulgaris biomass production and sugar utilization was investigated during mixotrophic cultivation on hydrolysates of two inexpensive and widely available recalcitrant agri-food waste biomasses: barley straw (BS) and citrus processing waste (CPW). CPW hydrolysate supported enhanced biomass production, compared to BS digestate, likely because of the presence, besides glucose, of significant amounts of galactose, which is rapidly metabolized by the algae. Notably, when pure monosaccharides were provided as sole organic carbon, growth stopped before complete sugar consumption. Arrested growth in presence of pure monosaccharides correlated with a drastic drop in extracellular pH, which appears to depend on both carbon and nitrogen sources. Our results show that mixotrophic cultivation of C. vulgaris on BS or CPW hydrolysates results in more efficient conversion of organic carbon into biomass, compared to growth on pure sugars, indicating that these agri-food by-products can be utilized as valid feedstocks for sustainable algal biomass production.

Renewable Carbohydrates: Advancements in Sustainable Glucose Production and Optimization

This study explores and optimizes glucose production through various biochemical processes and assesses the potential of diverse feedstock sources to meet the growing demand for renewable carbohydrates. It focuses on glucose production's significance in biological systems and industrial applications, analyzing pathways like enzymatic hydrolysis of polysaccharides and acid hydrolysis of biomass. The kinetics of glucose production are examined, encompassing kinetic models for enzymatic hydrolysis, acid hydrolysis, and fermentation processes. Factors influencing reaction kinetics are explored, and experimental techniques for kinetic parameter estimation are discussed. To address sustainability and resource utilization challenges, the study investigates locally sourced materials like agricultural residues, forest biomass, algal biomass, and food waste as renewable feedstock sources. Optimization strategies for glucose production are presented, using statistical design of experiments and response surface methodology. Techno-economic analysis and life cycle assessments provide a holistic evaluation of environmental and economic aspects associated with glucose production processes. The study's comprehensive approach to glucose production, encompassing both technological advancements and sustainability considerations, offers insights into enzymatic, acid hydrolysis, and fermentation processes, as well as comparing diverse feedstock sources. This knowledge can foster further advancements in the field, benefit industries, and encourage policymakers to promote the integration of renewable carbohydrates in the broader bioeconomy. The research contributes to the global shift towards a greener and more sustainable future, where glucose production plays a key role in building a resilient and eco-conscious society.

Exploring the potential of a new thermotolerant xylanase from Rasamsonia composticola (XylRc): production using agro-residues, biochemical studies, and application to sugarcane bagasse saccharification.

Xylanases from thermophilic fungi have a wide range of commercial applications in the bioconversion of lignocellulosic materials and biobleaching in the pulp and paper industry. In this study, an endoxylanase from the thermophilic fungus Rasamsonia composticola (XylRc) was produced using waste wheat bran and pretreated sugarcane bagasse (PSB) in solid-state fermentation. The enzyme was purified, biochemically characterized, and used for the saccharification of sugarcane bagasse. XylRc was purified 30.6-fold with a 22% yield. The analysis using sodium dodecyl sulphate-polyacrylamide gel electrophoresis revealed a molecular weight of 53kDa, with optimal temperature and pH values of 80°C and 5.5, respectively. Thin-layer chromatography suggests that the enzyme is an endoxylanase and belongs to the glycoside hydrolase 10 family. The enzyme was stimulated by the presence of K+, Ca2+, Mg2+, and Co2+ and remained stable in the presence of the surfactant Triton X-100. XylRc was also stimulated by organic solvents butanol (113%), ethanol (175%), isopropanol (176%), and acetone (185%). The Km and Vmax values for oat spelt and birchwood xylan were 6.7 ± 0.7mg/mL, 2.3 ± 0.59mg/mL, 446.7 ± 12.7µmol/min/mg, and 173.7 ± 6.5µmol/min/mg, respectively. XylRc was unaffected by different phenolic compounds: ferulic, tannic, cinnamic, benzoic, and coumaric acids at concentrations of 2.5-10mg/mL. The results of saccharification of PSB showed that supplementation of a commercial enzymatic cocktail (Cellic® CTec2) with XylRc (1:1 w/v) led to an increase in the degree of synergism (DS) in total reducing sugar (1.28) and glucose released (1.05) compared to the control (Cellic® HTec2). In summary, XylRc demonstrated significant potential for applications in lignocellulosic biomass hydrolysis, making it an attractive alternative for producing xylooligosaccharides and xylose, which can serve as precursors for biofuel production.

