Cellulase production using biomass feed stock and its application in lignocellulose saccharification for bio-ethanol production

A major constraint in the enzymatic saccharification of biomass for ethanol production is the cost of cellulase enzymes. Production cost of cellulases may be brought down by multifaceted approaches which include the use of cheap lignocellulosic substrates for fermentation production of the enzyme, and the use of cost efficient fermentation strategies like solid state fermentation (SSF). In the present study, cellulolytic enzymes for biomass hydrolysis were produced using solid state fermentation on wheat bran as substrate. Crude cellulase and a relatively glucose tolerant BGL were produced using fungi Trichoderma reesei RUT C30 and Aspergillus niger MTCC 7956, respectively. Saccharification of three different feed stock, i.e. sugar cane bagasse, rice straw and water hyacinth biomass was studied using the enzymes. Saccharification was performed with 50 FPU of cellulase and 10 U of β-glucosidase per gram of pretreated biomass. Highest yield of reducing sugars (26.3 g/L) was obtained from rice straw followed by sugar cane bagasse (17.79 g/L). The enzymatic hydrolysate of rice straw was used as substrate for ethanol production by Saccharomyces cerevisiae. The yield of ethanol was 0.093 g per gram of pretreated rice straw.     

Ethanol production from food residues

Food residues were converted to ethanol by simultaneous saccharification with an amylolytic enzyme complex (a mixture of amyloglucosidase, ??-amylase, and protease), and fermentation (SSF) with the yeast, Saccharomyces cerevisiae. About 36 g dm⁻³ of ethanol was obtained from 100 g dm⁻³ food residue in 48 h of fermentation. In the SSF with no nitrogen supplements, 25 g dm⁻³ of ethanol was produced from 100 g dm⁻³ food residues. In addition, none of the nutrient components except yeast extract from the SSF medium were found to affect ethanol production from food residues. This result indicates that food residues could be a good economic bioresource for ethanol production.     

Effect of nutrient supplementation on ethanol production in different strategies of saccharification and fermentation from acid pretreated rice straw

The effect of nutrient supplementation on ethanol production by recently selected thermotolerant yeast (Kluyveromyces marxianus NRRL Y-6860) was investigated in different strategies of saccharification and fermentation employing rice straw pretreated by dilute acid. Among the evaluated strategies, similar ethanol yields (Y_P/S ∼ 0.23 g g⁻¹) were obtained with or without nutrient addition. However, considering the whole process time, the strategy based on simultaneous saccharification and fermentation (SSF), without pre-hydrolysis, was assigned as the most suitable configuration due to the highest ethanol volumetric productivity (1.4 g L⁻¹ h⁻¹), about 2-fold higher in relation to the others. The impact of enzymatic preparation employed in this study was also evaluated on glucose fermentation in semi-synthetic medium. The enzymatic preparation affected both glucose consumption and ethanol production by K. marxianus NRRL Y-6860, but just in the absence of nutrients. Therefore, the enzyme type and loading should be carefully defined, not only by the capital costs involved, but also by the possibility of increasing the fermentation inhibitors.     

Bioethanol production from grape and sugar beet pomaces by solid-state fermentation

A suitable alternative to replace fossil fuels is the production of bioethanol from agroindustrial waste. Grape pomace is the most abundant residue in San Juan and sugar beet pomace could be important in the region. Solid-State Fermentation (SSF) is a technology that allows transforming agroindustrial waste into many valuable bioproducts, like ethanol. This work reports a laboratory scale SSF to obtain alcohol from grape and sugar beet pomace by means of Saccharomyces cerevisiae yeasts. The initial conditions of the culture medium were: sugars 16.5% (p/p); pH 4.5; humidity 68% (p/p). Cultures were inoculated with 10⁸ cells/g of pomace, and incubated in anaerobic environment, at 28 °C, during 96 h. SSF showed ethanol maximum concentrations at 48 h and ethanol yield on sugars consumed was more than 82%. Yield attained creates expectation about the use of SSF to obtain fuel alcohol.     

