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Background Bamboo, a lignocellulosic feedstock, is considered as a potentially excellent raw material and evaluated for lignocellulose degradation and bioethanol production, with a focus on using physical and chemical pre-treatment. However, studies reporting the biodegradation of bamboo lignocellulose using microbes such as bacteria and fungi are scarce. Results In the present study, Bacillus velezensis LC1 was isolated from Cyrtotrachelus buqueti, in which the symbiotic bacteria exhibited lignocellulose degradation ability and cellulase activities. We performed genome sequencing of B. velezensis LC1, which has a 3929,782-bp ring chromosome and 46.5% GC content. The total gene length was 3,502,596 bp using gene prediction, and the GC contents were 47.29% and 40.04% in the gene and intergene regions, respectively. The genome contains 4018 coding DNA sequences, and all have been assigned predicted functions. Carbohydrate-active enzyme annotation identified 136 genes annotated to CAZy families, including GH, GTs, CEs, PLs, AAs and CBMs. Genes involved in lignocellulose degradation were identified. After a 6-day treatment, the bamboo shoot cellulose degradation efficiency reached 39.32%, and the hydrolysate was subjected to ethanol fermentation with Saccharomyces cerevisiae and Escherichia coli KO11, yielding 7.2 g/L of ethanol at 96 h. Conclusions These findings provide an insight for B. velezensis strains in converting lignocellulose into ethanol. B. velezensis LC1, a symbiotic bacteria, can potentially degrade bamboo lignocellulose components and further transformation to ethanol, and expand the bamboo lignocellulosic bioethanol production.

Phenolic aldehydes from lignocellulose pretreatment harshly inhibit the viability and metabolism of ethanol fermenting strains. Direct conversion of phenolic aldehydes is usually incomplete due to their low water solubility and recalcitrance to bioconversion. Here we consolidated phenolic aldehydes bioconversion and ethanol fermentation in a typical ethanologenic bacterium Zymomonas mobilis by constructing an intracellular oxidative pathway. The gene PP_2680 encoding NAD+-dependent aldehyde dehydrogenase from Pseudomonas putida KT2440 was expressed in Z. mobilis ZM4. The expression significantly improved both aldehyde inhibitor conversion and ethanol fermentability in corn stover hydrolysate. The purified PP_2680 aldehyde dehydrogenase showed strong in vitro oxidative capacity on phenolic aldehydes and its in vivo expression significantly up-regulated the key genes in the ED pathway and the oxidative phosphorylation. This study provided an important concept of simultaneous biodetoxification and fermentation in ethanologenic strains for the improvement of ethanol fermentability.

Thermophilic methane fermentation was a valid approach for handling the stillage eluted from ethanol fermentation of waste paper and kitchen waste. The wide organic loading rate (OLR) range (2-14 g VTS/(L d)) for stable performance and relatively high energy recovery efficiency (79.0%) were achieved, and OLR of 8 g VTS/(L d) was optimum for achievement of highest biogas evolution and VTS removal efficiency. Microbial community analysis revealed that hydrolysis of cellulose was the critical step for methane production from the stillage.

Lignocellulosic biomass is a sustainable feedstock for fuel ethanol production, but it is characterized by low mass and energy densities, and distributed production with relatively small scales is more suitable for cellulosic ethanol, which can better balance cost for the feedstock logistics. Lignocellulosic biomass is recalcitrant to degradation, and pretreatment is needed, but more efficient pretreatment technologies should be developed based on an in-depth understanding of its biosynthesis and regulation for engineering plant cell walls with less recalcitrance. Simultaneous saccharification and co-fermentation has been developed for cellulosic ethanol production, but the concept has been mistakenly defined, since the saccharification and co-fermentation are by no means simultaneous. Lignin is unreactive, which not only occupies reactor spaces during the enzymatic hydrolysis of the cellulose component and ethanol fermentation thereafter, but also requires extra mixing, making high solid loading difficult for lignocellulosic biomass and ethanol titers substantially compromised, which consequently increases energy consumption for ethanol distillation and stillage discharge, presenting another challenge for cellulosic ethanol production. Pentose sugars released from the hydrolysis of hemicelluloses are not fermentable with Saccharomyces cerevisiae used for ethanol production from sugar- and starch-based feedstocks, and engineering the brewing yeast and other ethanologenic species such as Zymomonas mobilis with pentose metabolism has been performed within the past decades. However strategies for the simultaneous co-fermentation of pentose and hexose sugars that have been pursued overwhelmingly for strain development might be modified for robust ethanol production. Finally, unit integration and system optimization are needed to maximize economic and environmental benefits for cellulosic ethanol production. In this article, we critically reviewed updated progress, and highlighted challenges and strategies for solutions.

The objective of this study was to obtain two types of fuels, i.e., bioethanol and biogas, in a sequential combination of biochemical processes from lignocellulosic biomass (corn straw). Waste from the agricultural sector containing lignocellulose structures was used to obtain bioethanol, while the post-fermentation (cellulose stillage) residue obtained from ethanol fermentation was a raw material for the production of high-power biogas in the methane fermentation process. The studies on obtaining ethanol from lignocellulosic substrate were based on the simultaneous saccharification and fermentation (SSF) method, which is a simultaneous hydrolysis of enzymatic cellulose and fermentation of the obtained sugars. Saccharomyces cerevisiae (D-2) in the form of yeast cream was used for bioethanol production. The yeast strain D-2 originated from the collection of the Institute of Agricultural and Food Biotechnology. Volatile compounds identified in the distillates were measured using gas chromatography with flame ionization detector (GC-FID). CH4 and CO2 contained in the biogas were analyzed using a gas chromatograph in isothermal conditions, equipped with thermal conductivity detector (katharometer) with incandescent fiber. Our results show that simultaneous saccharification and fermentation enables production of bioethanol from agricultural residues with management of cellulose stillage in the methane fermentation process.

