Dilute-sulfuric acid pretreatment of corn stover in pilot-scale reactor

Corn stover is a domestic feedstock that has potential to produce significant quantities of fuel ethanol and other bioenergy and biobased products. However, comprehensive yield and carbon mass balance information and validated kinetic models for dilute-sulfuric acid (H₂SO₄) pretreatment of corn stover have not been available. This has hindered the estimation of process economics and also limited the ability to perform technoeconomic modeling to guide research. To better characterize pretreatment and assess its kinetics, we pretreated corn stover in a continuous 1 t/d reactor. Corn stover was pretreated at 20% (w/w) solids concentration over a range of conditions encompassing residence times of 3–12 min, temperatures of 165–195°C, and H₂SO₄ concentrations of 0.5–1.4% (w/w). Xylan conversion yield and carbon mass balance data were collected at each run condition. Performance results were used to estimate kinetic model parameters assuming biphasic hemicellulose hydrolysis and a hydrolysis mechanism incorporating formation of intermediate xylo-oligomers. In addition, some of the pretreated solids were tested in a simultaneous saccharification and fermentation (SSF) process to measure the reactivity of their cellulose component to enzymatic digestion by cellulase enzymes. Monomeric xylose yields of 69–71% and total xylose yields (monomers and oligomers) of 70–77% were achieved with performance level depending on pretreatment severity. Cellulose conversion yields in SSF of 80–87% were obtained for some of the most digestible pretreated solids.     

Influence of High Solid Concentration on Enzymatic Hydrolysis and Fermentation of Steam-Exploded Corn Stover Biomass

Steam-exploded corn stover biomass was used as the substrate for fed-batch separate enzymatic hydrolysis and fermentation (SHF) to investigate the solid concentration ranging from 10% to 30% (w/w) on the lignocellulose enzymatic hydrolysis and fermentation. The treatment of washing the steam-exploded material was also evaluated by experiments. The results showed that cellulose conversion changed little with increasing solid concentration, and fermentation by Saccharomyces cerevisiae revealed a nearly same ethanol yield with the water-washed steam-exploded corn stover. For the washed material at 30% substrate concentration, i.e., 30% water insoluble solids (WIS), enzymatic hydrolysis yielded 103.3 g/l glucose solution and a cellulose conversion of 72.5%, thus a high ethanol level up to 49.5 g/l. With the unwashed steam-exploded corn stover, though a cellulose conversion of 70.9% was obtained in hydrolysis at 30% solid concentration (27.9% WIS), its hydrolysate did not ferment at all, and the hydrolysate of 20% solid loading containing 3.3 g/l acetic acid and 145 mg/l furfural already exerted a strong inhibition on the fermentation and ethanol production.     

Kinetics of Lime Pretreatment of Sugarcane Bagasse to Enhance Enzymatic Hydrolysis

The objective of this work was to determine the optimum conditions of sugarcane bagasse pretreatment with lime to increase the enzymatic hydrolysis of the polysaccharide component and to study the delignification kinetics. The first stage was an evaluation of the influence of temperature, reaction time, and lime concentration in the pretreatment performance measured as glucose release after hydrolysis using a 2³ central composite design and response surface methodology. The maximum glucose yield was 228.45 mg/g raw biomass, corresponding to 409.9 mg/g raw biomass of total reducing sugars, with the pretreatment performed at 90°C, for 90 h, and with a lime loading of 0.4 g/g dry biomass. The enzymes loading was 5.0 FPU/dry pretreated biomass of cellulase and 1.0 CBU/dry pretreated biomass of β-glucosidase. Kinetic data of the pretreatment were evaluated at different temperatures (60°C, 70°C, 80°C, and 90°C), and a kinetic model for bagasse delignification with lime as a function of temperature was determined. Bagasse composition (cellulose, hemicellulose, and lignin) was measured, and the study has shown that 50% of the original material was solubilized, lignin and hemicellulose were selectively removed, but cellulose was not affected by lime pretreatment in mild temperatures (60–90°C). The delignification was highly dependent on temperature and duration of pretreatment.     

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.     