Production of biomethane, biohydrogen, and volatile fatty acids from Nordic phytoplankton biomass grown in blended wastewater

Upgrading carbon-negative microalgal biomass to biofuels and value-added products presents a three-pronged solution for waste treatment, carbon capture, and economically viable bioenergy production. Acidogenesis and methanogenesis are versatile processes at the core of anaerobic digestion systems, facilitating the conversion of diverse biogenic substrates into energy and a wide range of biobased products. The present study was conducted to integrate acidogenesis and methanogenesis for coproduction of biohydrogen, biomethane, and volatile fatty acids from Nordic phytoplankton consortia. For this purpose, microalgal consortia was cultivated in a raceway pond equipped with high surface area structures. Harvested microalgal biomass was subjected to thermoalkaline (2% NaOH solution at 121 °C) and enzymatic (cellulase) pretreatments. The hydrolysates of the pretreated biomass were inoculated with thermally treated sludge for acidogenic fermentation and with mixture of untreated sludge and cow dung (1:1 v/v ratio) for anerobic digestion. The acidogenic process produced a significant amount of biohydrogen (maximum 164.8 mL bioH2/VSload) along with volatile fatty acids (maximum 7.9 g COD/L), while methanogenesis resulted in biomethane production of maximum 210.7 mL bioCH4/VSload, accompanied by an ammonium recovery of 1278 mg NH4+/L. These maximum yields were all achieved by enzymatic pretreatment of the biomass fraction harvested from high surface area brush head structures inserted in the raceway pond. These results have important implications for designing phytoplankton cultivation systems and upstream pathways to optimize energy production from carbon-negative phytoplankton biomass.

Process development for fungal cellulase production and enzymatic saccharification of Parthenium hysterophorus for bioethanol production

Parthenium hysterophorus (PH), often known as parthenium weed, is one of the seventh most devastating weeds in the world.The primary aim of the study was to produce and optimize fungal FPase enzymes under solid-state fermentation (SSF) conditions for application in enzymatic hydrolysis to produce bioethanol. The impact of various physiological factors was verified through a systematic approach, initially using the one-factor-at-a-time (OFAT) method. Further optimization was carried out using statistical tools by response surface methodology (RSM), selecting three variables, i.e., lactose concentration, peptone concentration, and pH level. The outcomes of this research demonstrated that the RSM-based model led to the optimization of enzyme activity, resulting in 4.08U/ml of FPase, with a lactose concentration of 1.62 %, a peptone concentration of 1.5 %, and a pH level of 6 being the ideal conditions. When employing the FPase enzyme for biomass hydrolysis, the maximum yield of reducing sugar, at 0.37 ± 0.01 g/g, was achieved within a 96-hour period, using enzyme and substrate levels of 30 FPU/100 ml and 3 % (w/v), respectively. Fermentation of enzymatic hydrolysate was carried out by Saccharomyces cerevisiae at 30 °C, pH 6.0, and 120 rpm, which resulted in 0.45g/g ethanol yield after 48 h. Overall, our findings reveal thatPH biomass can be introduced into sugar-based biorefineries to produce biofuels and other valuable chemicals.

Genomic dissection of Niallia sp. for potential application in lignocellulose hydrolysis and bioremediation.