The exploitation of starchy wastes for the production of value-added products and the consolidated bioprocessing (CBP) have a positive environmental and cost-effective impact. For this reason, a cell factory (CF) was employed to perform three bioprocesses in one step (CBP) for alcoholic fermentation of starch without genetic modification. CF is a bilayer biocatalyst consisting of an inner layer of immobilized Saccharomyces cerevisiae on tubular cellulose (TC) and an external layer of immobilized Aspergillus awamori on alginates (ALG). In first, the suggested CF was proved to be more effective compared with co-immobilized S. cerevisiae and A. awamori on ALG beads for starch fermentation. Subsequently, the effect of the (i) S. cerevisiae concentration (g S. cerevisiae/g TC) during its immobilization, (ii) CaCl 2 concentration (% w/v), (iii) form of bilayer CF (wet or freeze-dried) and (iv) freeze-dried CF concentration on 5% (w/v) starch fermentation was examined. It was found that the higher ethanol production (32.17 mL/L), productivity (4.60 mL/L/d) and yield (0.51 g ethanol/g starch) reaching the theoretical one were obtained when the CF in freeze dried form fermented 100 mL of 5% (w/v) starch solution. Finally, two repeated fermentation batches were performed using the best CF. The ethanol yield was decreased during the repeated fermentation batches, but remained in acceptable levels. The successful preparation of CF was verified with FTIR spectroscopy, SEM and TEM analysis. The results indicated that the objective of designing CF was successfully achieved, and the results are promising to be the base for the exploitation of starchy wastes and the development of an innovation in brewing industry by eliminating the malting stage. This CF can be applied as model for different bioprocesses of White Biotechnology, e.g. substituting S. cerevisiae with the appropriate microorganism to produce other metabolites of added value.

A promising approach to help offset production costs for the cellulosic ethanol industry is to improve ethanol productivity while simultaneously generating value-added by-products. This study reports integration of an advanced fermentation approach (self-cycling fermentation) with the production of cellulose nanocrystals. Specifically, wood pulp was enzymatically hydrolyzed to yield dissolved sugars, which were fed to a self-cycling fermentation system for ethanol production, and residual solids were used for cellulose nanocrystals production via acid hydrolysis. Self-cycling fermentation achieved stable ethanol production for 10 cycles with significantly greater productivity than batch operation: ethanol volumetric productivity increased by 63–95% and annual ethanol productivity by 96 ± 5%. Additionally, the enzyme hydrolysis approach employed did not impede ethanol fermentation, and the cellulose nanocrystals generated displayed properties consistent with previous studies. Taken together, these results highlight the potential of this co-production strategy to produce both cellulosic ethanol and cellulose nanocrystals from a single feedstock.

Numerical analyses of energy production processes are important for practical applications. Here, we established a model of enzymatic hydrolysis and ethanol fermentation. The kinetic model of these reactions were represented by Langmuir adsorption, Michaelis-Menten, and Shuler models. In our model, ethanol fermentation and enzymatic hydrolysis models were fit to experimental data separately, and their model equations were then combined for fitting of experimental data, including the size of cellulose, amount of adsorbed protein, and decreases in glucose as the theoretical production values for SSF. From these kinetic models, the theoretical values for saccharification and fermentation were calculated, and optimal parameters were determined. Using these parameters, theoretical curves for simultaneous saccharification and fermentation were predicted. Additionally, we identified changes in the radius of cellulose and the concentrations of cellulose, cellobiose, glucose, and ethanol during saccharification and fermentation.

The aim of this study was to remove 5-hydroxymethyl furfural (5-HMF) and furfural, known as fermentation inhibitors, in acid pretreated hydrolysates (APH) obtained from Scenedesmus obliquus using activated carbon. Microwave-assisted pretreatment was used to produce APH containing glucose, xylose, and fermentation inhibitors (5-HMF, furfural). The response surface methodology was applied to optimize key detoxification variables such as temperature (16.5–58.5 °C), time (0.5–5.5 h), and solid–liquid (S-L) ratio of activated carbon (0.6–7.4 w/v%). Three variables showed significant effects on the removal of fermentation inhibitors. The optimum detoxification conditions with the maximum removal of fermentation inhibitors and the minimum loss of sugars (glucose and xylose) were as follows: temperature of 36.6 °C, extraction time of 3.86 h, and S-L ratio of 3.3 w/v%. Under these conditions, removal of 5-HMF, furfural, and sugars were 71.6, 83.1, and 2.44%, respectively, which agreed closely with the predicted values. When the APH and detoxified APH were used for ethanol fermentation by S. cerevisiae, the ethanol produced was 38.5% and 84.5% of the theoretical yields, respectively, which confirmed that detoxification using activated carbon was effective in removing fermentation inhibitors and increasing fermentation yield without significant removal of fermentable sugars.

Corn fiber is a co-product of commercial ethanol dry-grind plants, which is processed into distillers dried grains with solubles (DDGS) and used as animal feed, yet it holds high potential to be used as feedstock for additional ethanol production. Due to the tight structural make-up of corn fiber, a pretreatment step is necessary to make the cellulose and hemicellulose polymers in the solid fibrous matrix more accessible to the hydrolytic enzymes. A pretreatment process was developed in which whole corn kernels were soaked in aqueous solutions of 2.5, 5.0, 7.5, and 10.0 wt% ammonia at 105 °C for 24 h. The pretreated corn then was subjected to a conventional mashing procedure and subsequent ethanol fermentation using a commercial strain of natural Saccharomyces cerevisiae with addition of a commercial cellulase. Pretreatment of the corn with 7.5 wt% ammonia solution plus cellulase addition gave the highest ethanol production, which improved the yield in fermentation using 25 wt% solid from 334 g ethanol/kg corn obtained in the control (no pretreatment and no cellulase addition) to 379 g ethanol/kg corn (a 14% increase). The process developed can potentially be implemented in existing dry-grind ethanol facilities as a “bolt-on” process for additional ethanol production from corn fiber, and this additional ethanol can then qualify as “cellulosic ethanol” by the Environmental Protection Agency’s (EPA’s) Renewable Fuels Standard and thereby receive RINs (Renewable Identification Numbers).

