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.     

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.     

Conversion of paper sludge to ethanol by separate hydrolysis and fermentation (SHF) using Saccharomyces cerevisiae

The conversion of ethanol from paper sludge using the separate hydrolysis and fermentation (SHF) process with cellulase and Saccharomyces cerevisiae GIM-2 were investigated in this paper. Optimization strategy based on statistical experimental designs was employed to enhance degree of saccharification by enzymatic hydrolysis of paper sludge. Based on the Plackett-Burman design, hydrolysis time, substrate concentration and cellulase dosage were selected as the most significant variable on the degree of saccharification. Subsequently, the optimum combination of the selected factors was investigated by a Box-Behnken approach. A mathematical model was developed to show the effects of each factor and their combinatorial interactions on the degree of saccharification. The optimal conditions were hydrolysis time 82.7 h, substrate concentration 40.8 g L⁻¹ and cellulase dosage 18.1 FPU g⁻¹ substrate, and a degree of saccharification of 82.1% can be achieved. When hydrolysate was further fermented with S. cerevisiae GIM-2, the conversion rate of sugar to ethanol was 34.2% and the ethanol yield was 190 g kg⁻¹ of dry paper sludge, corresponding to an overall conversion yield of 56.3% of the available carbohydrates on the initial substrate. The results derived from this study indicate that the response surface methodology is a useful tool for optimizing the hydrolysis conditions to converse paper sludge to ethanol.     

Evaluation of fuel ethanol production from aqueous ammonia-treated rice straw via simultaneous saccharification and fermentation

Rice straw (RS) has been considered a promising feedstock for ethanol production in Asia. However, the recalcitrance of biomass, particularly the presence of lignin, hinders the enzymatic saccharification of polysaccharides in RS and consequently decreases the ethanol yield. Here, we used aqueous ammonia pretreatment to remove lignin from RS (aRS). The reaction conditions were a solid:liquid ratio of 1:12, an ammonia concentration of 27% (w w⁻¹), room temperature, and a 2-week incubation. We evaluated enzymatic digestibility and the ethanol production yield. A 42% reduction in lignin content increased the glucan conversion of aRS to glucose from 20 to 71% using a combination of Cellic Ctec2 cellulases and Cellic Htec2 xylanases at enzyme loads of 15 FPU +100 XU g⁻¹ solid. Scanning electron microscopy highlighted the extensive removal of external fibres and increased porosity of aRS, which aided the accessibility of cellulose for enzymes. Using the same enzyme dosage and a solid load of 100 g L⁻¹, simultaneous saccharification and fermentation using a monoculture of Saccharomyces cerevisiae and co-culture with Candida tropicalis yielded ethanol concentrations of 22 and 25 g L⁻¹, corresponding to fermentation efficiencies of 96 and 86% fermentation, respectively. The volumetric ethanol productivities for these systems were 0.45 and 0.52 g L⁻¹ h⁻¹. However, the ethanol yield based on the theoretical glucose and xylose concentrations was lower for the co-culture (0.44 g g⁻¹) than the monoculture (0.49 g g⁻¹) due to the low xylose consumption. Further research should optimise fermentation variables or select/improve microbial strains capable of fermenting xylose to increase the overall ethanol production yield.     

Evaluation the efficacy of extrusion pretreatment via enzymatic digestibility and simultaneous saccharification & fermentation with rapeseed straw

The efficacy of extrusion pretreatment was evaluated by enzymatic hydrolysis and simultaneous saccharification and fermentation (SSF) with straw of rapeseed, Brassica napus, an agricultural residue. An acceptable pretreatment result was obtained at a barrel temperature of 165 °C, acid concentration of 20 g L⁻¹, liquid feeding rate of 13.4 cm³ min⁻¹, solid feeding rate of 1.0 g min⁻¹, screw rotation speed of 6.3 rad s⁻¹, and residence time of 10.2 min, with a yield of xmg, representing the sum of the corresponding hydrolyzed sugars; xylose, mannose and galactose, of 794.3 g kg⁻¹ and a glucose release of 21.0 g kg⁻¹. These were calculated to be 963.0 g kg⁻¹ and 910.3 g kg⁻¹ based on cellulose and hemicellulose recoveries,respectively. The highest enzymatic digestibility of 781.0 g kg⁻¹was higher than that obtained from the batch pretreatment with dilute acid by 1.4-fold. The SSF process afforded an ethanol concentration of 16.0 g L⁻¹, corresponding to an ethanol yield of 790 g kg⁻¹ based on the total available cellulose in the pretreated rapeseed straw.     