Alkali (NaOH) Pretreatment of Switchgrass by Radio Frequency-based Dielectric Heating

Radio-frequency (RF)-based dielectric heating was used in the alkali (NaOH) pretreatment of switchgrass to enhance its enzymatic digestibility. Due to the unique features of RF heating (i.e., volumetric heat transfer, deep heat penetration of the samples, etc.), switchgrass could be treated on a large scale, high solid content, and uniform temperature profile. At 20% solid content, RF-assisted alkali pretreatment (at 0.1 g NaOH/g biomass loading and 90°C) resulted in a higher xylose yield than the conventional heating pretreatment. The enzymatic hydrolysis of RF-treated solids led to a higher glucose yield than the corresponding value obtained from conventional heating treatment. When the solid content exceeded 25%, conventional heating could not handle this high-solid sample due to the loss of fluidity, poor mixing, and heating transfer of the samples. As a result, there was a significantly lower sugar yield, but the sugar yield of the RF-based pretreatment process was still maintained at high levels. Furthermore, the optimal particle size and alkali loading in the RF pretreatment was determined as 0.25–0.50 mm and 0.25 g NaOH/g biomass, respectively. At alkali loading of 0.20–0.25 g NaOH/g biomass, heating temperature of 90^oC, and solid content of 20%, the glucose, xylose, and total sugar yield from the combined RF pretreatment and the enzymatic hydrolysis were 25.3, 21.2, and 46.5 g/g biomass, respectively.     

The effect of agitation speed,enzyme loading and substrate concentration on enzymatic hydrolysis of cellulose from brewer’s spent grain

Brewer’s spent grain components (cellulose, hemicellulose and lignin) were fractionated in a two-step chemical pretreatment process using dilute sulfuric acid and sodium hydroxide solutions. The cellulose pulp produced was hydrolyzed with a cellulolytic complex, Celluclast 1.5 L, at 45 °C to convert the cellulose into glucose. Several conditions were examined: agitation speed (100, 150 and 200 rpm), enzyme loading (5, 25 and 45 FPU/g substrate), and substrate concentration (2, 5 and 8% w/v), according to a 2³ full factorial design aiming to maximize the glucose yield. The obtained results were interpreted by analysis of variance and response surface methodology. The optimal conditions for enzymatic hydrolysis of brewer’s spent grain were identified as 100 rpm, 45 FPU/g and 2% w/v substrate. Under these conditions, a glucose yield of 93.1% and a cellulose conversion (into glucose and cellobiose) of 99.4% was achieved. The easiness of glucose release from BSG makes this substrate a raw material with great potential to be used in bioconversion processes.     

High solids simultaneous saccharification and fermentation of pretreated wheat straw to ethanol

Wheat straw was pretreated with dilute (0.5%) sulfuric acid at 140°C for 1 h. Pretreated straw solids were washed with deionized water to neutrality and then stored frozen at –20°C. The approximate composition of the pretreated straw solids was 64% cellulose, 33% lignin, and 2% xylan. The cellulose in the pretreated wheat straw solids was converted to ethanol in batch simultaneous saccharification and fermentation experiments at 37°C using cellulase enzyme fromTrichoderma reesei (Genencor 150 L) with or without supplementation with β–glucosidase fromAspergillus niger (Novozyme 188) to produce glucose sugar and the yeastSaccharomyces cerevisiae to ferment the glucose into ethanol. The initial cellulose concentrations were adjusted to 7.5, 10, 12.5, 15, 17.5, and 20% (w/w). Since wheat straw particles do not form slurries at these concentrations and cannot be mixed with conventional impeller mixers used in laboratory fermenters, a simple rotary fermenter was designed and fabricated for these experiments. The results of the simultaneous saccharification and fermentation (SSF) experiments indicate that the cellulose in pretreated wheat straw can be efficiently fermented into ethanol for up to a 15% cellulose concentration (24.4% straw concentration).     

Effect and Modeling of Glucose Inhibition and In Situ Glucose Removal During Enzymatic Hydrolysis of Pretreated Wheat Straw

The enzymatic hydrolysis of lignocellulosic biomass is known to be product-inhibited by glucose. In this study, the effects on cellulolytic glucose yields of glucose inhibition and in situ glucose removal were examined and modeled during extended treatment of heat-pretreated wheat straw with the cellulolytic enzyme system, Celluclast? 1.5 L, from Trichoderma reesei, supplemented with a β-glucosidase, Novozym? 188, from Aspergillus niger. Addition of glucose (0–40 g/L) significantly decreased the enzyme-catalyzed glucose formation rates and final glucose yields, in a dose-dependent manner, during 96 h of reaction. When glucose was removed by dialysis during the enzymatic hydrolysis, the cellulose conversion rates and glucose yields increased. In fact, with dialytic in situ glucose removal, the rate of enzyme-catalyzed glucose release during 48–72 h of reaction recovered from 20–40% to become ≈70% of the rate recorded during 6–24 h of reaction. Although Michaelis–Menten kinetics do not suffice to model the kinetics of the complex multi-enzymatic degradation of cellulose, the data for the glucose inhibition were surprisingly well described by simple Michaelis–Menten inhibition models without great significance of the inhibition mechanism. Moreover, the experimental in situ removal of glucose could be simulated by a Michaelis–Menten inhibition model. The data provide an important base for design of novel reactors and operating regimes which include continuous product removal during enzymatic hydrolysis of lignocellulose.     

