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.     

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.     

Enhanced bio-hydrogen production from cornstalk hydrolysate pretreated by alkaline-enzymolysis with orthogonal design method

Bio-hydrogen (H₂) production from renewable biomass has been accepted as a promising method to produce an alternative fuel for the future. In this study, fermentative hydrogen production from cornstalk (CS) hydrolysate pretreated by alkaline-enzymolysis method was investigated. Meanwhile, a five-factor and five-level orthogonal experimental array was designed to study the influences of Ca(OH)₂ concentration, alkaline hydrolysis time, alkaline hydrolysis temperature, cellulase and xylanase dosages on cornstalk pretreatment and hydrogen production. A maximum reducing sugar yield of 0.59 g/g-CS was obtained at Ca(OH)₂ 0.5%, hydrolysis temperature 115 °C, hydrolysis time 1.5 h, cellulase dosage 4000 U/g-CS and xylanase 4000 U/g-CS. Under this same condition, the maximum hydrogen yields of 168.9 mL/g-CS, 357.6 mL/g-CS, and 424.3 mL/g-CS were obtained at dark-fermentation, photo-fermentation, and two-stage fermentation respectively. It's also found that the significance of these five parameters on H₂ production followed from high to low order as: Ca(OH)₂ concentration, cellulase dosage, xylanase dosage, hydrolysis time, and hydrolysis temperature. By comparing the energy produced with the energy spent, the maximum Energy Sustainability Index (ESI) value of 1.11 was obtained at the two-stage fermentation. The results suggested that two-stage fermentation is a promising and efficient way for hydrogen production from lignocellulosic biomass.     

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.     

Comparative study on ethanol production from pretreated sugarcane bagasse using immobilized Saccharomyces cerevisiae on various matrices

Microwave alkali pretreated sugarcane bagasse was used as a substrate for production of cellulolytic enzymes, needed for biomass hydrolysis. The pretreated sugarcane bagasse was enzymatic hydrolyzed by crude unprocessed enzymes cellulase (Filter paper activity 9.4 FPU/g), endoglucanase (carboxymethylcellulase, 148 IU/g), β-glucosidase (116 IU/g) and xylanase (201 IU/g) produced by Aspergillus flavus using pretreated sugarcane bagasse as substrate under solid state fermentation. Concentrated enzymatic hydrolyzate was used for ethanol production using Saccharomyces cerevisiae immobilized on various matrices. The yield of ethanol was 0.44 g_p/g_s in case of yeast immobilized sugarcane bagasse, 0.38 g_p/g_s using Ca-alginate and 0.33 g_p/g_s using agar-agar as immobilization matrices. The immobilized yeast studied up to 10 cycles in case of immobilized sugarcane bagasse and up to 4 cycles in case of agar-agar and calcium alginate for ethanol production under repeated batch fermentation study.     

High-solid dark fermentation of cassava pulp and cassava processing wastewater for hydrogen production

Dark fermentation (DF) is a promising biological process for hydrogen production from biomass. However, low hydrogen yield (HY) is a major hurdle impeding its use at large-scale operation. A potential way to mitigate this problem is to increase the concentration of substrate in the process to increase hydrogen production. The present study investigated the possibility of using high-solid DF to produce hydrogen from cassava processing wastes, i.e., cassava pulp (CP) and cassava processing wastewater (CPW). CP was suspended in CPW and hydrolyzed enzymatically under optimum conditions of 150 g-CP/L, 29 U/g of α-amylase, 47 U/g of glucoamylase, and 60 FPU/g of cellulase. The hydrolysis performed at 50 °C for 24 h yielded a reducing sugar concentration of 117.7 ± 1.8 g/L, equivalent to 0.78 g-reducing-sugar/g-CP. Subsequent DF of CP-CPW enzymatic slurry, which contained 12.1% water insoluble solids, resulted in a cumulative production of 13.72 ± 0.22 L-H₂, equivalent to 225.2 ± 3.7 mL-H₂/g-VS. This was 83.1% of a maximum stoichiometric HY, based on carbohydrate content of CP and soluble metabolites production. The present study shows clearly the applicability of high-solid DF in the production of hydrogen from cassava processing wastes.     

