Enzymatic saccharification and fermentation of xylose-optimized dilute acid-treated lignocellulosics

The cellulose reactivity of two lignocellulosic feedstocks, switchgrass and poplar, was evaluated under straight saccharification (SS) and simultaneous saccharification and fermentation (SSF) conditions following dilute sulfuric acid pretreatments designed for optimum xylose yields. The optimum pretreatment conditions, within the constraints of the experimental system (Parr batch reactor), were 1.2% acid, 180°C, and 0.5 min for switchgrass and 1% acid, 180°C, and 0.56 min for poplar. The cellulase enzyme preparation was from Trichoderma reesei and fermentations were done with Saccharomyces cerevisiae. Time courses for SS were monitored as the sum of glucose and cellobiose; those for SSF as the sum of glucose, cellobiose, and ethanol. Percentage conversions under SS conditions were 79.1% and 91.4% for the pretreated poplar and switchgrass feedstocks, respectively. Analogous values under SSF conditions were 73.0% and 90.3% for pretreated poplar and switchgrass, respectively.     

Kinetic Study of Empty Fruit Bunch Using Hot Liquid Water and Dilute Acid

Empty fruit bunch (EFB), a residual product of the palm plantation, is an attractive biomass for biorefinery. As xylan is susceptible to high temperature pretreatment, it is important to setup a proper pretreatment condition to maximize the sugar recovery from EFB. Kinetic parameters of mathematical models were obtained in order to predict the concentration of xylose, glucose, furfural, and acetic acid in the hydrolysate and to find production conditions of xylose. We investigated the kinetics of hot liquid water and dilute sulfuric acid hydrolysis over a 40-min period using a self-designed setup by measuring the concentrations of released sugars (xylose, glucose) and degradation products (acetic acid and furfural). The reaction was performed within the range 160～180 °C, under reaction conditions of various concentration of sulfuric acid (0.1～0.2%) and 1:7 solid-liquid ratio in a batch reactor. The kinetic constants can be expressed by the Arrhenius equation with the activation energy for the hydrolysis of sugar and decomposition of sugar. The activation energy of xylose was determined to be 136.2187 kJ mol(-1).     

Consolidated Conversion of Hulled Barley into Fermentable Sugars Using Chemical,Thermal, and Enzymatic (CTE) Treatment

A novel process using chemical, thermal, and enzymatic treatment for conversion of hulled barley into fermentable sugars was developed. The purpose of this process is to convert both lignocellulosic polysaccharides and starch in hulled barley grains into fermentable sugars simultaneously without a need for grinding and hull separation. In this study, hulled barley grains were treated with 0.1 and 1.0 wt.-% sulfuric acid at various temperatures ranging from 110 to 170 °C in a 63-ml flow-through packed-bed stainless steel reactor. After sulfuric acid pretreatment, simultaneous conversion of lignocellulose and starch in the barley grains into fermentable sugars was performed using an enzyme cocktail, which included α-amylase, glucoamylase, cellulase, and β-glucosidase. Both starch and non-starch polysaccharides in the pre-treated barley grains were readily converted to fermentable sugars. The treated hulled barley grains, including their hull, were completely hydrolyzed to fermentable sugars with recovery of almost 100% of the available glucose and xylose. The pretreatment conditions of this chemical, thermal, and enzymatic (CTE) process for achieving maximum yield of fermentable sugars were 1.0 wt.% sulfuric acid and 110 °C. In addition to starch, the acid pretreatment also retained most of the available proteins in solid form, which is essential for subsequent production of fuel ethanol and high protein distiller’s dried grains with solubles co-product.     

Manufacture of xylose-based fermentation media from corncobs by posthydrolysis of autohydrolysis liquors

Milled corncob samples were mixed with water and heated to obtain a liquid phase containing oligosaccharides, sugars, and acetic acid as main reaction products (autohydrolysis reaction). To hydrolyze the sugar oligomers to the correspondent monomers, sulfuric acid was added to the autohydrolysis liquors to reach 0.5–2 wt% of solution, and the reaction media were heated at 101.5–135°C. With this operational procedure, sugar solutions suitable as fermentation media (containing xylose as the major component) were obtained. The kinetics of the posthydrolysis step was characterized on the basis of experimental data concerning the time courses of the concentrations of xylooligosaccharides, xylose, furfural, and acetic acid. The concentrations of other reaction byproducts (glucose or arabinose) were also measured.     