Arrowroot as a novel substrate for ethanol production by solid state simultaneous saccharification and fermentation

Ethanol production from Canna edulis Ker was successfully carried out by solid state simultaneous saccharification and fermentation. The enzymatic hydrolysis conditions of C. edulis were optimized by Plackett–Burman design. The effect of inert carrier (corncob and rice bran) on ethanol fermentation and the kinetics of solid state simultaneous saccharification and fermentation was investigated. It was found that C. edulis was an alternative substrate for ethanol production, 10.1% (v/v) of ethanol concentration can attained when 40 g corncob and 10 g rice bran per 100 g C. edulis powder were added for ethanol fermentation. No shortage of fermentable sugars was observed during solid state simultaneous saccharification and fermentation. There was no wastewater produced in the process of ethanol production from C. edulis with solid state simultaneous saccharification and fermentation and the ethanol yield of more than 0.28 tonne per one tonne feedstock was achieved. This is first report for ethanol production from C. edulis powder.     

Bioethanol production from pretreated Miscanthus floridulus biomass by simultaneous saccharification and fermentation

The production of ethanol from the fast-growing perennial C4 grass Miscanthus floridulus by simultaneous saccharification and fermentation (SSF) was investigated. M. floridulus biomass was composed of 36.3% glucan, 22.8% hemicellulose, and 21.3% lignin (based on dried mass). Prior to SSF, harvested stems of M. floridulus were pretreated separately by alkali treatment at room temperature, alkali treatment at 90 °C, steam explosion, and acid-catalyzed steam explosion. The delignification rates were determined to be 73.7%, 61.5%, 42.7%, and 63.5%, respectively, by these four methods, and the hemicellulose removal rates were 51.5%, 85.1%, 70.5%, and 97.3%, respectively. SSF of residual solids after various pretreatments was performed with dried yeast (Saccharomyces cerevisiae) and cellulases (Accellerase 1000) by using 10% water-insoluble solids (WIS) of the pretreated M. floridulus as the substrate. The ethanol yields from 72-h SSF of M. floridulus biomass after these pretreatments were 48.9 ± 3.5, 78.4 ± 1.0, 46.4 ± 0.1, and 69.0 ± 0.1% (w/w), respectively, while the ethanol concentrations after 72-h SSF were determined to be 15.4 ± 1.1, 27.5 ± 0.3, 13.9 ± 0.1, and 30.8 ± 0.1 g/L, respectively. Overall, the highest amount of ethanol (0.124 g/g-dried raw material) was generated from dried raw material of M. floridulus after alkaline pretreatment at 90 °C. The acid-catalyzed steam explosion pretreatment also resulted in a high ethanol yield (0.122 g/g-dried raw material). Pretreatment resulting in high lignin and hemicellulose removal rates could make biomass more accessible to enzyme hydrolysis and lead to higher ethanol production.     

Ethanol fermentation of sugar beet thick juice diluted with crude cheese whey by the flex yeast Kluyveromyces marxianus KD-15

Kluyveromyces marxianus KD-15, called flex yeast, is a strain that is insensitive to catabolite repression and has the capacity to produce ethanol efficiently from a mixture of beet molasses and whey powder. When a fermentation test was conducted in 50 mL of a medium containing 200 mg mL^?1 of sugar as sugar beet thick juice diluted with an arbitrary amount of crude whey, strain KD-15 produced over 99 mg mL^?1 ethanol in all the media tested, and ethanol formation decreased in proportion to the volume of whey by K. marxianus NBRC 1963, the parental strain of KD-15, and Saccharomyces cerevisiae NBRC 0224, the reference strain for conventional ethanol production. Fermentation of thick juice diluted with whey alone by strain KD-15 at 30 °C initially proceeded slower than that at 33 °C–37 °C but finally bore the highest level of ethanol. The maximum ethanol concentration obtained in 1.5 L of a medium using a 2-L fermentor was elevated by aeration of 15–50 mL min^?1and reduced by that in excess of 100 mL min^?1. Under optimized conditions in 72 h, strain KD-15 converted all of the sugars derived from thick juice and whey to ethanol at 102 mg mL^?1, corresponding to 92.9% of the theoretical yield.     

