Ethanol production from rice straw using optimized aqueous-ammonia soaking pretreatment and simultaneous saccharification and fermentation processes

Rice straw was pretreated using aqueous-ammonia solution at moderate temperatures to enable production of the maximum amount of fermentable sugars from enzymatic hydrolysis. The effects of various operating variables including pretreatment temperature, pretreatment time, the concentration of ammonia and the solid-to-liquid ratio on the degree of lignin removal and the enzymatic digestibility were optimized using response surface methodology. The optimal reaction conditions, which resulted in an enzymatic digestibility of 71.1%, were found to be 69 °C, 10 h and an ammonia concentration of 21% (w/w). The effects of different commercial cellulases and the additional effect of a non-cellulolytic enzyme, xylanase, were also evaluated. Additionally, simultaneous saccharification and fermentation was conducted with rice straw to assess the ethanol production yield and productivity.     

Low-liquid pretreatment of corn stover with aqueous ammonia

A low-liquid pretreatment method of corn stover using aqueous ammonia was studied to reduce the severity and liquid throughput associated with the pretreatment step for ethanol production. Corn stover was treated at 0.5-50.0 wt.% of ammonia loading, 1:0.2-5.0 (w/w) of solid-to-liquid ratio, 30 °C for 4-12 weeks. The effects of these conditions on the composition and enzyme digestibility of pretreated corn stover were investigated. Pretreatment of corn stover at 30 °C for four weeks using 50 wt.% of ammonia loading and 1:5 solid-to-liquid ratio resulted in 55% delignification and 86.5% glucan digestibility with 15 FPU cellulase + 30 CBU β-glucosidase/g-glucan.Simultaneous saccharification and fermentation of corn stover treated at 30 °C for four weeks using 50 wt.% ammonia loading and 1:2 solid-to-liquid ratio gave an ethanol yield of 73% of the theoretical maximum based on total carbohydrates (glucan + xylan) present in the untreated material.     

Liquid hot water and alkaline pretreatment of soybean straw for improving cellulose digestibility

Soybean straw was pretreated with either liquid hot water (LHW) (170-210 °C for 3-10 min) or alkaline soaking (4-40 g NaOH/100 g dry straw) at room temperature to evaluate the effects on cellulose digestibility. Nearly 100% cellulose was recovered in pretreated solids for both pretreatment methods. For LHW pretreatment, xylan dissolution from the raw material increased with pretreatment temperature and time. Cellulose digestibility was correlated with xylan dissolution. A maximal glucose yield of 70.76%, corresponding to 80% xylan removal, was obtained with soybean straw pretreated at 210 °C for 10 min. NaOH soaking at ambient conditions removed xylan up to 46.37% and the subsequent glucose yield of pretreated solids reached up to 64.55%. Our results indicated LHW pretreatment was more effective than NaOH soaking for improving cellulose digestibility of soybean straw.     

Xylose production from giant reed (Arundo donax L.): Modeling and optimization of dilute acid hydrolysis

Statistical modeling and optimization of dilute sulfuric acid hydrolysis of potential energy crop giant reed (Arundo donax L.) has been performed using response surface methodology. Central composite rotatable design was applied to assess the effect of acid concentration, reaction time and temperature on efficiency and selectivity of xylan polysaccharide conversion to xylose. Second-order polynomial model was fitted to experimental data to find the optimum reaction conditions by multiple regression analysis. The monomeric xylose recovery ca. 94% (vs. 93% predicted) was achieved under optimized hydrolysis conditions (1.27% acid concentration, 141.6 °C and 36.4 min), confirming the high validity of the developed model. The low content of glucose (2.7%) and monosaccharide degradation products (0.9% furfural and 0.7% 5-hydroxymethylfurfural) provided a high quality xylose-rich subtract, ready for subsequent biochemical conversion to value-added products. The solid xylan-free residue was easily converted to fermentable sugars resulting in cellulose digestibility of 70% vs. 9% for untreated biomass.     