High Xylose Yields from Dilute Acid Pretreatment of Corn Stover Under Process-Relevant Conditions

Pretreatment experiments were carried out to demonstrate high xylose yields at high solids loadings in two different batch pretreatment reactors under process-relevant conditions. Corn stover was pretreated with dilute sulfuric acid using a 4-l Steam Digester and a 4-l stirred ZipperClave® reactor. Solids were loaded at 45% dry matter (wt/wt) after sulfuric acid catalyst impregnation using nominal particle sizes of either 6 or 18 mm. Pretreatment was carried out at temperatures between 180 and 200 °C at residence times of either 90 or 105 s. Results demonstrate an ability to achieve high xylose yields (>80%) over a range of pretreatment conditions, with performance showing little dependence on particle size or pretreatment reactor type. The high xylose yields are attributed to effective catalyst impregnation and rapid rates of heat transfer during pretreatment.     

Comparative ethanol productivities of different Zymomonas recombinants fermenting oat hull hydrolysate

Iogen Corporation of Ottawa, Canada, has recently built a 50 t/d biomass-to-ethanol demonstration plant adjacent to its enzyme production facility. Iogen has partnered with the University of Toronto to test the C6/C5 cofermentation performance characteristics of National Renewable Energy Laboratory's metabolically engineered Zymomonas mobilis using its biomass hydrolysates. In this study, the biomass feedstock was an agricultural waste, namely oat hulls, which was hydrolyzed in a proprietary two-stage process involving pretreatment with dilute sulfuric acid at 200–250°C, followed by cellulase hydrolysis. The oat hull hydrolysate (OHH) contained glucose, xylose, and arabinose in a mass ratio of about 8:3:0.5. Fermentation media, prepared from diluted hydrolysate, were nutritionally amended with 2.5 mL/L of corn steep liquor (50% solids) and 1.2 g/L of diammonium phosphate. The estimated cost for large-scale ethanol production using this minimal level of nutrient supplementation was 4.4c/gal of ethanol. This work examined the growth and fermentation performance of xyloseutilizing, tetracycline-resistant, plasmid-bearing, patented, recombinant Z. mobilis cultures: CP4:pZB5, ZM4:pZB5, 39676:pZB4L, and a hardwood prehydrolysate-adapted variant of 39676:pZB4L (designated asthe “adapted” strain). In pH-stat batch fermentations with unconditioned OHH containing 6% (w/v) glucose, 3% xylose, and 0.75% acetic acid, rec Zm ZM4:pZB5 gave the best performance with a fermentation time of 30h, followed by CP4:pZB5 at 48h, with corresponding volumetric productivities of 1.4 and 0.89 g/(L·h), respectively. Based on the available glucose and xylose, the process ethanol yield for both strains was 0.47 g/g (92% conversion efficiency). At 48 h, the process yield for rec Zm 39676:pZB4L and the adapted strain was 0.32 and 0.34 g/g, respectively. None of the test strains was able to fermentarabinose. Acetic acid tolerance appeared to be a major determining factor in cofermentation performance.     

Evaluation of Ethanol Production from Corncob Using Scheffersomyces (Pichia) stipitis CBS 6054 by Volumetric Scale-up

In scale-up, the potential of ethanol production by dilute sulfuric acid pretreatment using corncob was investigated. Pretreatments were performed at 170 °C with various acid concentrations ranging from 0% to 1.656% based on oven dry weight. Following pretreatment, pretreated biomass yield ranged from 59% to 67%. More than 90% of xylan was removed at 0.828% of sulfuric acid. At same pretreatment condition, the highest glucose yield obtained from pretreated biomass by enzymatic hydrolysis was about 76%, based on a glucan content of 37/100 g. In hydrolysate obtained by pretreatment, glucose concentration was low, while xylose concentration was significantly increased above 0.368% of sulfuric acid. At 1.656% of sulfuric acid, xylose and glucose concentration was highest. In subsequent, fermentation with hydrolysate, maximal ethanol yield was attained after 24 h with 0.368% of sulfuric acid. The fermentation efficiency of hydrolysate obtained by enzymatic hydrolysis reached a maximum of 75% at an acid charge of 0.368%.     