Rheology of Dilute Acid Hydrolyzed Corn Stover at High Solids Concentration

The rheological properties of acid hydrolyzed corn stover at high solids concentration (20–35 wt.%) were investigated using torque rheometry. These materials are yield stress fluids whose rheological properties can be well represented by the Bingham model. Yield stresses increase with increasing solids concentration and decrease with increasing hydrolysis reaction temperature, acid concentration, and rheometer temperature. Plastic viscosities increase with increasing solids concentration and tend to decrease with increasing reaction temperature and acid concentration. The solids concentration dependence of the yield stress is consistent with that reported for other fibrous systems. The changes in yield stress with reaction conditions are consistent with observed changes in particle size. This study illustrates that torque rheometry can be used effectively to measure rheological properties of concentrated biomass.     

Effects of hemicellulose and lignin on enzymatic hydrolysis of cellulose from dairy manure

This study focused on the effect of hemicellulose and lignin on enzymatic hydrolysis of dairy manure and hydrolysis process optimization to improve sugar yield. It was found that hemicellulose and lignin in dairy manure, similar to their role in other lignocellulosic material, were major resistive factors to enzymatic hydrolysis and that the removal of either of them, or for best performance, both of them, improved the enzymatic hydrolysis of manure cellulose. This result combined with scanning electron microscope (SEM) pictures further proved that the accessibility of cellulose to cellulase was the most important feature to the hydrolysis. Quantitatively, fed-batch enzymatic hydrolysis of fiber without lignin and hemicellulose had a high glucose yield of 52% with respect to the glucose concentration of 17 g/L at a total enzyme loading of 1300 FPU/L and reaction time of 160 h, which was better than corresponding batch enzymatic hydrolysis.     

Effects of Substrate Loading on Enzymatic Hydrolysis and Viscosity of Pretreated Barley Straw

In this study, the applicability of a “fed-batch” strategy, that is, sequential loading of substrate or substrate plus enzymes during enzymatic hydrolysis was evaluated for hydrolysis of steam-pretreated barley straw. The specific aims were to achieve hydrolysis of high substrate levels, low viscosity during hydrolysis, and high glucose concentrations. An enzyme system comprising Celluclast and Novozyme 188, a commercial cellulase product derived from Trichoderma reesei and a β-glucosidase derived from Aspergillus niger, respectively, was used for the enzymatic hydrolysis. The highest final glucose concentration, 78 g/l, after 72 h of reaction, was obtained with an initial, full substrate loading of 15% dry matter weight/weight (w/w DM). Conversely, the glucose yields, in grams per gram of DM, were highest at lower substrate concentrations, with the highest glucose yield being 0.53 g/g DM for the reaction with a substrate loading of 5% w/w DM after 72 h. The reactions subjected to gradual loading of substrate or substrate plus enzymes to increase the substrate levels from 5 to 15% w/w DM, consistently provided lower concentrations of glucose after 72 h of reaction; however, the initial rates of conversion varied in the different reactions. Rapid cellulose degradation was accompanied by rapid decreases in viscosity before addition of extra substrate, but when extra substrate or substrate plus enzymes were added, the viscosities of the slurries increased and the hydrolytic efficiencies decreased temporarily.     

The effect of particle size on hydrolysis reaction rates and rheological properties in cellulosic slurries

The effect of varying initial particle sizes on enzymatic hydrolysis rates and rheological properties of sawdust slurries is investigated. Slurries with four particle size ranges (33 microm < x < or = 75 microm, 150 microm < x < or = 180 microm, 295 microm < x < or = 425 microm, and 590 microm < x < or = 850 microm) were subjected to enzymatic hydrolysis using an enzyme dosage of 15 filter paper units per gram of cellulose at 50 degrees C and 250 rpm in shaker flasks. At lower initial particle sizes, higher enzymatic reaction rates and conversions of cellulose to glucose were observed. After 72 h 50 and 55% more glucose was produced from the smallest size particles than the largest size ones, for initial solids concentration of 10 and 13% (w/w), respectively. The effect of initial particle size on viscosity over a range of shear was also investigated. For equivalent initial solids concentration, smaller particle sizes result in lower viscosities such that at a concentration of 10% (w/w), the viscosity decreased from 3000 cP for 150 microm < x < or = 180 microm particle size slurries to 61.4 cP for 33 microm < x < or = 75 microm particle size slurries. Results indicate particle size reduction may provide a means for reducing the long residence time required for the enzymatic hydrolysis step in the conversion of biomass to ethanol. Furthermore, the corresponding reduction in viscosity may allow for higher solids loading and reduced reactor sizes during large-scale processing.     