Background: Future biorefineries will focus on converting low value waste streams to chemical products that are derived from petroleum or refined sugars. Feedstock pretreatment in a simple, cost effective, agnostic manner is a major challenge. Methods: In this work, beechwood sawdust was delignified via an organosolv process, assisted by homogeneous inorganic acid catalysis. Mixtures of water and several organic solvents were evaluated for their performance. Specifically, ethanol (EtOH), acetone (AC), and methyl- isobutyl- ketone (MIBK) were tested with or without the use of homogeneous acid catalysis employing sulfuric, phosphoric, and oxalic acids under relatively mild temperature of 175 &deg, C for one hour. Results: Delignification degrees (DD) higher than 90% were achieved, where both AC and EtOH proved to be suitable solvents for this process. Both oxalic and especially phosphoric acid proved to be good alternative catalysts for replacing sulfuric acid. High gravity simultaneous saccharification and fermentation with an enzyme loading of 8.4 mg/gsolids at 20 wt.% initial solids content reached an ethanol yield of 8.0 w/v%. Conclusions: Efficient delignification combining common volatile solvents and mild acid catalysis allowed for the production of ethanol at high concentration in an efficient manner.

Об’єктом розробки є технологія виробництва біоетанолу з целюлозовмісної сировини. Мета: розробка модернізованої технології виробництва біоетанолу з целюлозовмісної сировини на базі відомої гідролізної технології. Розроблено проект технології виробництва біоетанолу з целюлозовмісної сировини з метою отримання біопалива, що відповідатиме нормам євростандарту і задовольнить потребу країні у біоетанолі. Технологічна схема базується на зброджуванні гідролізату целюлозовмісної сировини за використання дріжджів Saccharomyces cerevisiae, з подальшим виділення біоетанолу з бражки. Новизною проекту є технологічна стадія попередньої обробки сировини реактивом Фентона в водно-органічній суміші розчинників. Матеріали даної магістерської дисертації можна використовувати при проектуванні підприємств по виробництву спирту, які в якості сировини використовують целюлозовмісні відходи. Актуальність проекту полягає в вирішенні перш за все енергетичної проблеми – нестачі моторних палив в країні, а також частково економічної і екологічно. Master’s dissertation: pages,figures, tables, sources. The object of development is technology of bioethanol production from cellulose - containing materials. Objective: development an improved technology of bioethanol production from cellulose - containing materials based on known hydrolysis technology. A project of bioethanol production from cellulose - containing materials technology to obtain biofuel that meet the standards of standarts and meet the needs of the country in bioethanol was designed. Technological scheme is based on the fermentation of hydrolyzate from cellulose by Saccharomyces cerevisiae, with further receiving of bioethanol by rectification. The novelty of the project is using a new technological stage for cellulose organosolv pretreatment through Fenton reaction. Materials for this master’s dissertation can be used in the projection of ethanol-producing factories, that use cellulose as raw material. The topicality of the project is primarily a solving of energy problem – lacking of motor fuels in the country, and partly solving economic and environmental problem.

Choline acetate (ChOAc), a cholinium ionic liquid (IL), was compared with 1-ethyl-3-methylimidazolium acetate (EmimOAc) with regard to biomass pretreatment, inhibition on cellulase and yeast, residuals in pretreated biomass, and saccharification and fermentation of pretreated biomass. Irrespective of ChOAc and EmimOAc, cellulose and hemicellulose saccharification of the IL-pretreated bagasse were over 90% and 60%, respectively. Median effective concentrations (EC50) based on cellulase activity were 32 wt% and 16 wt% for ChOAc and EmimOAc, respectively. The EC50 based on yeast growth were 3.1 wt% and 0.3 wt% for ChOAc and EmimOAc respectively. The residuals in IL-pretreated bagasse were 10% and 23% for ChOAc and EmimOAc, respectively, when washed 2 times after pretreatment. Ethanol yield on a bagasse basis were 60% and 24% for ChOAc and EmimOAc, respectively, in the saccharification and fermentation of IL-pretreated bagasse when washed 2 times. ChOAc-pretreated bagasse could be saccharified and fermented with fewer wash times than EmimOAc-pretreated bagasse.

Several yeast strains have been engineered to express different cellulases to achieve simultaneous saccharification and fermentation of lignocellulosic materials. However, successes in these endeavors were modest, as demonstrated by the relatively low ethanol titers and the limited ability of the engineered yeast strains to grow using cellulosic materials as the sole carbon source. Recently, substantial enhancements to the breakdown of cellulosic substrates have been observed when lytic polysaccharide monooxygenases (LPMOs) were added to traditional cellulase cocktails. LPMOs are reported to cleave cellulose oxidatively in the presence of enzymatic electron donors such as cellobiose dehydrogenases. In this study, we coexpressed LPMOs and cellobiose dehydrogenases with cellobiohydrolases, endoglucanases, and β-glucosidases in Saccharomyces cerevisiae . These enzymes were secreted and docked onto surface-displayed miniscaffoldins through cohesin-dockerin interaction to generate pentafunctional minicellulosomes. The enzymes on the miniscaffoldins acted synergistically to boost the degradation of phosphoric acid swollen cellulose and increased the ethanol titers from our previously achieved levels of 1.8 to 2.7 g/liter. In addition, the newly developed recombinant yeast strain was also able to grow using phosphoric acid swollen cellulose as the sole carbon source. The results demonstrate the promise of the pentafunctional minicellulosomes for consolidated bioprocessing by yeast.