Production of lactic acid from pulp mill solid waste and xylose using Lactobacillus delbrueckii (NRRL B445)

Using the simultaneoussaccharification and fermentation (SSF) technique, pulp mill solid waste cellulose was converted into glucose using cellulase enzyme and glucose into lacticacid using NRRL B445. SSF experiments were conducted at various pH levels, temperatures, and nutrient concentrations, and the lactic acid yield ranged from 86 to 97%. The depletion of xylose in SSF was further investigated by inoculating NRRL B445 into a xylose-only medium. On prolonged incubation, depletion of xylose with lactic acid production was observed. An experimental procedure with a nonglucose medium was developed to eliminate the lag phase. From xylose fermentation, Lactobacillus delbrueckii yielded 88–92% lactic acid and 2–12% acetic acid.     

Two-Stage Acidic–Alkaline Hydrothermal Pretreatment of Lignocellulose for the High Recovery of Cellulose and Hemicellulose Sugars

The focus of this work was to develop a combined acid and alkaline hydrothermal pretreatment of lignocellulose that ensures high recovery of both hexose and pentose. Dilute sulfuric acid and lime pretreatments were employed sequentially. Process performance was optimized in terms of catalyst concentration, retention time, and temperature using response surface methodology. Medium operational conditions in the acid stage and harsh conditions in the alkaline stage were desirable with optimal performance at 0.73 wt% H₂SO₄, 150 °C, 6.1 min in the first stage, and 0.024 g lime/g biomass, 202 °C, 30 min in the second stage. In comparison to single-stage pretreatments with high recovery of either glucose or xylose, two-stage process showed great promises with >80 % glucose and >70 % xylose recovery. In addition, the method greatly improved ethanol fermentation with yields up to 0.145 g/g Miscanthus, due to significantly reduced formation of inhibitory by-products such as weak acids, furans, and phenols. Supplementing biomimetic acids would further increase glucose yield by up to 15 % and xylose yield by 25 %.     

Dilute Acid Pretreatment of Corncob for Efficient Sugar Production

Aqueous dilute acid pretreatments of corncob were conducted using cylindrical pressure vessels in an oil bath. Pretreatments were conducted in a temperature range of 160–190 °C with acid-solution-to-solid-corncob ratio of 2. The acid concentration (v/v) in the pretreatment solution was varied from 0% to 0.7%, depending on temperature. This gives acid charge on ovendry-weight corncob of 0–2.58%. It was found that optimal pretreatment temperature is between 160 and 170 °C based on total xylose and glucose yields and thermal energy consumption in pretreatment. At 170 °C and acid charge of 2.2% on cob, total glucose yield and xylose recovery were 97% and 75%, respectively, which resulted in an overall monomeric sugar recovery of about 88%. Xylose concentration in the hydrolysate was about 12%, with xylose-to-acetic-acid ratio of 8 and to furan (furfural and hydroxymethylfurfural) of about 15.     

Two-stage dilute-acid pretreatment of softwoods

Whole treechips obtained from softwood forest thinnings were pretreated via single-and two-stage dilute-sulfuric acid pretreatment. Whole-tree chips were impregnated with dilute sulfuric acid and steam treated in a 4-L steam explosion reactor. In single-stage pretreatment, wood chips were treated using a wide range of severity. In two-stage pretreatment, the first stage was carried out at low severity tomaximize hemicellulose recovery. Solubilized sugars were recovered from the first-stage prehydrolysate by washing with water. In the second stage, water-insoluble solids from first-stage prehydrolysate were impregnated with dilute sulfuric acid, then steam treated at more severe conditions to hydrolyze a portion of the remaining cellulose to glucose and to improve the enzyme digestibility. The total sugar yields obtained after enzymatic hydrolysis of two-stage dilute acid-pretreated samples were compared with sugar yields from single-stage pretreatment. The overall sugar yield from two-stage dilute-acid pretreatment was approx 10% higher, and the net enzyme requirement was reduced by about 50%. Simultaneous saccharification and fermentation using an adapted Saccharomyces cerevisiae yeast strain further improved cellulose conversion yield and lowered the enzyme requirement.     