Kinetics of glucose decomposition during dilute-acid hydrolysis of lignocellulosic biomass

Recent research work in-house both at Auburn University and National Renewable Energy Laboratory has demonstrated that extremely low concentrations of acid (e.g., 0.05–0.2 wt% sulfuric acid) and high temperatures (e.g., 200–230°C) are reaction conditions that can be effectively applied for hydrolysis of the cellulosic component of biomass. These conditions are far from those of the conventional dilute-acid hydrolysis processes, and the kinetic data for glucose decomposition are not currently available. We investigated the kinetics of glucose decomposition covering pH values of 1.5–2.2 and temperatures of 180–230°C using glass ampoule reactors. The primary factors controlling glucose decomposition are the reaction medium, acid concentration, and temperature. Based on the experimental data, a kinetic model was developed and the best-fit kinetic parameters were determined. However, a consistent discrepancy in the rate of glucose disappearance was found between that of the model based on pure glucose data and that observed during the actual process of lignocellulosic biomass hydrolysis. This was taken as an indication that glucose recombines with acid-soluble lignin during the hydrolysis process, and this conclusion was incorporated accordingly into the overall model of glucose decomposition.     

Cellulose hydrolysis under extremely low sulfuric acid and high-temperature conditions

The kinetics of cellulose hydrolysis under extremely low acid (ELA) conditions (0.07 wt%) and at temperatures >200°C was investigated using batch reactors and bed-shrinking flow-through (BSFT) reactors. The maximum yield of glucose obtained from batch reactor experiments was about 60% for α-cellulose, which occurred at 205 and 220°C. The maximum glucose yields from yellow poplar feedstockswere substantially lower, falling in the range of 26–50%. With yellow poplar feedstocks, a large amount of glucose was unaccounted for at the latter phase of the batch reactions. It appears that a substantial amount of released glucose condenses with nonglucosidic substances. in liquid. The rate of glucan hydrolysis under ELA was relatively insensitive to temperature in batch experiments for all three substrates. This contradicts the traditional concept of cellulose hydrolysis and implies that additional factors influence the hydrolysis of glucan under ELA. Inexperiments using BSFT reactors, the glucose yields of 87.5, 90,3, and 90.8% were obtained for yellow poplar feedstocks at 205, 220, and 235°C, respectively. The hydrolysis rate for glucan was about three times higher with the BSFT than with the batch reactors. The difference of observed kinetics and performance data between the BSFT and the batch reactors was far above that predicted by the reactor theory.     

Optimization of the process of chemical hydrolysis of cellulose to glucose

We studied the acid hydrolysis of cellulose in an aqueous medium with the aim of maximizing glucose yield and minimizing the formation of by-products. The influence of reaction parameters such as temperature, acid concentration, acid strength and type of cellulose precursor on glucose yield was investigated. We observed that moderate reaction temperature and low acid concentration resulted in the highest glucose yield with little formation of levulinic acid. Strong acid (pKa < 0) is required to achieve high glucose yield. The crystallite size of the cellulose also affects its reactivity; cellulose with higher crystallite size is more resistant to hydrolysis catalyzed by acid. The highest selectivity for glucose over levulinic acid was recorded at a reaction temperature of 413 K and a sulfuric acid concentration in the range of 0.2–0.5 mol/L. Under these reaction conditions, no levulinic acid was detected, but the glucose yield reached 20 % in only 2 h.     

Effects of a non-ionic surfactant,Tween 20, on adsorption/desorption of saccharification enzymes onto/from lignocelluloses and saccharification rate

In this work, we examined the role of a non-ionic surfactant, Tween 20, on enzymatic hydrolysis of lignocelluloses. Delignified lignocelluloses (pine wood chip) were used as model substrates. Effects of Tween 20 on adsorption/desorption onto/from lignocelluloses with and without hydrolysis were evaluated respectively. Tween 20 lowered the non-biospecific adsorption of β-glucosidase and enhanced the bio-specific adsorption of cellulase. Tween 20 did not affect the liquid phase reaction (cellobiose hydrolysis). However, for the solid surface reaction (cellulose hydrolysis), cellulose conversion for 72 hrs was increased 9–21% and 1–8.5% for samples with high lignin contents (PI) and low lignin contents (PIII) by injection of Tween 20 (0.024–0.24 mM), respectively. Moreover, Tween 20 increased the cellulose conversion rate substantially. It is suggested that the increase of cellulase amount adsorbed due to the increase of effective cellulose surface by Tween 20 contribute to the enhancement of cellulose conversion.     