Genus Niallia has recently been separated taxonomic group from the Bacillus based on conserved signature indels in the genome. Unlike bioremediation, its role in plant biomass hydrolysis has not garnered considerable attention. The present study investigates the genomic potential of a novel Niallia sp. CRN 25 for applications in lignocellulose hydrolysis, significant enzyme production, and bioremediation. The CRN 25 strain exhibits xylosidase, cellobiosidase, α-arabinosidase, and α-D-galactosidase activity as 0.03U/ml whereas β-D-glucosidase and glucuronidase as 0.06 U/ml and 0.01U/ml, respectively. Further genome sequencing reveals nine copies of GH43 gene coding for hemicellulose-specific xylanase enzyme attached to the CBM 6 domain for increased processivity. The presence of β-glucosidase and β-galactosidase indicates the possible application of CRN 25 in facilitating the valorization of plant biomass into value-added products. Apart from this, genes of FMN-dependent NADH-azoreductase, cytochrome P450, and nitrate reductase, playing a crucial role in bioremediation processes, were annotated. Biosynthetic gene clusters (BGCs), responsible for synthesizing specialized metabolites of terpenes and lasso peptides, were also found in the genome. Conclusively genomic sketch of Niallia sp. CRN 25 reveals versatile metabolic potential for diverse environmental applications.

Effective Degradation of Brewer Spent Grains by a Novel Thermostable GH10 Xylanase.

Brewer spent grains (BSGs) are one of the most abundant by-products in brewing industry. Due to microbiological instability and high perishability, the efficient degradation of BSGs is of environmental and economic importance. Streptomyces sp. F-3 could grow in the medium with BSGs as the only carbon and nitrogen source. Proteome mass spectrometry revealed that a GH10 xylanase SsXyn10A could be secreted in large quantities. SsXyn10A showed optimum activity at pH 7.0 and 60 °C. SsXyn10A exhibited excellent thermostability which retained approximately 100% and 58% after incubation for 5 h at 50 and 60 °C. SsXyn10A displayed high activity to beechwood xylan (BX) and wheat arabinoxylan (WAX). SsXyn10A is active against xylotetracose (X4), xylopentose (X5), and xylohexose (X6) to produce main products xylobiose (X2) and xylotriose (X3). Ssxyn10A showed synergistic effects with commercial cellulase on BSGs hydrolyzing into soluble sugar. In addition, the steam explosion pretreatment of BSGs as the substrate produced twice as much reducing sugar as the degradation of the original substrate. This study will contribute to efficient utilization of BSGs and provide a thermostable GH10 xylanase which has potential application in biomass hydrolysis.

Synergistic Effects of Torrefaction and Alkaline Pretreatment on Sugar and Bioethanol Production from Wood Waste

Abundant availability of lignocellulosic biomass (LCB) coupled with diverse pretreatment methods have made it a promising option for energy production. However, it faces several challenges, some of which can be overcome by integrating pretreatment processes. The present study aims to optimize the integration of two different pretreatment methods—torrefaction (to reduce moisture content and fractionate biomass) and alkaline pretreatment of wood waste (to delignify biomass)—and utilize it for bioethanol production. Pretreatment performance was evaluated based on delignification, biomass hydrolysis, and bioethanol production. Initially, torrefaction was performed in a continuous reactor at a temperature range of 225–300 °C, followed by optimization of the critical parameters of alkaline pretreatment of torrefied wood waste (TWW), that is, the temperature, reaction time, solid–liquid ratio, and alkali concentration. Subsequently, the chemical and carbohydrate compositions of raw wood waste (RWW) and TWW were studied, followed by enzymatic hydrolysis and bioethanol fermentation. Integrated pretreatment positively impacted the cellulose and glucose contents of raw and torrefied biomass at lower temperatures. The enzymatic hydrolysis of TWW treated with alkali produced higher levels of glucose and bioethanol than (stand-alone) TWW. These results can be used as a basis for choosing the most suitable pretreatment for enhanced biomass conversion.

Erwiniaceae bacteria play defensive and nutritional roles in two widespread ambrosia beetles.

Ambrosia beetles are fungal-growing insects excavating galleries deep inside the wood. Their success as invaders increased scientific interest towards them. However, most studies on their microbiota targeted their fungal associates whereas the role of bacterial associates is understudied. To explore the role of abundant microbial associates, we isolated bacteria from active galleries of two widespread ambrosia beetles, Xylosandrus crassiusculus and X. germanus. These isolates were classified within the Erwiniaceae family and through a phylogenetic analysis including isolates from other insects we showed that they clustered with isolates obtained from ambrosia and bark beetles, including Erwinia typographi. The whole genome analysis of the isolate from active galleries of X. crassiusculus suggested that this bacterium plays both a nutritional role, by providing essential amino acids and enzymes for the hydrolysis of plant biomass, and a defensive role, by producing antibiotics. This defensive role was also tested in vitro against fungi, including mutualists, common associates, and parasites. The bacteria inhibited the growth of some of the common associates and parasites but did not affect mutualists. Our study supported the hypothesis of a mutualist role of Erwiniaceae bacteria in ambrosia beetles and highlighed the importance of bacteria in maintaining the symbiosis of their host with nutritional fungi.