Optimum utilization of fermentable sugars from lignocellulosic biomass to deliver multiple products under biorefinery concept has been reported in this work. Alcohol fermentation has been carried out with multiple cell recycling of Kluyveromyces marxianus IIPE453. The yeast utilized xylose-rich fraction from acid and steam treated biomass for cell generation and xylitol production with an average yield of 0.315±0.01g/g while the entire glucose rich saccharified fraction had been fermented to ethanol with high productivity of 0.9±0.08g/L/h. A detailed insight into its genome illustrated the strain's complete set of genes associated with sugar transport and metabolism for high-temperature fermentation. A set flocculation proteins were identified that aided in high cell recovery in successive fermentation cycles to achieve alcohols with high productivity. We have brought biomass derived sugars, yeast cell biomass generation, and ethanol and xylitol fermentation in one platform and validated the overall material balance. 2kg sugarcane bagasse yielded 193.4g yeast cell, and with multiple times cell recycling generated 125.56g xylitol and 289.2g ethanol (366mL).

Several papers have reported various technical aspects of lignocellulosic bioethanol production. Recalcitrance to saccharification is a major limitation for conversion of lignocellulosic biomass to ethanol. The biological process for converting lignocellulose to fuel ethanol includes delignification in order to liberate cellulose and hemicelluloses, depolymerization of carbohydrate polymers to produce free sugars, and sugar fermentation to produce ethanol. Access of plant cell wall polysaccharides to chemical, enzymatic and microbial digestion is limited by many factors, including the presence of lignin and hemicellulose that cover cellulose microfibrils. An effort to support the fuel ethanol fermentation industry using the Indonesian woody plant species Shorea uliginosa Foxw., was undertaken with regard to the established efficient bioethanol production process. This paper relates to a method for producing bioethanol from the lignocellulose of S. uliginosa Foxw. by saccharification and fermentation of xylem. A literature study of previous research on cellulose hydrolysis as a method for producing bioethanol was necessary. The objective of this study was to gain a deeper understanding of the degradation mechanisms of cellulose by enzymes through a study of previous research, which were then compared to the new method.

Direct ethanol fermentation from amorphous cellulose was achieved using an engineered industrial Saccharomyces cerevisiae strain. Two cellulase genes endoglucanase (eg3) and β-glucosidase (bgl1) were obtained from Trichoderma viride and integrated into the genome of S. cerevisiae. These two cellulases could be constitutively coexpressed and secreted by the recombinant strain S. cerevisiae-eb. The enzyme activities were analyzed in the culture supernatants, with the highest endoglucanase activity of 2.34 units/ml and β-glucosidase activity of 0.95 units/ml. The effects of pH, temperature and metal ions on enzyme activities were analyzed. The coexpression strain S. cerevisiae-eb could grow in carboxymethyl cellulose (CMC) and utilize it as the single carbon source. The 20 g/L CMC as a model substrate of amorphous cellulose was used in fermentation. The ethanol production reached 4.63 g/L in 24 h, with the conversion ratio of 64.2% compared with the theoretical concentration. This study demonstrated that the engineered industrial strain S. cerevisiae-eb could convert amorphous cellulose to ethanol simultaneously and achieve consolidated bioprocessing (CBP) directly.

The yeast Kluyveromyces marxianus is considered as a potential alternative to Saccharomyces cerevisiae in producing ethanol as a biofuel. In this study, we investigated the ethanol fermentation properties of novel K. marxianus strain DMB1, isolated from bagasse hydrolysates. This strain utilized sorbitol as well as various pentoses and hexoses as single carbon sources under aerobic conditions and produced ethanol from glucose in hydrolysates of the Japanese cedar at 42 °C. Reference strains K. marxianus NBRC1777 and S. cerevisiae BY4743 did not assimilate sorbitol or ferment lignocellulosic hydrolysates to ethanol at this temperature. Thus strain DMB1 appears to be optimal for producing bioethanol at high temperatures, and might provide a valuable means of increasing the efficiency of ethanol fermentation.

This study aimed to produce a cellulase blend and to evaluate its application in a simultaneous saccharification and fermentation (SSF) process for second generation ethanol production from sugar cane bagasse. The sugar cane bagasse was subjected to pretreatments (diluted acid and alkaline), as for disorganizing the ligocellulosic complex, and making the cellulose component more amenable to enzymatic hydrolysis. The residual solid fraction was named sugar cane bagasse partially delignified cellulignin (PDC), and was used for enzyme production and ethanol fermentation. The enzyme production was performed in a bioreactor with two inoculum concentrations (5 and 10% v/v). The fermentation inoculated with higher inoculum size reduced the time for maximum enzyme production (from 72 to 48). The enzyme extract was concentrated using tangential ultrafiltration in hollow fiber membranes, and the produced cellulase blend was evaluated for its stability at 37°C, operation temperature of the simultaneous SSF process, and at 50°C, optimum temperature of cellulase blend activity. The cellulolytic preparation was stable for at least 300h at both 37°C and 50°C. The ethanol production was carried out by PDC fed-batch SSF process, using the onsite cellulase blend. The feeding strategy circumvented the classic problems of diffusion limitations by diminishing the presence of a high solid:liquid ratio at any time, resulting in high ethanol concentration at the end of the process (100g/L), which corresponded to a fermentation efficiency of 78% of the maximum obtainable theoretically. The experimental results led to the ratio of 380L of ethanol per ton of sugar cane bagasse PDC.