Alkali-stable cellulase from a halophilic isolate,Gracilibacillus sp. SK1 and its application in lignocellulosic saccharification for ethanol production

A halophilic strain SK1 showing cellulolytic activity was isolated from Yuncheng Salt Lake, and was identified as the genus of Gracilibacillus by 16S rRNA gene sequence analysis. Cellulase production was strongly influenced by the salinity of culture medium with maximal level in the presence of 10% NaCl. Substrate specificity test indicated the crude cellulase was a multi-component enzyme system, showing a combined activity of endoglucanase, exoglucanase and β-glucosidase. Zymogram analysis indicated six different endoglucanases were secreted by this strain. The crude enzyme was highly active and stable over broad ranges of temperature (40–70 °C), pH (6.0–10.0) and NaCl concentration (7.5–17.5%), with an optimum at 60 °C, pH 8.0 and 12.5% NaCl, which showed excellent thermostable, alkali-stable and halostable properties. Moreover, it displayed high stability in the presence of hydrophobic organic solvents. Saccharification of corn stover and rice straw by the cellulase resulted in respective yields of 0.678 and 0.502 g g⁻¹ dry substrate of reducing sugars. The enzymatic hydrolysates of corn stover were then used as the substrate for ethanol production by Saccharomyces cerevisiae. The yield of ethanol was 0.186 g g⁻¹ dry substrate, and the efficiency of reducing sugars conversion to ethanol was about 52.8%, which suggested the prospects of the crude enzyme from Gracilibacillus sp. SK1 in application for bio-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.     

ABE fermentation from enzymatic hydrolysate of NaOH-pretreated corncobs

Acetone butanol ethanol (ABE) was produced from enzymatic-hydrolyzed corncobs by Clostridium saccharobutylicum DSM 13864. Pretreatment of corncobs was carried out with 0.5 mol L⁻¹ NaOH followed by enzymatic hydrolysis. The yield of total reducing sugars was 917 g kg⁻¹ pretreated (de-lignified) and washed corncobs. The hydrolysate was used without sediments removal for ABE fermentation. A solvent production of 19.44 g L⁻¹ with 12.27 g L⁻¹ butanol was obtained from 55.22 g L⁻¹ sugars, resulting in an ABE yield of 350 g kg⁻¹ and a production rate of 0.54 g L⁻¹ h⁻¹. A control experiment using 55.3 g L⁻¹ mixed sugars resulted in an ABE production of 16.81 g L⁻¹ with 10.26 g L⁻¹ butanol, corresponding to an ABE yield of 300 g kg⁻¹ and a production rate of 0.47 g L⁻¹ h⁻¹, indicating that the enzymatic hydrolysates may contain stimulating compounds that can improve the ABE fermentation.     

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.     

Bioethanol production from thick juice as intermediate of sugar beet processing

The aims of this study were to investigate the bioethanol production of thick juice as intermediate from sugar beet processing in batch culture by free Saccharomyces cerevisiae cells and the effect of sugar concentration on ethanol yield and CO₂ weight loss rate. Thick juice and molasses of sugar beet from a domestic sugar factory were diluted with distilled water to give a total sugar concentration of 5, 10, 15, 20 and 25% (w w^?1). Initial concentration of fermentable sugars of 20% (w w^?1) in culture medium can be taken as optimal, enabling maximal ethanol yield (68%) and maximal CO₂ evolution rate was realized, amounting to more than 90 g L^?1 h^?1. The optimal concentration of fermentable sugar from thick juice for bioethanol production by free S. cerevisiae cells was 20% (w w^?1) at 30 °C, pH 5 and agitation rate 200 rpm gave maximum ethanol concentration of 12% (v v^?1).     

Efficient ethanol production from inulin by two-stage aerate strategy

A major concern for ethanol production from inulin-containing materials, is the higher unconverted sugar, which increases the cost of ethanol production and wastewater treatment. Some key factors, such as inulinase, biomass or aeration rates, were studied to solve the problems in the process of ethanol fermentation from inulin. It was showed that more inulinase and increasing inoculum size can shorten the fermentation time, but could not reduce residual sugars. Two-stage aerate strategy was developed to utilize the remained sugars: keep the aeration at 5 h⁻¹ at the first 12 h, and drop it to 1.2 h⁻¹. Under this condition, contradiction between fermentation time and high ethanol yield was solved (60 h and 0.43 g g⁻¹), and the final residual sugar concentration decreased to about 10 g L⁻¹ with 98 g L⁻¹ ethanol. The ethanol productivity was up to 1.63 g L⁻¹ h⁻¹, which is the highest productivity of ethanol fermentations from inulin-containing materials.     