Valorization of Eucalyptus wood by glycerol-organosolv pretreatment within the biorefinery concept: An integrated and intensified approach

The efficient utilization of lignocellulosic biomass and the reduction of production cost are mandatory to attain a cost-effective lignocellulose-to-ethanol process. The selection of suitable pretreatment that allows an effective fractionation of biomass and the use of pretreated material at high-solid loadings on saccharification and fermentation (SSF) processes are considered promising strategies for that purpose. Eucalyptus globulus wood was fractionated by organosolv process at 200 °C for 69 min using 56% of glycerol-water. A 99% of cellulose remained in pretreated biomass and 65% of lignin was solubilized. Precipitated lignin was characterized for chemical composition and thermal behavior, showing similar features to commercial lignin. In order to produce lignocellulosic ethanol at high-gravity, a full factory design was carried to assess the liquid to solid ratio (3–9 g/g) and enzyme to solid ratio (8–16 FPU/g) on SSF of delignified Eucalyptus. High ethanol concentration (94 g/L) corresponding to 77% of conversion at 16FPU/g and LSR = 3 g/g using an industrial and thermotolerant Saccharomyces cerevisiae strain was successfully produced from pretreated biomass. Process integration of a suitable pretreatment, which allows for whole biomass valorization, with intensified saccharification-fermentation stages was shown to be feasible strategy for the co-production of high ethanol titers, oligosaccharides and lignin paving the way for cost-effective Eucalyptus biorefinery.     

Sweet sorghum as a whole-crop feedstock for ethanol production

The potential of sweet sorghum as an alternative crop for ethanol production was investigated in this study. Initially, the enzymatic hydrolysis of sorghum grains was optimized, and the hydrolysate produced under optimal conditions was used for ethanol production with an industrial strain of Saccharomyces cerevisiae, resulting in an ethanol concentration of 87 g L⁻¹. From the sugary fraction (sweet sorghum juice), 72 g L⁻¹ ethanol was produced. The sweet sorghum bagasse was submitted to acid pretreatment for hemicellulose removal and hydrolysis, and a flocculant strain of Scheffersomyces stipitis was used to evaluate the fermentability of the hemicellulosic hydrolysate. This process yielded an ethanol concentration of 30 g L⁻¹ at 23 h of fermentation. After acid pretreatment, the remaining solid underwent an alkaline extraction for lignin removal. This partially delignified material, known as partially delignified lignin (PDC), was enriched with nutrients in a solid/liquid ratio of 1 g/3.33 mL and subjected to simultaneous saccharification and fermentation (SSF) process, resulting in an ethanol concentration of 85 g L⁻¹ at 21 h of fermentation. Thus, from the conversion of starchy, sugary and lignocellulosic fractions approximately 160 L ethanol.ton⁻¹ sweet sorghum was obtained. This amount corresponds to 13,600 L ethanol.ha⁻¹.     

Cellulolytic enzymes produced by a newly isolated soil fungus Penicillium sp. TG2 with potential for use in cellulosic ethanol production

A newly isolated soil fungus, Penicillium sp. TG2, had cellulase activities that were comparable to those of Trichoderma reesei RUT-C30, a common commercial strain used for cellulase production. The maximal and specific activities were 1.27 U/mL and 2.28 U/mg for endoglucanase, 0.31 U/mL and 0.56 U/mg for exoglucanase, 0.54 U/mL and 1.03 U/mg for β-glucosidase, and 0.45 U/mL and 0.81 U/mg for filter paper cellulase (FPase), respectively. Optimal FPase activity was at pH 5.0 and 50 °C. We used a simultaneous saccharification and fermentation (SSF) process, which employed the yeast Kluyveromyces marxianus and Penicillium sp. TG2 cellulolytic enzymes, to produce ethanol from empty palm fruit bunches (EFBs), a waste product from the palm oil industry. The present findings indicate that Penicillium sp. TG2 has great potential as an alternative source of enzymes for saccharification of lignocellulosic biomass.     