Pretreatment of yellow poplar sawdust by pressure cooking in water

The pretreatment of yellow poplar wood sawdust using liquid water at temperatures above 220°C enhances enzyme hydrolysis. This paper reviews our prior research and describes the laboratory reactor system currently in use for cooking wood sawdust at temperatures ranging from 220 to 260°C. The wood sawdust at a 6–6.6% solid/liquid slurry was treated in a 2 L, 304 SS, Parr reactor with three turbine propeller agitators and a proportional integral derivative (PID) controller, which controlled temperature within ±1°C. Heat-up times to the final temperatures of 220, 240, or 260°C were achieved in 60–70 min. Hold time at the final temperature was less than 1 min. A serpentine cooling coil, through which tap water was circulated at the completion of the run, cooled the reactor’s contents within 3 min after the maximum temperature was attained. A bottoms port, as well as ports in the reactor’s head plate, facilitated sampling of the slurry and measuring the pH, which changes from an initial value of 5 before cooking to a value of approx 3 after cooking. Enzyme hydrolysis gave 80–90% conversion of cellulose in the pretreated wood to glucose. Simultaneous saccharification and fermentation of washed, pretreated lignocellulose gave an ethanol yield that was 55% of theoretical. Untreated wood sawdust gave less than 5% hydrolysis under the same conditions.     

Ethanol Production from Residual Wood Chips of Cellulose Industry: Acid Pretreatment Investigation,Hemicellulosic Hydrolysate Fermentation,and Remaining Solid Fraction Fermentation by SSF Process

Current research indicates the ethanol fuel production from lignocellulosic materials, such as residual wood chips from the cellulose industry, as new emerging technology. This work aimed at evaluating the ethanol production from hemicellulose of eucalyptus chips by diluted acid pretreatment and the subsequent fermentation of the generated hydrolysate by a flocculating strain of Pichia stipitis. The remaining solid fraction generated after pretreatment was subjected to enzymatic hydrolysis, which was carried out simultaneously with glucose fermentation [saccharification and fermentation (SSF) process] using a strain of Saccharomyces cerevisiae. The acid pretreatment was evaluated using a central composite design for sulfuric acid concentration (1.0–4.0 v/v) and solid to liquid ratio (1:2–1:4, grams to milliliter) as independent variables. A maximum xylose concentration of 50 g/L was obtained in the hemicellulosic hydrolysate. The fermentation of hemicellulosic hydrolysate and the SSF process were performed in bioreactors and the final ethanol concentrations of 15.3 g/L and 28.7 g/L were obtained, respectively.     

Influence of inhibitory compounds and minor sugars on xylitol production by <Emphasis Type="Italic">Debaryomyces hansenii</Emphasis>

To obtain in-depth information on the overall metabolic behavior of the new good xylitol producer Debaryomyces hansenii UFV-170, batch bioconversions were carried out using semisynthetic media with compositions simulating those of typical acidic hemicellulose hydrolysates of sugarcane bagasse. For this purpose, we used media containing glucose (4.3–6.5 g/L), xylose (60.1–92.1 g/L), or arabinose (5.9–9.2 g/L), or binary or ternary mixtures of them in either the presence or absence of typical inhibitors of acidic hydrolysates, such as furfural (1.0–5.0 g/L), hydroxymethylfurfural (0.01–0.30 g/L), acetic acid (0.5–3.0 g/L), and vanillin (0.5–3.0 g/L). D. hansenii exhibited a good tolerance to high sugar concentrations as well as to the presence of inhibiting compounds in the fermentation media. It was able to produce xylitol only from xylose, arabitol from arabinose, and no glucitol from glucose. Arabinose metabolization was incomplete, while ethanol was mainly produced from glucose and, to a lesser less extent, from xylose and arabinose. The results suggest potential application of this strain in xyloseto-xylitol bioconversion from complex xylose media from lignocellulosic materials.     