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.     

Energy self-sufficient production of bioethanol from a mixture of hemp straw and triticale seeds: Life-cycle analysis

Industrial hemp shows exceptional potential for cellulosic ethanol production, especially regarding yields per hectare, costs and environmental impact. Additionally, co-products, such as high-value food-grade oil, increase the value of this plant. In this work, hemp straw was steam-exploded for 45 min at 155 °C and hydrolysed with a cellulase/xylanase mixture. Up to 0.79 g g⁻¹ of cellulose was degraded and subsequent simultaneous-saccharification-and-fermentation with added triticale grist resulted in >0.90 g g⁻¹ fermentation of cellulose. Hemp straw is very suitable, as it contains 0.63 g g⁻¹ of cellulose and only 0.142 g g⁻¹ of hemicellulose.A 2000 m³ a⁻¹ ethanol biorefinery requires a land use of 3 km² each for hemp and for triticale. A total of 2630 kg ethanol and 150 kg hemp oil can be gained from 1 ha. Slurry and triticale straw serve as raw material for the biogas fermenter or as animal feed. Biogas supplies thermal and electric energy in combined heat and power. Ethanol will remain at 0.66 € dm⁻³ based on market prices. In addition, data have been calculated for market prices plus and minus 30% market prices (0.51–0.81 € dm⁻³). Carbon dioxide (CO₂) abatement for ethanol achieves 121 g MJ⁻¹ CO_2eq for a combined ethanol/biogas plant. The CO₂ abatement costs vary from 38 € to 262 € t⁻¹ CO_2eq.     

Enhanced enzymatic conversion with freeze pretreatment of rice straw

Production of bioethanol by the conversion of lignocellulosic waste has attracted much interest in recent years, because of its low cost and great potential availability. The pretreatment process is important for increasing the enzymatic digestibility of lignocellulosic materials. Enzymatic conversion with freeze pretreatment of rice straw was evaluated in this study. The freeze pretreatment was found to significantly increase the enzyme digestibility of rice straw from 48% to 84%. According to the results, enzymatic hydrolysis of unpretreated rice straw with 150 U cellulase and 100 U xylanase for 48 h yielded 226.77 g kg⁻¹ and 93.84 g kg⁻¹ substrate-reducing sugars respectively. However, the reducing sugar yields from freeze pretreatment under the same conditions were 417.27 g kg⁻¹ and 138.77 g kg⁻¹ substrate, respectively. In addition, hydrolyzates analysis showed that the highest glucose yield obtained during the enzymatic hydrolysis step in the present study was 371.91 g kg⁻¹ of dry rice straw, following pretreatment. Therefore, the enhanced enzymatic conversion with freeze pretreatment of rice straw was observed in this study. This indicated that freeze pretreatment was highly effective for enzymatic hydrolysis and low environmental impact.     

Ethanol production from halophyte Juncus maritimus using freezing and thawing biomass pretreatment

Juncus maritimus contains (41.5 ± 0.3)% cellulose and (31.34 ± 0.2)% hemicellulose on dry solid (DS) basis and has the potential to serve as a low cost feedstock for ethanol production. Dilute acid or freezing/thawing pretreatments and enzymatic saccharification were evaluated for conversion of halophyte plant from J. maritimus cellulose and hemicelluloses to monomeric sugars. The maximum concentration of released glucose from J. maritimus (53.78 ± 3.24) g L⁻¹) by Freezing/thawing pretreatment and enzymatic saccharification (55 °C, pH 5.0 and 48 h) using CellicCTec2 from Novozymes and (49.14 ± 5.24) g L⁻¹ obtained by dilute acid pretreatment. The maximum yield of ethanol from acid pretreated enzyme saccharified J. maritimus hydrolyzate by Saccharomyces cerevisiae strain was (84.28 ± 5.11)% of the theoretical yield with a productivity of (0.88 ± 0.16)g L⁻¹ h⁻¹. It was (90.87 ± 1.94)% of the theoretical yield with a productivity of (1.04 ± 0.10) g L⁻¹h⁻¹ for freezing/thawing pretreated plant and enzymatic hydrolysis by CellicCTec2.     

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.     