Evaluation of the Char Formation During the Hydrothermal Treatment of Wooden Balls.

With wooden balls, a visualization of the hydrothermal carbonization to show the progress of the conversion to char is presented. In the present study, the balls represent the particles of biomass to investigate the differences in conversion outside and inside of biomass particles, during hydrothermal carbonization. A special focus is on hydrochar and pyrochar formation. The wooden balls are treated in subcritical water at 220°C for holding times between 0 and 960min. Even after 960min, hydrolysis of the original biomass is incomplete as cellulose and hemicellulose are linked by lignin, inhibiting the reaction with water. Moreover, two different pathways of char production can be observed. Inside of the wooden ball pyrochar is formed as any water got that deep in, on the surface hydrochar is fixed, originated from the surrounding liquid. On the ground of the HTC reactor, a thin, brittle precipitate of likely hydrochar or humins can be found either from the precipitation of loosely attached compounds on the surface of the biomass or direct precipitation from the liquid.

Microfluidic reactor designed for time-lapsed imaging of pretreatment and enzymatic hydrolysis of lignocellulosic biomass

The effect of tissue-specific biochemical heterogeneities of lignocellulosic biomass on biomass deconstruction is best understood through confocal laser scanning microscopy (CLSM) combined with immunohistochemistry. However, this process can be challenging, given the fragility of plant materials, and is generally not able to observe changes in the same section of biomass during both pretreatment and enzymatic hydrolysis. To overcome this challenge, a custom polydimethylsiloxane (PDMS) microfluidic imaging reactor was constructed using standard photolithographic techniques. As proof of concept, CLSM was performed on 60 μm-thick corn stem sections during pretreatment and enzymatic hydrolysis using the imaging reactor. Based on the fluorescence images, the less lignified parenchyma cell walls were more susceptible to pretreatment than the lignin-rich vascular bundles. During enzymatic hydrolysis, the highly lignified protoxylem cell wall was the most resistant, remaining unhydrolyzed even after 48 h. Therefore, imaging thin whole biomass sections was useful to obtain tissue-specific changes during biomass deconstruction.

Highly efficient synergistic activity of an α-L-arabinofuranosidase for degradation of arabinoxylan in barley/wheat.

Here, an α-L-arabinofuranosidase (termed TtAbf62) from Thermothelomyces thermophilus is described, which efficiently removes arabinofuranosyl side chains and facilitates arabinoxylan digestion. The specific activity of TtAbf62 (179.07 U/mg) toward wheat arabinoxylan was the highest among all characterized glycoside hydrolase family 62 enzymes. TtAbf62 in combination with endoxylanase and β-xylosidase strongly promoted hydrolysis of barley and wheat. The release of reducing sugars was significantly higher for the three-enzyme combination relative to the sum of single-enzyme treatments: 85.71% for barley hydrolysis and 33.33% for wheat hydrolysis. HPLC analysis showed that TtAbf62 acted selectively on monosubstituted (C-2 or C-3) xylopyranosyl residues rather than double-substituted residues. Site-directed mutagenesis and interactional analyses of enzyme-substrate binding structures revealed the catalytic sites of TtAbf62 formed different polysaccharide-catalytic binding modes with arabinoxylo-oligosaccharides. Our findings demonstrate a "multienzyme cocktail" formed by TtAbf62 with other hydrolases strongly improves the efficiency of hemicellulose conversion and increases biomass hydrolysis through synergistic interaction.