Performance of immobilized Zymomonas mobilis 31821 (pZB5) on actual hydrolysates produced by arkenol technology

By applying the Arkenol process using highly concentrated sulfuric acid, various biomass feedstocks, including cedar tree, rice straw, newspaper, and bagasse, were successfully processed and converted into glucose and xylose for fermentation usage in a flash fermentation reactor in which the performance of National Renewable Energy Laboratory’s patented rec-Zymomonas mobilis 31821 (pZB5) after immobilization was investigated. The immobilization medium is a photocrosslinked resin made from polyethylene glycols or polypropylene glycols. Recombinant or rec-Z. mobilis used in the study has been shown to efficiently ferment glucose and xylose at a relatively high concentration (12–15%), that is a typical hydrolysate produced from cellulosic feedstocks. The application of immobilized rec-Z. mobilis and flash fermentation technology, together with concentrated acid technology producing a high concentration sugar solution, promises to speed the development of the cellulose-to-ethanol industry.     

Steam pretreatment of acid-sprayed and acid-soaked barley straw for production of ethanol

Barley is an abundant crop in Europe, which makes its straw residues an interesting cellulose source for ethanol production. Steam pretreatment of the straw followed by enzymatic hydrolysis converts the cellulose to fermentable sugars. Prior to pretreatment the material is impregnated with a catalyst, for example, H₂SO₄, to enhance enzymatic digestibility of the pretreated straw. Different impregnation techniques can be applied. In this study, soaking and spraying were investigated and compared at the same pretreatment condition in terms of overall yield of glucose and xylose. The overall yield includes the soluble sugars in the liquid from pretreatment, including soluble oligomers, and monomer sugars obtained in the enzymatic hydrolysis. The yields obtained differed for the impregnation techniques. Acid-soaked barley straw gave the highest overall yield of glucose, regardless of impregnation time (10 or 30 min) or acid concentration (0.2 or 1.0 wt%). For xylose, soaking gave the highest overall yield at 0.2 wt% H₂SO₄. An increase in acid concentration resulted in a decrease in xylose yield for both acid-soaked and acid-sprayed barley straw. Optimization of the pretreatment conditions for acid-sprayed barley straw was performed to obtain yields using spraying that were as high as those with soaking. For acid-sprayed barley straw the optimum pretreatment condition for glucose, 1.0 wt% H₂SO₄ and 220°C for 5 min, gave an overall glucose yield of 92% of theoretical based on the composition of the raw material. Pretreatment with 0.2wt% H₂SO₄ at 190°C for 5 min resulted in the highest overall xylose yield, 67% of theoretical based on the composition of the raw material.     

Adding value to the Brazilian sisal: acid hydrolysis of its pulp seeking production of sugars and materials

The present work is inserted into the broad context of the upgrading of lignocellulosic fibers. Sisal was chosen in the present study because more than 50% of the world’s sisal is cultivated in Brazil, it has a short life cycle and its fiber has a high cellulose content. Specifically, in the present study, the subject addressed was the hydrolysis of the sisal pulp, using sulfuric acid as the catalyst. To assess the influence of parameters such as the concentration of the sulfuric acid and the temperature during this process, the pulp was hydrolyzed with various concentrations of sulfuric acid (30–50%) at 70 °C and with 30% acid (v/v) at various temperatures (60–100 °C). During hydrolysis, aliquots were withdrawn from the reaction media, and the solid (non-hydrolyzed pulp) was separated from the liquid (liquor) by filtering each aliquot. The sugar composition of the liquor was analyzed by HPLC, and the non-hydrolyzed pulps were characterized by viscometry (average molar mass), and X-ray diffraction (crystallinity). The results support the following conclusions: acid hydrolysis using 30% H₂SO₄ at 100 °C can produce sisal microcrystalline cellulose and the conditions that led to the largest glucose yield and lowest decomposition rate were 50% H₂SO₄ at 70 °C. In summary, the study of sisal pulp hydrolysis using concentrated acid showed that certain conditions are suitable for high recovery of xylose and good yield of glucose. Moreover, the unreacted cellulose can be targeted for different applications in bio-based materials. A kinetic study based on the glucose yield was performed for all reaction conditions using the kinetic model proposed by Saeman. The results showed that the model adjusted to all 30–35% H₂SO₄ reactions but not to greater concentrations of sulfuric acid. The present study is part of an ongoing research program, and the results reported here will be used as a comparison against the results obtained when using treated sisal pulp as the starting material.     