Biological hydrogen production from corn stover by moderately thermophile Thermoanaerobacterium thermosaccharolyticum W16

This study addressed the utilization of an agro-waste, corn stover, as a renewable lignocellulosic feedstock for the fermentative H₂ production by the moderate thermophile Thermoanaerobacterium thermosaccharolyticum W16. The corn stover was first hydrolyzed by cellulase with supplementation of xylanase after delignification with 2% NaOH. It produced reducing sugar at a yield of 11.2 g L⁻¹ glucose, 3.4 g L⁻¹ xylose and 0.5 g L⁻¹ arabinose under the optimum condition of cellulase dosage 25 U g⁻¹ substrate with supplement xylanase 30 U g⁻¹ substrate. The hydrolyzed corn stover was sequentially introduced to fermentation by strain W16, where, the cell density and the maximum H₂ production rate was comparable to that on simulated medium, which has the same concentration of reducing sugars with hydrolysate. The present results suggest a promising combined hydrogen production process from corn stover with enzymatic hydrolysis stage and fermentation stage using W16.     

Application of low-intensity pulsed ultrasound to increase bio-ethanol production

We explored the application of Low-Intensity Pulsed Ultrasound (LIPUS) technology to improve the metabolic activity of microorganisms. In this study we showed that LIPUS improves bio-ethanol production from lignocellulosic biomass. We determined specific LIPUS conditions to increase the metabolic activity of both the cellulose degrading fungus, Trichoderma reesei Rut C-30 and the ethanol producing yeast, Saccharomyces cerevisiae. LIPUS conditions of 1.5 MHz, 20% duty cycle, 80 mW/cm² intensity, 5 min exposure and 12 exposures per day were found to improve the activity of the organisms the most. These LIPUS treatment conditions increased cellulase production by T. reesei by 16 ± 6%. The same LIPUS treatment conditions induced a 31 ± 10% increase in ethanol production by S. cerevisiae which implies a cumulative improvement of 52 ± 16% in lignocellulosic bio-ethanol production with LIPUS. This observation shows a new potential for LIPUS in the improvement of lignocellulosic bioethanol production to make it a sustainable energy source.     

Bioethanol production from sweet potato by co-immobilization of saccharolytic molds and Saccharomyces cerevisiae

To investigate the bioethanol production from sweet potato, the saccharification and fermentation conditions of co-immobilization of saccharolytic molds (Aspergillus oryzae and Monascus purpureus) with Saccharomyces cerevisiae were analyzed. The immobilized yeast cells showed that at 10% glucose YPD (yeast extract peptone dextrose) the maximum fermentation rate was 80.23%. Viability of yeasts cells were 95.70% at a final ethanol concentration of 6%. Immobilization enhanced the ethanol tolerance of yeast cells. In co-immobilization of S. cerevisiae with A. oryzae or M. purpureus, the optimal hardening time of gel beads was between 15 and 60 min. Bioethanol production was 3.05-3.17% (v v⁻¹) and the Y_E/s (yield of ethanol production/starch consumption) was 0.31-0.37 at pH 4, 30 °C and 150 rpm during 13 days fermentation period. Co-immobilization of S. cerevisiae with a mixed cultures of A. oryzae and M. purpureus at a ratio of 2:1, the bioethanol production was 3.84% (v v⁻¹), and the Y_E/s was 0.39 for a 11 days incubation. However a ratio of A. oryzae and M. purpureus at 1:2 resulted a bioethanol production rate of 4.08% (v v⁻¹), and a Y_E/s of 0.41 after 9 days of fermentation.     