The effect of time and pressure on the selective extraction of sugar beet pectin using steam pre-treatment on unprocessed Sugar Beet Pulp was evaluated using a design of experiments approach. This process gave the highest solubilisation of pectin oligomers at a relatively low pressure and longer time (5 Bar, 24 min), whilst leaving the majority of the cellulose fraction intact. This method of steam pre-treatment fits into the concept of a sugar beet biorefinery as it valorises an existing waste stream without requiring any further physical processing such as milling or dilution with water. The residual cellulose fraction was enriched in cellulose and could be effectively fermented into ethanol by yeast after enzymatic digestion, producing 0.48 g ethanol per gram of glucose.

Accurate mass balance and conversion data from integrated operation is needed to fully elucidate the economics of biofuel production processes. This study explored integrated conversion of corn stover to ethanol and highlights techniques for accurate yield calculations. Acid pretreated corn stover (PCS) produced in a pilot-scale reactor was enzymatically hydrolyzed and the resulting sugars were fermented to ethanol by the glucose–xylose fermenting bacteria, Zymomonas mobilis 8b. The calculations presented here account for high solids operation and oligomeric sugars produced during pretreatment, enzymatic hydrolysis, and fermentation, which, if not accounted for, leads to overestimating ethanol yields. The calculations are illustrated for enzymatic hydrolysis and fermentation of PCS at 17.5% and 20.0% total solids achieving 80.1% and 77.9% conversion of cellulose and xylan to ethanol and ethanol titers of 63 g/L and 69 g/L, respectively. These procedures will be employed in the future and the resulting information used for techno-economic analysis.

The potential of cashew apple bagasse as a source of sugars for ethanol production by Kluyveromyces marxianus CE025 was evaluated in this work. This strain was preliminarily cultivated in a synthetic medium containing glucose and xylose and was able to produce ethanol and xylitol at pH 4.5. Next, cashew apple bagasse hydrolysate (CABH) was prepared by a diluted sulfuric acid pretreatment and used as fermentation media. This hydrolysate is rich in glucose, xylose, and arabinose and contains traces of formic acid and acetic acid. In batch fermentations of CABH at pH 4.5, the strain produced only ethanol. The effects of temperature on the kinetic parameters of ethanol fermentation by K. marxianus CE025 using CABH were also evaluated. Maximum specific growth rate (μmax), overall yields of ethanol based on glucose consumption \(Y_{{P \mathord{\left/ {\vphantom {P {S_1 }}} \right. \kern-\nulldelimiterspace} {S_1 }}}^{\text{G}}\) and based on glucose + xylose consumption (YP/S), overall yield of ethanol based on biomass (YP/X), and ethanol productivity (PE) were determined as a function of temperature. Best results of ethanol production were achieved at 30°C, which is also quite close to the optimum temperature for the formation of biomass. The process yielded 12.36 ± 0.06 g l−1 of ethanol with a volumetric production rate of 0.257 ± 0.002 g l−1 h−1 and an ethanol yield of 0.417 ± 0.003 g g−1 glucose.

Sugarcane bagasse is one of the most promising agricultural by-products for conversion to biofuels. Here, ethanol fermentation from bagasse has been achieved using an integrated process combining mechanical pretreatment by ball milling, with enzymatic hydrolysis and fermentation. Ball milling for 2 h was sufficient for nearly complete cellulose structural transformation to an accessible amorphous form. The pretreated cellulosic residues were hydrolyzed by a crude enzyme preparation from Penicillium chrysogenum BCC4504 containing cellulase activity combined with Aspergillus flavus BCC7179 preparation containing complementary beta-glucosidase activity. Saccharification yields of 84.0% and 70.4% for glucose and xylose, respectively, were obtained after hydrolysis at 45 degrees C, pH 5 for 72 h, which were slightly higher than those obtained with a commercial enzyme mixture containing Acremonium cellulase and Optimash BG. A high conversion yield of undetoxified pretreated bagasse (5%, w/v) hydrolysate to ethanol was attained by separate hydrolysis and fermentation processes using Pichia stipitis BCC15191, at pH 5.5, 30 degrees C for 24 h resulting in an ethanol concentration of 8.4 g/l, corresponding to a conversion yield of 0.29 g ethanol/g available fermentable sugars. Comparable ethanol conversion efficiency was obtained by a simultaneous saccharification and fermentation process which led to production of 8.0 g/l ethanol after 72 h fermentation under the same conditions. This study thus demonstrated the potential use of a simple integrated process with minimal environmental impact with the use of promising alternative on-site enzymes and yeast for the production of ethanol from this potent lignocellulosic biomass.

This research aims at developing a biorefinery platform to convert lignocellulosic corn fiber into fermentable sugars at a moderate temperature (37 °C) with minimal use of chemicals. White-rot (Phanerochaete chrysosporium), brown-rot (Gloeophyllum trabeum), and soft-rot (Trichoderma reesei) fungi were used for in situ enzyme production to hydrolyze cellulosic and hemicellulosic components of corn fiber into fermentable sugars. Solid-substrate fermentation of corn fiber by either white- or brown-rot fungi followed by simultaneous saccharification and fermentation (SSF) with coculture of Saccharomyces cerevisiae has shown a possibility of enhancing wood rot saccharification of corn fiber for ethanol fermentation. The laboratory-scale fungal saccharification and fermentation process incorporated in situ cellulolytic enzyme induction, which enhanced overall enzymatic hydrolysis of hemi/cellulose components of corn fiber into simple sugars (mono-, di-, and trisaccharides). The yeast fermentation of the hydrolyzate yielded 7.8, 8.6, and 4.9 g ethanol per 100 g corn fiber when saccharified with the white-, brown-, and soft-rot fungi, respectively. The highest ethanol yield (8.6 g ethanol per 100 g initial corn fiber) is equivalent to 35% of the theoretical ethanol yield from starch and cellulose in corn fiber. This research has significant commercial potential to increase net ethanol production per bushel of corn through the utilization of corn fiber. There is also a great research opportunity to evaluate the remaining biomass residue (enriched with fungal protein) as animal feed.