Pretreatment with ammonia water for enzymatic hydrolysis of corn husk, bagasse, and switchgrass

Bagasse, corn husk, and switchgrass were pretreated with ammonia water to enhance enzymatic hydrolysis. The sample (2 g) was mixed with 1–6 mL ammonia water (25–28% ammonia) and autoclaved at 120°C for 20 min. After treatment, the product was vacuum-dried to remove ammonia gas. The dried solid could be used immediately in the enzymatic hydrolysis without washing. The enzymatic hydrolysis was effectively improved with more than 0.5 and 1 mL ammonia water/g for corn husk and bagasse, respectively. In bagasse, glucose, xylose, and xylobiose were the main products. The adsorption of CMCase and xylanase was related to the initial rate of enzymatic hydrolysis. In corn husks, arabinoxylan extracted by pretreatment was substantially unhydrolyzed because of the high ratio of arabinose to xylose (0.6). The carbohydrate yields from cellulose and hemicellulose were 72.9% and 82.4% in bagasse, and 86.2% and 91.9% in corn husk, respectively. The ammonia/water pretreatment also benefited from switchgrass (Miscanthus sinensis and Solidago altissima L.) hydrolysis.     

Stagewise Dilute-Acid Pretreatment and Enzyme Hydrolysis of Distillers’ Grains and Corn Fiber

Distillers’ grains and corn fiber are the coproducts of the corn dry grind and wet milling industries, respectively. Availability of distillers’ grains and corn fiber at the ethanol plant and their high levels of lignocellulosic material make these coproducts attractive feedstocks for conversion to ethanol. In this study, dilute sulfuric acid hydrolysis of these coproducts was investigated in a multistage scheme. After the completion of each pretreatment stage, the liquid substrate was separated and reused in the succeeding pretreatment stage with a fresh substrate. The substrate from each stage was also subjected to enzyme hydrolysis in a separate experiment. The sulfuric acid concentration and the substrate loading were maintained at 1.0 vol% and 15.0 wt.%, respectively, and the temperature was maintained at 120 °C in all the experiments. Experiments were also performed to study the effect of removing oil from the samples prior to the pretreatment. The highest concentration of monomeric sugars (MS) was observed when three stages of pretreatment were followed by the enzyme reaction. The enzyme hydrolysis of the three-stage pretreated dried distillers’ grains and corn fiber yielded 122.6 ± 5.8 and 184.5 ± 4.1 mg/mL of MS, respectively. The formation of inhibitory products was also monitored.     

Optimizing Dilute-Acid Pretreatment of Rapeseed Straw for Extraction of Hemicellulose

Biological conversion of biomass into fuels and chemicals requires hydrolysis of the polysaccharide fraction into monomeric sugars prior to fermentation. Hydrolysis can be performed enzymatically or with mineral acids. In this study, dilute sulfuric acid was used as a catalyst for the pretreatment of rapeseed straw. The purpose of this study is to optimize the pretreatment process in a 15-mL bomb tube reactor and investigate the effects of the acid concentration, temperature, and reaction time. These parameters influence hemicellulose removal and production of sugars (xylose, glucose, and arabinose) in the hydrolyzate as well as the formation of by-products (furfural, 5-hydroxymethylfurfural, and acetic acid). Statistical analysis was based on a model composition corresponding to a 3³ orthogonal factorial design and employed the response surface methodology to optimize the pretreatment conditions, aiming to attain maximum xylan, mannan, and galactan (XMG) extraction from hemicellulose of rapeseed straw. The obtained optimum conditions were: H₂SO₄ concentration of 1.76% and temperature of 152.6 °C with a reaction time of 21 min. Under these optimal conditions, 85.5% of the total sugar was recovered after acid hydrolysis (78.9% XMG and 6.6% glucan). The hydrolyzate contained 1.60 g/L glucose, 0.61 g/L arabinose, 10.49 g/L xylose, mannose, and galactose, 0.39 g/L cellobiose, 0.94 g/L fructose, 0.02 g/L 1,6-anhydro-glucose, 1.17 g/L formic acid, 2.94 g/L acetic acid, 0.04 g/L levulinic acid, 0.04 g/L 5-hydroxymethylfurfural, and 0.98 g/L furfural.