Biobutanol production from fiber-enhanced DDGS pretreated with electrolyzed water

DDGS (distiller's dried grains with solubles) is a major co-product in dry-grind ethanol production from corn. A recently developed physical process separates DDGS into two value-added components: a fiber-enriched DDGS and a portion that is rich in oil and protein. Electrolyzed water, a new pretreatment catalyst was employed to pretreat fiber-enriched DDGS. Four temperatures (130, 145, 160, and 175 °C) and three treatment times (10, 20, and 30 min) were examined in the pretreatment with a solid loading of 20% w/w. Other pretreatment methods, such as diluted sulfuric acid, alkaline solution, and hot water, were also tested for comparison purposes. Fifteen FPU cellulase/g cellulose, 40 units β-glucosidase/g cellulose, and 50 units xylanase/g dry biomass were used in the enzymatic hydrolysis at 50 °C and 10% solid loading. The hydrolyzates were fermented by Clostridium beijerinckii BA 101 at 35 °C in an auto-controlled Six-fors fermentor with continuous mixing. The highest sugar yield was achieved when using the acidic electrolyzed water treatment at 175 °C for 10 min, with 23.25 g glucose, xylose and arabinose released from 100 g fiber-enriched DDGS. The C. beijerinckii fermentation produced 5.35 g ABE (acetone, butanol, and ethanol) from 100 g dry fiber-enhanced DDGS. This study demonstrated that DDGS pretreated with electrolyzed water and hydrolyzed with commercial enzymes could be used to produce biobutanol without detoxification.     

Simultaneous enzymatic saccharification and ABE fermentation using pretreated oil palm empty fruit bunch as substrate to produce butanol and hydrogen as biofuel

Simultaneous saccharification and acetone–ethanol–butanol (ABE) fermentation was conducted in order to reduce the number of steps involved in the conversion of lignocellulosic biomass into butanol. Enzymatic saccharification of pretreated oil palm empty fruit bunch (OPEFB) by cellulase produced 31.58 g/l of fermentable sugar. This saccharification was conducted at conditions similar to the conditions required for ABE fermentation. The simultaneous process by Clostridium acetobutylicum ATCC 824 produced 4.45 g/l of ABE with butanol concentration of 2.75 g/l. The butanol yield of 0.11 g/g and ABE yield of 0.18 g/g were obtained from this simultaneous process as compared to the two-step process (0.10 g/g of butanol yield and 0.14 g/g of ABE yield). In addition, the simultaneous process also produced higher cumulative hydrogen (282.42 ml) than to the two-step process (222.02 ml) after 96 h of fermentation time. This study suggested that the simultaneous process has the potential to be implemented for the integrated production of butanol and hydrogen from lignocellulosic biomass.     

Pretreatment and hydrolysis of cellulosic agricultural wastes with a cellulase-producing Streptomyces for bioethanol production

Production of reducing sugar by hydrolysis of corncob material with Streptomyces sp. cellulase and ethanol fermentation of cellulosic hydrolysate was investigated. Cultures of Streptomyces sp. T3-1 improved reducing sugar yields with the production of CMCase, Avicelase and ??-glucosidase activity of 3.8, 3.9 and 3.8 IU/ml, respectively. CMCase, Avicelase, and ??-glucosidase produced by the Streptomyces sp. T3-1 favored the conversion of cellulose to glucose. It was recognized that the synergistic interaction of endoglucanase, exoglucanase and ??-glucosidase resulted in efficient hydrolysis of cellulosic substrate. After 5 d of incubation, the overall reducing sugar yield reached 53.1 g/100 g dried substrate. Further fermentation of cellulosic hydrolysate containing 40.5 g/l glucose was performed using Saccharomyces cerevisiae BCRC 21812, 14.6 g/l biomass and 24.6 g/l ethanol was obtained within 3 d. The results have significant implications and future applications regarding to production of fuel ethanol from agricultural cellulosic waste.     