Simultaneous saccharification and fermentation (SSF) of very high gravity (VHG) potato mash for the production of ethanol

Simultaneous saccharification and fermentation (SSF) of very high gravity (VHG) potato mash, containing 304 g L^?1 of dissolved carbohydrates, was carried out for ethanol production. Potato tubers were ground into a mash, which was highly viscous. Mash viscosity was reduced by the pretreatment with mixed enzyme preparations of pectinase, cellulase and hemicellulase. The enzymatic pretreatment established the use of VHG mash with a suitable viscosity. Starch in the pretreated mash was liquefied to maltodextrins by the action of thermo-stable α-amylase at 85 °C. SSF of liquefied mash was performed at 30 °C with the simultaneous addition of glucoamylase, yeast (Saccharomyces cerevisiae) and ammonium sulfate as a nitrogen source for the yeast. The optimal glucoamylase loading, ammonium sulfate concentration and fermentation time were 1.65 AGU g^?1, 30.2 mM and 61.5 h, respectively, obtained using the response surface methodology (RSM). Ammonium sulfate supplementation was necessary to avoid stuck fermentation under VHG condition. Using the optimized condition, ethanol yield of 16.61% (v/v) was achieved, which was equivalent to 89.7% of the theoretical yield.     

Simultaneous saccharification and fermentation (SSF) of non-starch polysaccharides and starch from fresh tuber of Canna edulis ker at a high solid content for ethanol production

Canna edulis ker is a potential feedstock for ethanol production because of its low nutrition requirements and the high starch content of its tubers. The processing of C. edulis is limited by the high viscosity of the biomass. In this study, cell wall degrading enzymes (CWDEs) containing acid xylanase and β-glucanase were successful in reducing the viscosity (from 167.30 Pa s to 8.66 Pa s) at 50 °C for 120 min. The effect of CWDEs on simultaneous saccharification and fermentation (SSF) was investigated. Addition of CWDEs before SSF, resulted in an increase in total sugar and fermentable sugar. Meanwhile, the viscosity decreased sharply from approximately 200.00 Pa s to 2.98 Pa s, thereby improving the fermentation parameters and the mass fraction of the theoretical ethanol yield was 94.5%. Only special demand of nutritional ingredients was nitrogen, urea at 750 mg kg⁻¹ was found to be suitable for this purpose. In the verification experiments, the mass fraction of the theoretical ethanol yield in a 5 L fermentor was 98.3%. In conclusion, the pretreatment with CWDEs has significant effect on high level ethanol production using roots and tubers on an industrial scale from the biomass utilization efficiency and economic standpoint.     

Aerobic and anaerobic sequential culture fermentation (AASF) to produce bio-hydrogen from steam-exploded cornstalk

A novel aerobic and anaerobic sequential culture fermentation (AASF) method was designed to improve the conversion efficiency of steam-exploded cornstalk during bio-hydrogen production. The enzyme activities of cellulase and β-glucosidase produced by Trichoderma viride ACCC 30169 were 76.79 FPU g⁻¹ dry weight and 45.23 IU g⁻¹ dry weight after 6-days steam-exploded cornstalk fermentation, respectively. The aerobic fermentation residue was used as the substrate for bio-hydrogen production by Clostridium butyricum AS1.209 anaerobic fermentation. The optimum solid-to-liquid ratio of the anaerobic fermentation substrate was 1:5. The maximum bio-hydrogen yield was attained on the medium with addition of 0.1 g g⁻¹ substrate urea after 2 days of aerobic fermentation. Compared with simultaneous saccharification and fermentation (SSF), AASF for bio-hydrogen production could shorten the fermentation period by at least 66% and the hydrogen yield reached 83% of the total volume after 24 h of anaerobic fermentation. AASF from steam-exploded cornstalk was an effective way for bio-hydrogen production without additional commercial cellulase.     