Hydrothermal treatment can be applied to pretreatment of ethanol fermentation process. What is important in the pretreatment step is that lignocellulosic material be effectively hydrolyzed to smaller saccharides and not be further converted to other substances. In this study we developed reaction kinetics of cellulose hydrolysis to glucose for evaluation of hydrothermal treatment condition.

A simultaneous saccharification and fermentation (SSF) process was investigated to produce ethanol using two kinds of cellulose carriers that were respectively suitable for immobilization of Aspergillus awamori and Saccharomyces pastorianus. The maximum ethanol concentration attained by the batch operation was 25.5 g l−1. Under suitable conditions, both cellulose carriers with immobilized cells could be reused efficiently for three cycles. The total amount of ethanol production was 66.0 g (per 1 l working volume) after the repeated operation. Ethanol productivity mainly depends on a saccharification process. There is a limit in durability in the repeated batch operation, and it is important to maintain high activity of the fungus in order to produce ethanol efficiently. Journal of Industrial Microbiology & Biotechnology (2001) 27, 52–57.

In order to convert cellulosic biomass to raw materials for alcohol fermentation, cellulose and a chinquapin, a hard wood, were hydrothermolyzed with hot compressed water in a batch reactor at temperatures between 250 and 405°C.Cellulose was quickly solubilized to water, and nearly 40% of cellulose could be sac-charified to hexose by adjusting the heating time in the range of 13-30s at the heating temperature of 305-405°C. As a result of fermentation test of the water soluble frac-tion, the fermentation rate was found to be remarkably improved by removing furfurals in the solution through an active carbon treatment, and further, good mass balance was confirmed between the consumed hexose and formed ethanol.In the case of chinquapin, the hydrothermolysis started at lower temperature than pure cellulse. Active lignin was first decomposed, and then hemicellulose followed by cellulose was hydrolyzed to water solubles (WS), although stable lignin was hard to de-compose and remained as solid residue (methanol insolubles, MI). An increase in the heating temperature caused the decrease in the yield of MI, but did not the increase in the yield of WS because of the second decomposition of WS. However, the WS includ-ing over 20% of hexose was obtained in the yield of near 30% by optimizing the heating time. Since species from lignin were obtained as methanol solubles separately from WS, the WS were suitable for the fermentation and ethanol was successfully produced with saccharomyces.

Background Numerous studies have examined the direct fermentation of cellulosic materials by cellulase-expressing yeast; however, ethanol productivity in these systems has not yet reached an industrial level. Certain microorganisms, such as the cellulolytic fungus Trichoderma reesei, produce expansin-like proteins, which have a cellulose-loosening effect that may increase the breakdown of cellulose. Here, to improve the direct conversion of cellulose to ethanol, yeast Saccharomyces cerevisiae co-displaying cellulase and expansin-like protein on the cell surface were constructed and examined for direct ethanol fermentation performance. Results The cellulase and expansin-like protein co-expressing strain showed 246 mU/g-wet cell of phosphoric acid swollen cellulose (PASC) degradation activity, which corresponded to 2.9-fold higher activity than that of a cellulase-expressing strain. This result clearly demonstrated that yeast cell-surface expressed cellulase and expansin-like protein act synergistically to breakdown cellulose. In fermentation experiments examining direct ethanol production from PASC, the cellulase and expansin-like protein co-expressing strain produced 3.4 g/L ethanol after 96 h of fermentation, a concentration that was 1.4-fold higher than that achieved by the cellulase-expressing strain (2.5 g/L). Conclusions The PASC degradation and fermentation ability of an engineered yeast strain was markedly improved by co-expressing cellulase and expansin-like protein on the cell surface. To our knowledge, this is the first report to demonstrate the synergetic effect of co-expressing cellulase and expansin-like protein on a yeast cell surface, which may be a promising strategy for constructing direct ethanol fermenting yeast from cellulose.

A cellulose hydrolysate from Aspen wood, containing mainly glucose, was fermented into ethanol by a thermotolerant strain MSN77 of Zymomonas mobilis. The effect of the hydrolysate concentration on fermentation parameters was investigated. Growth parameters (specific growth rate and biomass yield) were inhibited at high hydrolysate concentrations. Catabolic parameters (specific glucose uptake rate, specific ethanol productivity and ethanol yield) were not affected. These effects could be explained by the increase in medium osmolality. The results are similar to those described for molasses based media. Strain MSN77 could efficiently ferment glucose from Aspen wood up to a concentration of 60 g/l. At higher concentration, growth was inhibited.

Growth and ethanol production by three strains (MSN77, thermotolerant, SBE15, osmotolerant and wild type ZM4) of the bacterium Zymomonas mobilis were tested in a rich medium containing the hexose fraction from a cellulose hydrolysate (Aspen wood). The variations of yield and kinetic parameters with fermentation time revealed an inhibition of growth by the ethanol produced. This inhibition may result from the increase in medium osmolality due to ethanol formation from glucose.