Ethanol production by Mucor indicus and Rhizopus oryzae from rice straw by separate hydrolysis and fermentation

Rice straw was successfully converted to ethanol by separate enzymatic hydrolysis and fermentation by Mucor indicus, Rhizopus oryzae, and Saccharomyces cerevisiae. The hydrolysis temperature and pH of commercial cellulase and β-glucosidase enzymes were first investigated and their best performance obtained at 45 °C and pH 5.0. The pretreatment of the straw with dilute-acid hydrolysis resulted in 0.72 g g^?1 sugar yield during 48 h enzymatic hydrolysis, which was higher than steam-pretreated (0.60 g g^?1) and untreated straw (0.46 g g^?1). Furthermore, increasing the concentration of the dilute-acid pretreated straw from 20 to 50 and 100 g L^?1 resulted in 13% and 16% lower sugar yield, respectively. Anaerobic cultivation of the hydrolyzates with M. indicus resulted in 0.36–0.43 g g^?1 ethanol, 0.11–0.17 g g^?1 biomass, and 0.04–0.06 g g^?1 glycerol, which is comparable with the corresponding yields by S. cerevisiae (0.37–0.45 g g^?1 ethanol, 0.04–0.10 g g^?1 biomass and 0.05–0.07 glycerol). These two fungi produced no other major metabolite from the straw and completed the cultivation in less than 25 h. However, R. oryzae produced lactic acid as the major by-product with yield of 0.05–0.09 g g^?1. This fungus had ethanol, biomass and glycerol yields of 0.33–0.41, 0.06–0.12, and 0.03–0.04 g g^?1, respectively.     

Production and optimization of high grade cellulase from waste date seeds by Cellulomonas uda NCIM 2353 for biohydrogen production

Production of high grade cellulolytic enzymes from waste agricultural biomass would valorise these wastes to valuable products as well as avoid the pollution problems associated with landfilling of the biomass. In the present study, waste date palm (Phoenix dactylifera) seeds were valorised for cellulase production from Cellulomonas uda NCIM 2353 and for its subsequent usage in biohydrogen production. Optimization of key operational parameters such as date seed concentration, xylose, casein and initial media pH were performed using central composite design to obtain the maximum enzyme yield. The optimum values obtained were (g/L): date seed concentration 30.65, xylose concentration 0.55, casein 7.00 and pH 7.40 for a determination coefficient of 0.999. The results demonstrated a higher prediction accuracy of response surface methodology as the cellulase activity increased six fold (175.96 IU/mL) after optimization. The optimum pH and temperature of purified cellulase was 7 and 50 °C respectively where the enzyme retained nearly 80% of activity upto 180 min. Enzymatic hydrolysis studies showed that a high saccharification efficiency of 60.5% was obtained for acid pretreated sugarcane bagasse by the indigenous cellulase, equivalent to the performance of commercial cellulase. Further, the as-obtained reducing sugars were decomposed by Clostridium thermocellum to produce biohydrogen of maximum concentration 187.44 mmol/L at end of 24 h of fermentation. Results show that date seed substrate based cellulase protein can be employed for industrial processes of biohydrogen production.     

Biological saccharification by Clostridium thermocellum and two-stage hydrogen and methane production from hydrogen peroxide-acetic acid pretreated sugarcane bagasse

The present work aimed at establishing an efficient degradation and energy recovery system form sugarcane bagasse (SCB) through hydrogen peroxide-acetic acid (HPAC) pretreatment, thermophilic hydrogen production and mesophilic methane production. The degradation ratio of HPAC pretreated SCB (HPAC-SCB, 2%, w/v) exceeded 90% under the biological hydrolysis of C. thermocellum without enzyme addition. The hydrogen yield in the co-culture fermentation of T. thermosaccharolyticum and C. thermocellum from HPAC-SCB (2%, w/v) reached 226 mL/g substrate. The long-term hydrogen fermentation was successfully established with 1.59 L/(L·d), 0.159 L/g substrate for average hydrogen productivity and yield, respectively. Methane production of 0.341 L/g COD (chemical oxygen demand)_added was recovered by semi-continuous methane fermentation from hydrogen-producing effluent at 12 days of hydraulic retention time (HRT). Average energy recovery of 8.79 MJ/kg SCB was obtained under the optimal conditions. The present work indicated the promising application of the established process in valorization of lignocellulosic bio-waste.     