Fuel ethanol production from steam-pretreated corn stover using SSF at higher dry matter content

Replacing fossil fuels by bio-fuels has many advantages, such as the reduction of CO₂-emission to the atmosphere, the possibility for non-oil-producing countries to be self-sufficient in fuel, and increased local job opportunities. Bio-ethanol is such a promising renewable fuel. However, today it is produced from sugar or starch—raw materials that are relatively expensive. To lower the production cost of bio-ethanol the cost of the raw material must be reduced and the production process made more efficient. The production of bio-ethanol from corn stover using simultaneous saccharification and fermentation (SSF) at high dry matter content addresses both issues. Corn stover is an agricultural by-product and thus has a low economic value. SSF at high dry matter content results in a high ethanol concentration in the fermented slurry, thereby decreasing the energy demand in the subsequent distillation step.In this study, SSF was performed on steam-pretreated corn stover at 5, 7.5 and 10% water-insoluble solids (WIS) with 2 g/L hexose-fermenting Saccharomyces cerevisiae (ordinary compressed baker's yeast). SSF at 10% WIS resulted in an ethanol yield of 74% based on the glucose content in the raw material and an ethanol concentration of 25 g/L. Neither higher yeast concentration (5 g/L) nor yeast cultivated on the liquid after the pretreatment resulted, under these conditions, in a higher overall ethanol yield.     

Effects of rice husk particle size on biohydrogen production under solid state fermentation

As a renewable energy source bio-hydrogen production from lignocellulosic wastes is a promising approach which can produce clean fuel with no CO₂ emissions. Utilization of agro-industrial residues in solid state fermentation (SSF) is offering a solution to solid wastes disposal and providing an economical process of value-added products such as hydrogen.In this study three different particle size of rice husk (<2000 μm, <300 μm, <74 μm) was subjected to batch SSF with a Clostridium termitidis: Clostridium intestinale ratio of 5:1. C. termitidis is a cellulolytic microorganism that has the ability to hydrolyze cellulosic substances and C. intestinale is able to grow on glucose having a potential of enhancing hydrogen production when used in the co-culture. 5 g dw rice husk with 75% humidity was used as substrate in SSF under mesophilic conditions. The highest HF Volume (29.26 mL) and the highest yield (5.9 mL H₂ g⁻¹ substrate) were obtained with the smallest particle size (<74 μm). The main metabolites obtained from the fermentation media were acetic, butyric, propionic and lactic acids. The second best production yield (3.99 mL H₂ g⁻¹ substrate) was obtained with the middle particle size (<300 μm) rice husk with a HF of 19.71 mL.     

Hydrolysis of pretreated rice straw by an enzyme cocktail comprising acidic xylanase from Aspergillus sp. for bioethanol production

The most crucial enzyme involved in xylan hydrolysis is endoxylanase which cleaves the internal glycosidic bonds of xylan. The aim of this work was to study the production of extracellular xylanase by a locally isolated strain of Aspergillus sp. under solid-state fermentation (SSF) and to evaluate the potential of the enzyme in enzymatic hydrolysis of pretreated rice straw. Xylanase production reached maximum with incubation period (96 h), moisture level (80%), inoculum size (3 × 10⁶ spores/mL), pH (4.8), temperature (25 °C), carbon source (wheat bran) and nitrogen source (yeast extract). Under optimized conditions, xylanase production reached to 5059 IU/gds. Crude xylanase was used for supplementing the enzyme cocktail comprising cellulases (Zytex, India), β-glucosidase (In-house) and xylanase (In-house) for the saccharification of alkali-pretreated rice straw to get the maximum reducing sugar production. The cocktail containing the three enzymes resulted a maximum of 574.8 mg/g of total reducing sugars in comparison to 430.2 mg/g sugars by the cocktail without xylanase. These results proved that the crude xylanase preparation from Aspergillus sp. could be a potent candidate for the enzyme cocktail preparation for biomass hydrolysis in lignocellulosic bioethanol program.     

The addition of hydrolyzed rice straw in xylose fermentation by Pichia stipitis to increase bioethanol production at the pilot-scale

The pretreatment of agricultural biomass by diluted acid is often employed to facilitate the release of monosaccharide for the subsequent enzyme hydrolysis for lignocellulosic ethanol production. However, furfural and hydroxymethylfurfural are usually generated and markedly decrease the yield of pentose fermentation during this pretreatment. In the present study, the enhancement of lignocellulosic ethanol production was successfully demonstrated at pilot scale with extra addition of hydrolyzed rice straw into pentose fermentation by Pichia stiptis. This way has resulted into the increase of P. stiptis cell mass was shown to play a positive role. The ethanol yield, 0.45 g_p/g_s, with the addition of hydrolyzed rice straw in hemicellulosic hydrolysate from plywood, bagasse and bamboo were increase 20–51% to demonstrate the applicability of this technology in a variety of lignocellulosic ethanol processes due to the efficient conversion of xylose.     