Ethanol production from oil palm trunks treated with aqueous ammonia and cellulase

Oil palm trunks are a possible lignocellulosic source for ethanol production. Low enzymatic digestibility of this type of material (11.9% of the theoretical glucose yield) makes pretreatment necessary. An enzymatic digestibility of 95.4% with insoluble solids recovery of 49.8% was achieved after soaking shredded oil palm trunks in ammonia under optimum conditions (80 °C, 1:12 solid-to-liquid ratio, 8 h and 7% (w/w) ammonia solution). Treatment with 60 FPU of commercial cellulase (Accellerase 1000) per gram of glucan and fermentation with Saccharomyces cerevisiae D₅A resulted in an ethanol concentration of 13.3 g/L and an ethanol yield of 78.3% (based on the theoretical maximum) after 96 h. These results indicate that oil palm trunks are a biomass feedstock that can be used for bioethanol production.     

Conversion of olive tree biomass into fermentable sugars by dilute acid pretreatment and enzymatic saccharification

The production of fermentable sugars from olive tree biomass was studied by dilute acid pretreatment and further saccharification of the pretreated solid residues. Pretreatment was performed at 0.2%, 0.6%, 1.0% and 1.4% (w/w) sulphuric acid concentrations while temperature was in the range 170-210 degrees C. Attention is paid to sugar recovery both in the liquid fraction issued from pretreatment (prehydrolysate) and that in the water-insoluble solid (WIS). As a maximum, 83% of hemicellulosic sugars in the raw material were recovered in the prehydrolysate obtained at 170 degrees C, 1% sulphuric acid concentration, but the enzyme accessibility of the corresponding pretreated solid was not very high. In turn, the maximum enzymatic hydrolysis yield (76.5%) was attained from a pretreated solid (at 210 degrees C, 1.4% acid concentration) in which cellulose solubilization was detected; moreover, sugar recovery in the prehydrolysate was the poorest one among all the experiments performed. To take account of fermentable sugars generated by pretreatment and the glucose released by enzymatic hydrolysis, an overall sugar yield was calculated. The maximum value (36.3 g sugar/100 g raw material) was obtained when pretreating olive tree biomass at 180 degrees C and 1% sulphuric acid concentration, representing 75% of all sugars in the raw material. Dilute acid pretreatment improves results compared to water pretreatment.     

Comparing the Recalcitrance of Eucalyptus, Pine, and Switchgrass Using Ionic Liquid and Dilute Acid Pretreatments

Pine, eucalyptus, and switchgrass were evaluated for the production of fermentable sugars via ionic liquid and dilute acid pretreatments and subsequent enzymatic hydrolysis. The results show that among the three feedstocks, switchgrass has the highest sugar yields and faster hydrolysis rates for both pretreatment technologies by achieving 48 % (dilute acid) and 96 % (ionic liquid) sugar yields after 24 h. Of the two wood species, eucalyptus has a higher and faster sugar recovery after ionic liquid pretreatment than pine (93 vs. 62 % in 24 h) under 160 °C for 3 h with [C₂mim][OAc]. Pretreatment of pine and eucalyptus is observed to be ineffective under 1.2 % dilute acid condition and 160 °C for 15 min, indicating that further enhancement of reaction temperature or acid concentration is necessary to increase the digestibility of pretreated materials. Raman spectroscopy data show that the extent of lignin depolymerization that occurs during pretreatment also varies for the three different feedstocks. Under similar hemicellulose removal conditions, lignin removal in ionic liquid pretreatment can help improve cellulose conversion. This finding may help explain the observed variation in the saccharification yields and kinetics. These results indicate that ionic liquid pretreatment not only improved saccharification over dilute acid for all three feedstocks but also better dealt with the differences among them, suggesting better tolerance to feedstock variability.     

Effect of pretreatment severity on the conversion of barley straw to fermentable substrates and the release of inhibitory compounds

The production of fermentable substrates from barley straw under various process conditions was studied. Pretreatment included chemical pretreatment with dilute-acid followed by enzymatic hydrolysis; the pretreatment conditions were expressed in a combined severity factor, CS, which ranged in the present study from −1.6 to 1.1. Considering the production of fermentable sugars and the release of inhibitory compounds, the optimal pretreatment conditions were 170 °C, 0% sulfuric acid and 60 min, corresponding to CS −0.4. Under these conditions, 21.4 g glucose/L, 8.5 g xylose/L, and 0.5 g arabinose/L were produced, while 0.1 g HMF/L, 0.4 g furfural/L, 0.0 g levulinic acid/L, 0.0 g formic acid/L, and 2.1 g acetic acid/L were released. The ratio of Σsugars/Σinhibitors proved to be a good tool for evaluating the suitability of a hydrolysate for fermentation purposes.     