Treatment with dilute sulfuric acid (H2SO4) or calcium hydroxide (Ca(OH)2) at 121C and 103.4 kPa was used to improve the efficiency of the cellulose digestion of purple guinea grass. Cellulase hydrolysis of the dilute H2SO4-pretreated purple guinea grass under optimized conditions (6% (w/v) in 3% (w/v) H2SO4 for 30 min) yielded a slightly higher level of reducing sugars than that from the Ca(OH)2 pretreatment under optimized conditions (6% (w/v) in 4% (w/v) Ca(OH)2 for 5 min). However, the level of glucose released from the Ca(OH)2-pretreated purple guinea grass was slightly higher than that from the dilute H2SO4 pretreatment. Ethanol fermentation, via the separate hydrolysis and fermentation (SHF) process using Saccharomyces cerevisiae, of the Ca(OH)2-pretreated purple guinea grass and then hydrolyzed with commercial cellulase (9 PFU/g, dry wt.) for 6 h yielded ethanol at 0.44 g/g glucose (0.21 g/g cellulose) within 48 h, while that from the simultaneous saccharification and fermentation process yielded 14.3% less ethanol at 0.18 g/g cellulose within 96 h (including the 6 h saccharification time). The ethanol yield from the SHF process increased 1.14-fold to 0.497 g/g glucose (0.24 g/g cellulose) when the fermentation was performed in a 5 L fermentor.

não consta número Ethanol can be produced from cellulosic biomass in a process known as simultaneous saccharification and fermentation (SSF). The presence of yeast together with the cellulolytic enzyme complex reduces the accumulation of sugars within the reactor, increasing the ethanol yield and saccharification rate. This paper reports the isolation of Saccharomyces cerevisiae LBM-1, a strain capable of growth at 42 °C. In addition, S. cerevisiae LBM-1 and Kluyveromyces marxianus UFV-3 were able to ferment sugar cane bagasse in SSF processes at 37 and 42 °C. Higher ethanol yields were observed when fermentation was initiated after presaccharification at 50 °C than at 37 or 42 °C. Furthermore, the volumetric productivity of fermentation increased with presaccharification time, from 0.43 g/L/h at 0 h to 1.79 g/L/h after 72 h of presaccharification. The results suggest that the use of thermotolerant yeasts and a presaccharification stage are key toincreasing yields in this process.

Reed is a widespread-growing, inexpensive, and readily available lignocellulosic material source in northeast China. The objective of this study is to evaluate the liquid hot water (LHW) pretreatment efficiency of reed based on the enzymatic digestibility and ethanol fermentability of water-insoluble solids (WISs) from reed after the LHW pretreatment. Several variables in the LHW pretreatment and enzymatic hydrolysis process were optimized. The conversion of glucan to glucose and glucose concentrations are considered as response variables in different conditions. The optimum conditions for the LHW pretreatment of reed area temperature of 180°C for 20min and a solid-to-liquid ratio of 1 : 10. These optimum conditions for the LHW pretreatment of reed resulted in a cellulose conversion rate of 82.59% in the subsequent enzymatic hydrolysis at 50°C for 72 h with a cellulase loading of 30 filter paper unit per gram of oven-dried WIS. Increasing the pretreatment temperature resulted in a higher enzymatic digestibility of the WIS from reed. Separate hydrolysis and fermentation of WIS showed that the conversion of glucan to ethanol reached 99.5% of the theoretical yield. The LHW pretreatment of reed is a suitable method to acquire a high recovery of fermentable sugars and high ethanol conversion yield.

Dilute sulfuric acid pretreatment of the Jatropha curcas fruit hull at high temperatures (140°C to 180°C) performed in a 110-mL stainless steel reactor was investigated to enhance the enzymatic digestibility of its lignocellulosic components. Carbohydrates accounted for 43% of the dry matter of the J. curcas fruit hull biomass. The goal of the study was to optimize the pretreatment conditions (acid concentration, time, and temperature) in order to obtain the highest sugar yield after subsequent enzymatic hydrolysis. A Box-Behnken design was applied to the experimental setup in order to reduce the number of experiments. The optimal pretreatment conditions are 30-min incubations at a temperature of 178°C with a sulfuric acid concentration of 0.9% (w/v). Using these pretreatment conditions for a fruit solid loading of 9.52% followed by a 24-h enzymatic hydrolysis resulted in a liberation of 100% of all pentoses present (71% yield and 29% degradation to furfural) and 83% of the hexoses (78% yield and 5% degradation to 5-hydroxymethylfurfural). The simultaneous saccharification and fermentation experiment showed that acid-pretreated fruit hull can be used as a substrate for Saccharomyces cerevisiae to produce ethanol.

Consolidated bioprocessing represents an attractive approach to converting cellulosic materials into bioethanol, yet is practically unavailable. We developed a ventilation-mediated, simultaneous ethanol fermentation and recovery system. Running the system under air-supplied conditions, apparently pure ethanol (28g) was recovered from cellobiose (100g) by growing recombinant Kluyveromyces marxianus expressing β-glucosidase.

Growth and ethanol production by three strains of the bacterium Zymomonas mobilis were tested, at 40°C, in a medium containing cellulose hydrolysate (hexose fraction) as carbon source. The thermotolerant mutant C107 exhibited the best growth compared to wild type ZM4 and to the osmotolerant mutant SBE15. When cultivated in media supplemented with various nutrients, growth was only observed in presence of yeast extract (10 g/l) which acted both as a vitamin supplier and pH stabilizer. Using calcium pantothenate instead of yeast extract and sodium acetate to control pH resulted in growth inhibition by the high medium osmolality. Batch fermentation with pH control (KOH addition) showed good growth and ethanol production with the mineral medium.

The effectiveness of ball milling (BM) and wet disk milling (WDM) on treating sugarcane bagasse and straw were compared. Pretreated materials were characterized by wide angle X-ray diffraction analysis, particle-size distribution and scanning electron microscopy and the effectiveness of pretreatments was evaluated by enzymatic hydrolysis and fermentation. Glucose and xylose hydrolysis yields at optimum conditions for BM-treated bagasse and straw were 78.7% and 72.1% and 77.6% and 56.8%, respectively. Maximum glucose and xylose yields for bagasse and straw using WDM were 49.3% and 36.7% and 68.0% and 44.9%, respectively. BM improved the enzymatic hydrolysis by decreasing the crystallinity, while the defibrillation effect observed for WDM samples seems to have favored enzymatic conversion. Bagasse and straw BM hydrolysates were fermented by Saccharomyces cerevisiae strains. Ethanol yields from total fermentable sugars using a C6-fermenting strain reached 89.8% and 91.8% for bagasse and straw hydrolysates, respectively, and 82% and 78% when using a C6/C5 fermenting strain.