Engineered co-culture for consolidated production of phenylpropanoids directly from aromatic-rich biomass

Consolidated bioprocesses for the in situ hydrolysis and conversion of biomass feedstocks into value-added products offers great potential for both process and cost reduction. However, to date few consolidated bioprocesses have been developed that target aromatic rich feedstock fractions. Reported here is the development of synthetic co-cultivation for the consolidated hydrolysis and valorisation of corncob hydroxycinnamic acids. Biomass hydrolysis was achieved via a secretion module developed in B. subtilis using a genetically encoded biosensor-actuator to secrete hydrolytic enzymes. Conversion was achieved via a biotransformation module developed in E. coli using a suite of plug-and-play encoded enzymes to convert the released hydroxycinnamic acids into high-value phenylpropanoid target compounds. Finally, employing cellulolytic pre-treatment, extractive fermentation and in situ product recovery multiple aromatic products, coniferol and chavicol, were isolated from the same process in high purity.

A Comparison of Dilute Aqueous Isethionic Acid and Sulfuric Acid in Hydrolysis of Three Different Untreated Lignocellulosic Biomass Varieties.

Efficient catalytic hydrolysis of lignocellulosic biomass to sugars is a major challenge in the production of sustainable biofuels and chemical feedstocks. In this study isethionic acid was compared with H2SO4 for hydrolysis of polysaccharides in corn stover, switch grass, and poplar. The catalytic activities of acids were compared by analysis of total reducing sugar (TRS) and glucose yields in a sequence of experiments in water at 90-190 °C using 0.050 mol of H+/L isethionic acid and H2SO4. In comparison to using H2SO4, the use of isethionic acid catalyst lowered the maximum TRS percent yield temperatures by 25, 24, and 21% for corn stover, switch grass, and poplar. A similar effect was observed for glucose percent yields as well. This temperature reduction is due to lowering of the activation energy in the polysaccharide depolymerization reaction and most likely due to hydrogen-bonding-type dipolar interactions between the isethionic acid -OH group and -OH groups in biomass polysaccharides.

Improvement of Laccase Activity in Co-Culture of Panus lecomtei and Sporidiobolus pararoseus and Its Application as an Enzymatic Additive in Biomass Hydrolysis and Dye Decolorization

This work investigates the effects of the co-culture between the filamentous fungus Panus lecomtei and the yeast Sporidiobolus pararoseus in the production of laccases. The variations of time interval and inoculum volume of S. pararoseus in co-cultures with P. lecomtei stimulated laccase production, reaching its highest activity at nearly 2960.7 ± 244 U/mL with a maximum time point of 120 h and 2.0% (v/v), respectively. Further application in the pretreated sugarcane bagasse hydrolysis was performed, using P. lecomtei and S. pararoseus extract added to an enzyme mixture from the co-culture of P. lecomtei and Trichoderma reesei that positively favored the hydrolysis efficiency by 66.87%. Furthermore, the addition of P. lecomtei and S. pararoseus extract increased the degradation of industrial anthraquinone Remazol Brilliant Blue R by 78.98%. As a result, the extract derived from the co-culture of P. lecomtei and S. pararoseus rich in laccases presents potential in biotechnological applications, being suitable in the hydrolysis of lignocellulosic biomass and the degradation of unwanted dyes released in the environment.

Near-complete genome sequence of Lipomyces tetrasporous NRRL Y-64009, an oleaginous yeast capable of growing on lignocellulosic hydrolysates.

Lipomyces tetrasporous is an oleaginous yeast that can utilize a variety of plant-based sugars. It accumulates lipids during growth on lignocellulosic biomass hydrolysates. We present the annotated genome sequence of L. tetrasporous NRRL Y-64009 to aid in its development as a platform organism for producing lipids and lipid-based bioproducts.

Characterization of a novel acidophilic, ethanol tolerant and halophilic GH12 β-1,4-endoglucanase from Trichoderma asperellum ND-1 and its synergistic hydrolysis of lignocellulosic biomass