Biochemical conversion of sugarcane bagasse into bioproducts

The ground sugarcane bagasse conversions were examined through chemical treatment methods employing soaking in aqueous ammonia (SAA), and ethyl-hydro-oxides (EHOs). To characterize a chemical treatment method, both generated solvent based extract and pulp were examined. The generated pulps were evaluated through chemical composition and enzymatic saccharification. The enzyme mixtures were investigated including Trichoderma reesei Rut C-30 originated cellulase, T. reesei Rut C-30 originated cellulase with external added β-glucosidase, Accellerase^® 1500, and Cellic^® CTec2. The physiochemical effects of chemical treatments on the structural-chemical properties of treated-bagasse were also analyzed at high substrate enzymatic saccharification. The substrate loadings (using both SAA-treated and EHOs-treated bagasse) of 125, 150, 175, 200, and 225 g L⁻¹ were examined during enzymatic saccharification process. The generated phenolic compounds were characterized based on density, antioxidant activity, and anticancer activity. All findings are discussed in relation to developing a self-sustainable integrated biorefinery.     

Optimization of alkaline pretreatment on corn stover for enhanced production of 1.3-propanediol and 2,3-butanediol by Klebsiella pneumoniae AJ4

Corn stover is one of the most promising lignocellulosic biomass that can be utilized for producing 1,3-propanediol and 2,3-butanediol. The pretreatment and enzymatic hydrolysis steps are essential for the bioconversion of lignocellulosic biomass to diols. For optimizing the pretreatment step, temperature, time, and NaOH concentration were evaluated based on total sugar recovery. Enzymatic hydrolysis for cellulose and hemicellulose were investigated at different solid-to-liquid ratios. The optimum conditions were found to be alkaline pretreatment with 0.25 mol dm⁻³ NaOH for 1 h at 60 °C followed by enzymatic hydrolysis at 50 °C for 48 h, with a solid slurry concentration of 100 g dm⁻³. Under these conditions, conversion rates of 92.55% and 78.82% were obtained from glucan and xylan, respectively. Diol production from fermentable sugars was 14.8 g dm⁻³, with a conversion yield and productivity of 0.46 g g⁻¹, and 0.98 g dm⁻³ h⁻¹, respectively. Our results are similar for diol production obtained using pure sugars under the same conditions. Therefore, mild alkaline pretreatment of corn stover facilitates delignification, significantly improving the rate of enzymatic saccharification and sugar recovery.     

Steam pretreatment of dry and ensiled industrial hemp for ethanol production

Biomass can be converted into liquid and gaseous biofuels with good efficiency. In this study, the conversion of industrial hemp (Cannabis sativa L.), a biomass source that can be cultivated with a high biomass yield per hectare, was used. Steam pretreatment of dry and ensiled hemp was investigated prior to ethanol production. The pretreatment efficiency was evaluated in terms of sugar recovery and polysaccharide conversion in the enzymatic hydrolysis step. For both materials, impregnation with 2% SO₂ followed by steam pretreatment at 210 °C for 5 min were found to be the optimal conditions leading to the highest overall yield of glucose. Simultaneous saccharification and fermentation experiments carried out with optimised pretreatment conditions resulted in ethanol yields of 163 g kg^?1 ensiled hemp (dry matter) (71% of the theoretical maximum) and 171 g kg^?1 dry hemp (74%), which corresponds to 206–216 l Mg^?1 ethanol based on initial dry material.     

Enhanced saccharification of SO2 catalyzed steam-exploded corn stover by polyethylene glycol addition

Corn stover is a renewable, low cost and abundant feedstock in China. Its effective utilization is crucial for providing bioenergy, releasing environmental pollution and increasing farmers’ income. This aim of this study was to obtain the efficient saccharification of SO₂ catalyzed steam-exploded corn stover (SSECS) by polyethylene glycol (PEG) addition. According to the results, adding PEG6000 could lower the enzyme loading by 33.3%. With 20% solid loading, the highest glucose concentration of 102 g L⁻¹ and 91.3% saccharification yield were obtained using 30 CBU (g glucan)⁻¹ ??-glucosidase and 10 FPU (g glucan)⁻¹ cellulase in presence of PEG6000. In addition, protein and enzyme activities assays in the supernatants revealed that PEG could facilitate the desorption of enzyme protein from lignocellulose. These indicated that PEG addition not only can enhance enzymatic saccharification at high substrate concentration, but also can improve enzyme recycling by reducing the enzyme activity loss caused by adsorption during the hydrolysis.