Selective production of hemicellulose-derived carbohydrates from wheat straw using dilute HCl or FeCl3 solutions under mild conditions. X-ray and thermo-gravimetric analysis of the solid residues

The present work explores the combined production of hemicellulose-derived carbohydrates and an upgraded solid residue from wheat straw using a dilute-acid pretreatment at mild temperature. Dilute aqueous HCl solutions were studied at temperatures of 100 and 120 °C, and they were compared to dilute FeCl₃ under the same conditions. Comparable yields of soluble sugars and acetic acid were obtained, affording an almost complete removal of pentoses when using 200 mM aqueous solutions at 120 °C. The solid residues of pretreatment were characterized showing a preserved crystallinity of the cellulose, and a almost complete removal of ash forming matter other than Si. Results showed upgraded characteristic of the residues for thermal conversion applications compared to the untreated wheat straw.     

Optimization of thermal-dilute sulfuric acid pretreatment for enhancement of methane production from cassava residues

In this study, the pretreatment of cassava residues by thermal-dilute sulfuric acid (TDSA) hydrolysis was investigated by means of a statistically designed set of experiments. A three-factor central composite design (CCD) was employed to identify the optimum pretreatment condition of cassava residues for methane production. The individual and interactive effects of temperature, H₂SO₄ concentration and reaction time on increase of methane yield (IMY) were evaluated by applying response surface methodology (RSM). After optimization, the resulting optimum pretreatment condition was 157.84 °C, utilizing 2.99% (w/w TS) H₂SO₄ for 20.15 min, where the maximum methane yield (248 mL/g VS) was 56.96% higher than the control (158 mL/g VS), which was very close to the predict value 56.53%. These results indicate the model obtained through RSM analysis is suit to predict the optimum pretreatment condition and there is great potential of using TDSA pretreatment of cassava residues to enhance methane yield.     

Addition of ammonia and/or oxygen to an ionic liquid for delignification of miscanthus

Ammonia and/or oxygen were used to enhance the delignification of miscanthus dissolved in 1-ethyl-3-methylimidazolium acetate at 140 °C. After dissolution of the gas at 9 bar, water was added as antisolvent to regenerate the dissolved biomass. In a next step, an acetone/water mixture was used to remove carbohydrate-free lignin from the regenerated biomass. The lignin content in the final product was around 10%, much lower than the ca. 23% lignin content of the raw dry miscanthus. This lignin reduction is achieved without diminution of cellulose or of total carbohydrates recovered, relative to the recovery achieved with the ionic liquid pretreatment in contact with air or nitrogen.     

Thermo-mechanical extrusion pretreatment for conversion of soybean hulls to fermentable sugars

Thermo-mechanical extrusion pretreatment for lignocellulosic biomass was investigated using soybean hulls as the substrate. The enzyme cocktail used to hydrolyze pretreated soybean hulls to fermentable sugars was optimized using response surface methodology (RSM). Structural changes in substrate and sugar yields from thermo-mechanical processing were compared with two traditional pretreatment methods that utilized dilute acid (1% sulfuric acid) and alkali (1% sodium hydroxide). Extrusion processing parameters (barrel temperature, in-barrel moisture, screw speed) and processing aids (starch, ethylene glycol) were studied with respect to reducing sugar and glucose yields. The conditions resulting in the highest cellulose to glucose conversion (95%) were screw speed 350 rpm, maximum barrel temperature 80 °C and in-barrel moisture content 40% wb. Compared with untreated soybean hulls, glucose yield from enzymatic hydrolysis of soybean hulls increased by 69.6%, 128.7% and 132.2%, respectively, when pretreated with dilute acid, alkali and extrusion.     

Optimization of microwave-assisted FeCl3 pretreatment conditions of rice straw and utilization of Trichoderma viride and Bacillus pumilus for production of reducing sugars

In this study, Box-Behnken design (BBD) and response surface methodology (RSM) were used to optimize microwave-assisted FeCl₃ pretreatment conditions of rice straw with respect to FeCl₃ concentration, microwave intensity, irradiation time and substrate concentration. When rice straw was pretreated at the optimal conditions of FeCl₃ concentration, 0.14 mol/L; microwave intensity, 160 °C; irradiation time, 19 min; substrate concentration, 109 g/L; and inoculated with Trichoderma viride and Bacillus pumilus, the production of reducing sugars was 6.62 g/L. This yield was 2.9 times higher than that obtained with untreated rice straw. The microorganisms degraded 37.8% of pretreated rice straw after 72 h. The structural characteristic analyses suggest that microwave-assisted FeCl₃ pretreatment damaged the silicified waxy surface of rice straw, disrupted almost all the ether linkages between lignin and carbohydrates, and removed lignin.     