A novel acidophilic GH5 β-1,4-endoglucanase (TaCel12) from Trichoderma asperellum ND-1 was efficiently expressed in Pichia pastoris (a 1.5-fold increase). Deglycosylated TaCel12 migrated as a single band (26.5 kDa) in SDS-PAGE. TaCel12 was acidophilic with a pH optimum of 4.0 and displayed great pH stability (>80 % activity over pH 3.0–5.0). TaCel12 exhibited considerable activity towards sodium carboxymethyl cellulose and sodium alginate with Vmax values of 197.97 μmol/min/mg and 119.06 μmol/min/mg, respectively. Moreover, TaCel12 maintained >80 % activity in the presence of 20 % ethanol and 4.28 M NaCl. Additionally, Mn2+, Pb2+ and Cu2+ negatively affected TaCel12 activity, while the presence of 5 mM Co2+ significantly increased the enzyme activity. Analysis of action mode revealed that TaCel12 required at least four glucose (cellotetraose) residues for hydrolysis to yield cellobiose and cellotriose. Site-directed mutagenesis results suggested that Glu133 and Glu217 of TaCel12 are crucial catalytic residues, with Asp116 displaying an auxiliary function. Production of soluble sugars from lignocellulose is a crucial step in bioethanol development, and it is noteworthy that TaCel12 could synergistically yield fermentable sugars from corn stover and bagasse, respectively. Thus TaCel12 with excellent properties will be considered a potential biocatalyst for applications in various industries, especially for lignocellulosic biomass conversion.

Biorefinery concept for Cylindrospermum alatosporum CCALA 988 extracting multiple high-value compounds and residue utilization by P. pastoris fermentation producing phytase

Economically feasible biorefinery concepts rely on the production of multiple high-value products and valorization even of residual side streams. This generally holds true for biomass from agriculture, forestry, and aquaculture. Cyanobacteria are seen as promising organisms for this purpose. The potential for commercialization, however, strongly depends on the strain being used and the range of products it offers. The cyanobacterial strain Cylindrospermum alatosporum CCALA 988 was reported to produce high amounts of the cyclic lipopeptides puwainaphycins (PUWs) and minutisamides (MINs) as high-value products. Hence, this study investigated the biomass of the cyanobacterial strain as a potential feedstock for a biorefinery concept. The study also explored the enzymatic hydrolysis of the residual biomass and its utilization as a medium supplement for appA E. coli phytase expression with Pichia pastoris. Analysis of the cyanobacterial biomass revealed the following composition: ash 1.9 %, lipids 4.1 %, starch 2.4 %, structural saccharides 8.7 %, and protein 52.4 %. Further, a sequential extraction concept and a bench-scale mass balance were investigated, and products were proposed with 56.2 % of biomass being utilized and 43.8 % being left as residue. PUWs (21.6 mg/g), MINs (101.5 mg/g), phycobiliproteins (34.5 mg/g), and pigments (6.4 mg/g) were selected as products and quantified. The utilization of hydrolysate by fermentation of P. pastoris for appA E. coli phytase expression was studied in a 1 L system. In batch mode, the hydrolysate allowed to achieve comparable results to a rich complex medium, with over 500 U/mL of appA E. coli phytase. The obtained results provide a basis for further development of this biorefinery concept.

Bioethanol production from mixed sugars at a semi-pilot scale through two-step repeated sequential fermentation

Assessing the industrial suitability of sequential culture for efficient conversion of mixed sugars from plant biomass hydrolysate is essential. In the present study, a two-stage sequential fermentation of mixed sugar-rich sugar cane bagasse hydrolysate was performed to produce bioethanol. In the first stage, glucose fermentation using Z. mobilis was carried out, followed by xylose fermentation using S. stipitis in the second. However, xylose fermentation was done only after completely separating primary cells and residual ethanol. Sequential fermentation of mixed sugar-rich bagasse hydrolysate yielded an ethanol titer of 77.7 g.L−1 with an average ethanol yield of 0.48 g.g−1 and overall ethanol productivity of 1.38 g.L−1.h−1. Further, repeated sequential fermentation (RSF) of mixed sugars was performed for two cycles. RSF assessed the fermentation performance of high cell density sequential culture achieved through sequential cell recycling. The maximum ethanol productivity achieved from glucose and xylose was 6.92 g.L−1.h−1 and 2.28 g.L−1.h−1, respectively. In addition, a techno-economic analysis found that the minimum selling price of the ethanol (MSP) produced from sequential fermentation of mixed sugar-rich bagasse hydrolysate (scenario I) was up to 1.7 US$. L−1. On the other hand, the MSP calculated after omitting reagent costs of synthetic glucose and xylose of the first scenario was up to 0.52 US$. L−1.