Feasibility of reed for biobutanol production hydrolyzed by crude cellulase

The aim of this study was to efficiently utilize reed for both cellulase and biobutanol production. The unprocessed cellulase blend produced under solid-state fermentation using reed as the substrate showed a similar reducing sugar yield using Whatman filter paper to the commercial enzyme blend (38.61%). Organosolv pretreatment method could efficiently reduce hemicellulose (29.3%–14.6%) and lignin (17.2%–14.1%) content and increase cellulose content (42.5%–62.3%) from reed. Enzymatic hydrolysis of organosolv-pretreated reed using the crude cellulase with enzyme loading of 25 FPU/g reed, 20% solid content at 50 °C and pH 5.5 resulted in a reed hydrolysate containing 40.01 g/L glucose and 3.55 g/L xylose after 72 h. Fermentation of the hydrolysate medium by Clostridium acetobutylicum produced 9.07 and 14.24 g/L of biobutanol and ABE with yield of 0.21 g/g and 0.33 g/g, respectively. This study proved that crude cellulase complex produced under solid state fermentation and organsolv pretreatment can efficiently provide reed hydrolysate that can be converted to biobutanol without any commercial cellulase usage.     

Enzymatic saccharification of pretreated rice straw by cellulase produced from Bacillus carboniphilus CAS 3 utilizing lignocellulosic wastes through statistical optimization

A marine bacterium, Bacillus carboniphilus CAS 3 was subjected to optimization for cellulase production utilizing cellulosic waste through response surface methodology. Plackett – Burman and Central composite design was employed and the optimal medium constituents for maximum cellulase production (4040.45 U/mL) were determined as rice bran, yeast extract, MgSO₄·7H₂O and KH₂PO₄ at 6.27, 2.52, 0.57 and 0.39 g/L, respectively. The cellulase produced was purified to the specific activity of 434.94 U/mg and 11.46% of recovery with the molecular weight of 56 kDa. The optimum temperature, pH and NaCl for enzyme activity was determined as 50 °C, 9 and 30% and more than 70% of its original activity was retained even at 80 °C, 12 and 35% respectively. Further, enzymatic saccharification of pretreated rice straw yielded about 15.56 g/L of reducing sugar at 96 h, suggesting that the purified cellulase could be useful for production of reducing sugars from cellulosic biomass into ethanol.     

Enzymatic hydrolysis and fermentation of crushed whole sugar beets

Pectinase and cellulase enzymes were used for hydrolysis of whole sugar beets and the hydrolyzates were fermented with Escherichia coli KO11 and Saccharomyces cerevisiae via simultaneous saccharification and fermentation (SSF). Ethanol production rate was significantly higher for S. cerevisiae than for E. coli KO11. The combined effect of pectinase and cellulase loadings on ethanol production as well as residual galacturonic acid and arabinose concentrations were modeled for fermentations with S. cerevisiae. Ethanol yields of more than 92% were reached with moderate to high cellulase and pectinase loadings at 0.51 FPU g⁻¹ and 51 U g⁻¹ of dry biomass, respectively. Ethanol yields of 85% were achieved without any enzyme addition. However, addition of cellulase and pectinase enzymes increased effluent arabinose and galacturonic acid concentrations and reduced total suspended solids. This study demonstrated the yield potential of fermentation of crushed, whole sugar beets with or without the addition of cellulase and pectinase enzymes.     

The preparation and application of crude cellulase for cellulose-hydrogen production by anaerobic fermentation

Strategies were adopted to cost-efficiently produce cellulose-hydrogen by anaerobic fermentation in this paper. First, cellulase used for hydrolyzing cellulose was prepared by solid-state fermentation (SSF) on cheap biomass from Trichoderma viride. Several cultural conditions for cellulase production on cheap biomass such as moisture content, inoculum size and culture time were studied. And the components of solid-state medium were optimized using statistical methods to further improve cellulase capability. Second, the crude cellulase was applied to cellulose-hydrogen process directly. The maximal hydrogen yield of 122 ml/g-TVS was obtained at the substrate concentration of 20 g/L and cultured time of 53 h. The value was about 45-fold than that of raw corn stalk wastes. The hydrogen content in the biogas was 44–57%(v/v) and there was no significant methane gas observed.     

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.