Effect of chip size on steam explosion pretreatment of softwood

Although considerable progress has been made in technology for converting lignocellulosic biomass into ethanol, substantial opportunities still exist toreduce production costs. In biomass pretreatment, reducing milling power is a technological improvement that will substantially lower production costs for ethanol. Improving sugar yield from hemicellulose hydrolysis would also reduce ethanol production costs. Thus, it would be desirable to test innovative pretreatment conditions to improve the economics by reducing electrical power of the milling stage and by optimizing pretreatment recovery of hemicellulose, as well as to enhance cellulose hydrolysis. The objective of this study was to evaluate the effect of chip size (2–5, 5–8, and 8–12 mm) on steam-explosion pretreatment (190 and 210°C, 4 and 8 min) of softwood (Pinus pinater).     

Interactive metabolic regulations during biomethanation ofLeucaena leucocephala

Biomethanation of leaves of the legumeL. leucocephala operated in batch reactors at different input volatile solids (12–18 g/L) proceeded in distinct metabolic phases. An initial cellulolytic phase of 4 d was followed by an early and active methanogenic phases (5–21 d) and a terminal phase of low-rate methanogenesis. Hydrolysis of cellulose was concentration-dependent and resulted in increased volatile acid levels. The trend of changes showed some variations at different input volatile solids. The changes in the levels of volatile acids followed an oscillatory pattern. The controlled rate of cellulose hydrolysis, levels of volatile acids, and steady-state levels of soluble carbohydrates and reducing sugars observed during active methanogenesis are indicative of interactive metabolic regulations.     

Cellulose hydrolysis using zinc chloride as a solvent and catalyst

Cellulose gel with < 10% of crystallinity was prepared by treatment of microcrystalline cellulose, Avicel, with zinc chloride solution at a ratio of zinc chloride to cellulose from 1.5 to 18 (w/w). The presence of zinc ions in the cellulose gels enhanced the rate of hydrolysis and glucose yield. The evidence obtained from X-ray diffraction, iodine absorption experiments; and Nuclear Magnetic Resonance spectra analysis suggested the presence of zinc-cellulose complex after Avicel was treated with zinc chloride. Zinc-cellulose complex was more susceptible to hydrolysis than amorphous cellulose. Under the experimental condition, cellulose gels with zinc ions were hyrolyzed to glucose with 95% theoretical yield and a concentration of 14% (w/v) by cellulases within 20 h. The same gel was hydrolyzed by acid to glucose with 91.5% yield and a concentration of 13.4% (w/v).     

Xylanase and cellulase production byAspergillus fumigatus

When grown on a purified cellulose such as CF11 cellulose,Aspergillus fumigatus produces mainly exoglucanase (Avicelase) and endoglucanase (CMCase) with small amounts of Β-glucosidase and xylanase. In such cultures, the pH drops to 3–3.5 after 2 d incubation, which may account for the low levels of Β-glucosidase. The amounts of extracellular enzymes produced are larger when the organism is grown on hay or straw than when grown on CF11 cellulose. In particular, CMCase levels increase approximately seven times and xylanase levels increase 40–50 times. In such cultures the pH remains fairly constant at 6–7 over the 10-d incubation period used and so Β-glucosidase levels are also increased. Extraction of the hay or straw substrate with ethanol had little effect on enzyme production and so there appears to be no soluble material present that influences enzyme production. The organism produced elevated levels of CMCase and xylanase on barley straw, oat straw, and wheat straw, there being little difference between the varieties of each tested. However, grasses dried at elevated temperatures (260–500‡C) gave enzyme levels similar to CF11 cellulose. Similarly, chemical delignification of hay or straw gave enzyme levels similar to CF11 cellulose. Thus, both these treatments must lead to degradation of the hemicellulose present in the substrate. A. fumigatus was able to grow on a number of laboratory prepared and commercially available xylans (hay, barley straw, oat straw, and larch) as a pelletted mycelium. In all cases xylanase levels were increased 10–30 times over CF11 cellulose as substrate, but CMCase levels were similar to those with CF11 cellulose as substrate. Β-Glucosidase in most cases was not detectable, probably because the pH fell to 3–3.5 during incubation. Thus it appears that cellulase and xylanase can be independently induced in this organism. The optimum incubation time at 37‡C for xylanase production was 4–7 d and the optimum concentration of hay as substrate was 4–5%, even though this produces a very thick slurry that does not shake well.