Release of monomeric sugars from Miscanthus sinensis by microwave-assisted ammonia and phosphoric acid treatments

Microwave-assisted ammonium hydroxide (NH₄OH) followed by phosphoric acid (H₃PO₄) treatments were used to release monomeric sugars from Miscanthus sinensis grown in Cha-Chueng-Sao province, Thailand. Treatment with 1.0% (w/v) NH₄OH, 15:1 liquid-to-solid ratio (LSR) at 120 °C temperature for 15 min liberated 2.9 g of monomeric sugars per 100 g of dried biomass, whereas the corresponding yield for a treatment with 1.78% v/v H₃PO₄, 15:1 LSR at 140 °C for 30 min was 62.3 g/100 g. The two-stage pretreatment, treatment with NH₄OH at 120 °C temperature for 15 min followed by treatment with H₃PO₄ at 140 °C for 30 min, impressively provided the highest total monomeric sugar yield of 71.6 g/100 g dried biomass.     

Preparation of levoglucosenone through sulfuric acid promoted pyrolysis of bagasse at low temperature

Fast pyrolysis of bagasse pretreated by sulfuric acid was conducted in a fixed bed reactor to prepare levoglucosenone (LGO), a very important anhydrosugar for organic synthesis. The liquid yield and LGO yield were studied at temperatures from 240 to 350 °C and sulfuric acid loadings from 0.92 to 7.10 wt.%. An optimal LGO yield of 7.58 wt.% was obtained at 270 °C with a sulfuric acid pretreatment concentration of 0.05 M (corresponding to 4.28 wt.% sulfuric acid loading). For comparison, microcrystalline cellulose pretreated by 0.05 M sulfuric acid solution was pyrolyzed at temperature from 270 °C to 320 °C, and bagasse loaded with 3-5 wt.% phosphoric acid was pyrolyzed at temperature from 270 °C to 350 °C. The highest yield of LGO from bagasse was 30% higher than that from microcrystalline cellulose, and treatment with sulfuric acid allowed a 21% higher yield than treatment with phosphoric acid.     

Simplified process for ethanol production from sugarcane bagasse using hydrolysate-resistant Escherichia coli strain MM160

Hexose and pentose sugars from phosphoric acid pretreated sugarcane bagasse were co-fermented to ethanol in a single vessel (SScF), eliminating process steps for solid-liquid separation and sugar cleanup. An initial liquefaction step (L) with cellulase was included to improve mixing and saccharification (L + SScF), analogous to a corn ethanol process. Fermentation was enabled by the development of a hydrolysate-resistant mutant of Escherichia coli LY180, designated MM160. Strain MM160 was more resistant than the parent to inhibitors (furfural, 5-hydroxymethylfurfural, and acetate) formed during pretreatment. Bagasse slurries containing 10% and 14% dry weight (fiber plus solubles) were tested using pretreatment temperatures of 160-190 °C (1% phosphoric acid, 10 min). Enzymatic saccharification and inhibitor production both increased with pretreatment temperature. The highest titer (30 g/L ethanol) and yield (0.21 g ethanol/g bagasse dry weight) were obtained after incubation for 122 h using 14% dry weight slurries of pretreated bagasse (180 °C).     

Geographic differences on accumulation of sugars and polyols in locust eggs in response to cold acclimation

The accumulation of low molecular weight sugars and polyols is one of major mechanisms hypothesized to increase cold tolerance in overwintering insects. But little is known about whether these sugars and polyols are involved in geographic variation of cold tolerance. In this study, we investigated accumulation patterns of eight low molecular weight sugars and polyols of eggs in tropical and temperate populations of the migratory locust, which exhibits between-population variation in cold tolerance, in response to cold acclimation (5, 0 and −5 °C). Excluding erythritol, the other seven carbohydrates were identified as possible cryoprotectants in locust eggs. Basal maximal and minimal concentrations were 45 μg/g wet weight for trehalose and 0.59 μg/g wet weight for glycerol. Most sugars and polyols were elevated after a −5 °C exposure. In a tropical population, fructose, glucose, sorbitol and myo-inositol were significantly accumulated by low temperature treatments, but glycerol was not. In the temperate population, glycerol, glucose, mannitol, sorbitol, myo-inositol were significantly accumulated but trehalose did not increase. Our results suggest different accumulation patterns of these carbohydrates of locust eggs between tropical and temperate populations and highlighted possible roles for them in geographic variation of cold tolerance in the migratory locust.     