Coproduction of hydrogen,ethanol and 2,3-butanediol from agro-industrial residues by the Antarctic psychrophilic GA0F bacterium

In this study, the simultaneous production of hydrogen, ethanol, and 2,3-butanediol was assessed using three agro-industrial residues: cheese whey powder (CWP), wheat straw hydrolysate (WSH) and sugarcane molasses (SCM), by the Antarctic psychrophilic GA0F bacterium [EU636050], which is closely related to Pseudomonas antarctica [KX186936.1]. The main soluble metabolites produced in all the fermentations were ethanol and 2,3-butanediol. CWP demonstrated to be the most effective carbon source, since fermentation of this substrate resulted in the highest yields of H₂ (73.5 ± 10 cm³ g⁻¹), ethanol (0.24 ± 0.03 g g⁻¹) and 2,3-butanediol (0.42 ± 0.04 g g⁻¹), followed by the use of SCM, whereas WSH showed to have an inhibitory effect during the fermentation process, showing the lowest production values. Our results demonstrated the ability of the Antarctic psychrophilic GA0F bacterium to produce valuable products using low-cost substrates at room temperature conditions.     

Lignocellulosic ethanol production employing immobilized Saccharomyces cerevisiae in packed bed reactor

Most of ethanol production processes are limited by lower ethanol production rate and recyclability problem of ethanologenic organism. In the present study, immobilized co-fermenting Saccharomyces cerevisiae GSE1618 was employed for ethanol fermentation using rice straw enzymatic hydrolysate in a packed bed reactor (PBR). The immobilization of S. cerevisiae was performed by entrapment in Ca-alginate for optimization of ethanol production by varying alginic acid concentration, bead size, glucose concentration, temperature and hardening time. Remarkably, extra hardened beads (EHB) immobilized with S. cerevisiae could be used up to repeated 40 fermentation batches. In continuous PBR, maximum 81.82 g L⁻¹ ethanol was obtained with 29.95 g L⁻¹ h⁻¹ productivity with initial glucose concentration of 180 g L⁻¹ in feed at dilution rate of 0.37 h⁻¹. However, maximum ethanol concentration of 40.33 g L⁻¹ (99% yield) with 24.61 g L⁻¹ h⁻¹ productivity was attained at 0.61 h⁻¹ dilution rate in fermentation of un-detoxified rice straw enzymatic hydrolysate (REH). At commercial scale, EHB has great potential for continuous ethanol production with high productivity using lignocellulosic hydrolysate in PBR.     

Modeling semi-simultaneous saccharification and fermentation of ethanol production from cellulose

Using a highly refined standard cellulose (microcrystalline cellulose, (Avicel PH101)), the kinetics of the simultaneous saccharification and fermentation (SSF) of cellulose to ethanol was studied with a prior hydrolysis phase (semi-simultaneous saccharification and fermentation (SSSF)) conducted under optimal conditions for enzymatic hydrolysis. Four cases have been studied: 24-h pre-hydrolysis + 48-h SSF (SSSF 24), 12-h pre-hydrolysis + 60-h SSF (SSSF 12), 72-h SSF, and 48-h hydrolysis + 24-h fermentation. SSSF 24 produced higher yield and higher productivity of ethanol than the other operating modes. A coupled set of differential equations were developed to describe the change rates of cellobiose, glucose, microorganism, ethanol, glycerol, acetic acid, and lactic acid concentrations in the batch operation of separate hydrolysis and SSF of ethanol production from Avicel PH101. The model parameters were determined by a MATLAB program based on the batch experimental data of the SSSF. The analysis of the reaction rates of cellobiose, glucose, cell, and ethanol using the model showed that the conversion of cellulose to cellobiose was the rate-controlling step in the SSSF process of ethanol production from cellulose. The batch SSSF model was extended to the continuous and fed-batch operating modes. For the continuous operation in the SSSF, the productivity of SSSF 24 was much higher than that of SSSF 12 though the ethanol concentrations of both cases have not a great difference.