Lignocellosic ethanol production is now at a stage where commercial or semi-commercial plants are coming online and, provided cost effective production can be achieved, lignocellulosic ethanol will become an important part of the world bio economy. However, challenges are still to be overcome throughout the process and particularly for the fermentation of the complex sugar mixtures resulting from the hydrolysis of hemicellulose. Here we describe the continuous fermentation of glucose, xylose and arabinose from non-detoxified pretreated wheat straw, birch, corn cob, sugar cane bagasse, cardboard, mixed bio waste, oil palm empty fruit bunch and frond, sugar cane syrup and sugar cane molasses using the anaerobic, thermophilic bacterium Thermoanaerobacter Pentocrobe 411. All fermentations resulted in close to maximum theoretical ethanol yields of 0.47-0.49 g/g (based on glucose, xylose, and arabinose), volumetric ethanol productivities of 1.2-2.7 g/L/h and a total sugar conversion of 90-99% including glucose, xylose and arabinose. The results solidify the potential of Thermoanaerobacter strains as candidates for lignocellulose bioconversion.

Fuel ethanol production from plant biomass hydrolysates by Saccharomyces cerevisiae is of great economic and environmental significance. This paper reviews the current status with respect to alcoholic fermentation of the main plant biomass-derived monosaccharides by this yeast. Wild-type S. cerevisiae strains readily ferment glucose, mannose and fructose via the Embden-Meyerhof pathway of glycolysis, while galactose is fermented via the Leloir pathway. Construction of yeast strains that efficiently convert other potentially fermentable substrates in plant biomass hydrolysates into ethanol is a major challenge in metabolic engineering. The most abundant of these compounds is xylose. Recent metabolic and evolutionary engineering studies on S. cerevisiae strains that express a fungal xylose isomerase have enabled the rapid and efficient anaerobic fermentation of this pentose. L: -Arabinose fermentation, based on the expression of a prokaryotic pathway in S. cerevisiae, has also been established, but needs further optimization before it can be considered for industrial implementation. In addition to these already investigated strategies, possible approaches for metabolic engineering of galacturonic acid and rhamnose fermentation by S. cerevisiae are discussed. An emerging and major challenge is to achieve the rapid transition from proof-of-principle experiments under 'academic' conditions (synthetic media, single substrates or simple substrate mixtures, absence of toxic inhibitors) towards efficient conversion of complex industrial substrate mixtures that contain synergistically acting inhibitors.

The effect of the non-ionic surfactants on the ethanol fermentation was greatly dependent on the surfactant added. While Tween 20 and Tween 80 slightly enhanced ethanol fermentation, Triton X-100 which exhibited the inghest increase in the enzymatic saccharification had a negative effect on the ethanol fermentation. The negative effect of Triton X-100 on ethanol production was the most pronounced when the cellulosic hydrolyzates were used. Tween 80 showed the best performance for the ethanol production from steam exploded wood hydrolyzate.

Cassava pulp, a solid by-product from starch processing, is a promising and underused biomass that can be converted to biofuels and other value-added bio-products. In this study, an alternative cassava pulp saccharification process, which utilizes the multi-activity enzyme from Aspergillus niger BCC17849 and obviates the need for a pre-gelatinization step, was developed. The crude multi-enzyme composed of non-starch polysaccharide hydrolyzing enzyme activities, including cellulase, pectinase and hemicellulase act cooperatively to release the trapped starch granules from the fibrous cell wall structure for subsequent saccharification by raw starch degrading activity. A high yield of fermentable sugars, equivalent to 716 mg glucose and 67 mg xylose/g of cassava pulp, was obtained after 48 h incubation at 40 degrees C and pH 5 using the multi-enzyme, which was greater than the yield obtained from the optimized combinations of the corresponding commercial enzymes. The multi-enzyme saccharification reaction can be performed simultaneously with the ethanol fermentation process using a thermotolerant yeast Candida tropicalis BCC7755. The combined process produced 14.3 g/l ethanol from 4% (w/v) cassava pulp after 30 h of fermentation. The productivity rate of 0.48 g/l/h is equivalent to 93.7% of the theoretical yield based on total starch and cellulose, or 85.4% based on total fermentable sugars. The non-thermal enzymatic saccharification process described is more energy efficient and yields more fermentable sugar than the conventional enzymatic process. Furthermore, the process is applicable for production of various bio-products of economic importance.

The reducing sugars, glucose, and ethanol produced during growth of the anaerobes Clostridium thermocellum and Acetivibrio cellulolyticus on cellulose were assayed. Zymomonas mobilis was grown under similar conditions and could ferment glucose to ethanol. The ethanol production by the cellulolytic bacteria alone and in co-culture with Zymomonas is described. Approximately 27% of a 1% cellulose substrate could be converted to ethanol by this co-culture.

Ethanol fermentation studies were conducted with Saccharomyces cerevisiae ATCC "4126, to determine the optimal conditions of oxygen tension and feed sugar concentration. In long-term continuous culture maximum ethanol production was found to occur at 0.07 mmHg oxygen tension and 10% glucose feed concentration. Preliminary process design and cost studies are developed for industrial scale fermentations to produce ethanol and torula yeast from sugars obtained by enzymatic hydrolysis of newsprint.