Recovery of Sugars from Ionic Liquid Biomass Liquor by Solvent Extraction

The dissolution of biomass into ionic liquids (ILs) has been shown to be a promising alternative biomass pretreatment technology, facilitating faster breakdown of cellulose through the disruption of lignin and the decrystallization of cellulose. Both biological and chemical catalysis have been employed to enhance the conversion of IL-treated biomass polysaccharides into monomeric sugars. However, biomass-dissolving ILs, sugar monomers, and smaller carbohydrate oligomers are all soluble in water. This reduces the overall sugar content in the recovered solid biomass and complicates the recovery and recycle of the IL. Near-complete recovery of the IL and the holocellulose is essential for an IL-based pretreatment technology to be economically feasible. To address this, a solvent extraction technique, based on the chemical affinity of boronates such as phenylboronic acid and naphthalene-2-boronic acid for sugars, was applied to the extraction of glucose, xylose, and cellobiose from aqueous mixtures of 1-ethyl-3-methylimidazolium acetate. It was shown that boronate complexes could extract up to 90% of mono- and disaccharides from aqueous IL solutions, 100% IL systems, and hydrolysates of corn stover containing IL. The use of boronate complexes shows significant potential as a way to recover sugars at several stages in ionic liquid biomass pretreatment processes, delivering a concentrated solution of fermentable sugars, minimizing toxic byproducts, and facilitating ionic liquid cleanup and recycle.     

Enhanced enzymatic saccharification of kenaf powder after ultrasonic pretreatment in ionic liquids at room temperature

This study demonstrates for the first time that the enzymatic hydrolysis of cellulose is drastically enhanced following ultrasonic pretreatment of lignocellulosic material in ionic liquids (ILs) when compared to conventional thermal pretreatment. Five types of ILs, 1-buthyl-3-methylimidazolium chloride (BmimCl), 1-allyl-3-methylimidazolium chloride (AmimCl), 1-ethyl-3-methylimidazolium chloride (EmimCl), 1-ethyl-3-methylimidazolium diethyl phosphate (EmimDep), and 1-ethyl-3-methylimidazolium acetate (EmimOAc) were tested. Cellulose saccharification ratio was about 20% for kenaf powders pretreated in BmimCl, AmimCl, EmimCl, and EmimDep by conventional heating at 110 °C for 120 min. Conversely, 60-95% of cellulose was hydrolyzed to glucose, subsequent to ultrasonic pretreatment in the same ILs for 120 min at 25 °C. The cellulose saccharification ratio of kenaf powder in EmimOAc was 86% after only 15 min of the ultrasonic pretreatment at 25 °C, compared to only 47% in that case of thermal pretreatment in the IL.     

Enzymatic hydrolysis of cellulose by the cellobiohydrolase domain of CelB from the hyperthermophilic bacterium Caldicellulosiruptor saccharolyticus

The celB gene of Caldicellulosiruptor saccharolyticus was cloned and expressed in Escherichia coli to create a recombinant biocatalyst for hydrolyzing lignocellulosic biomass at high temperature. The GH5 domain of CelB hydrolyzed 4-nitrophenyl-β-d-cellobioside and carboxymethyl cellulose with optimum activity at pH 4.7-5.5 and 80 °C. The recombinant GH5 and CBM3-GH5 constructs were both stable at 80 °C with half-lives of 23 h and 39 h, respectively, and retained >94% activity after 48 h at 70 °C. Enzymatic hydrolysis of corn stover and cellulose pretreated with the ionic liquid 1-ethyl-3-methylimidazolium acetate showed that GH5 and CBM3-GH5 primarily produce cellobiose, with product yields for CBM3-GH5 being 1.2- to 2-fold higher than those for GH5. Confocal microscopy of bound protein on cellulose confirmed tighter binding of CBM3-GH5 to cellulose than GH5, indicating that the enhancement of enzymatic activity on solid substrates may be due to the substrate binding activity of CBM3 domain.