Effect of corn stover concentration on rheological characteristics

Corn stover, a well-known example of lignocellulosic biomass, is a potential renewable feed for bioethanol production. Dilute sulfuric acid pretreatment removes hemicellulose and makes the cellulose more susceptible to bacterial digestion. The rheologic properties of corn stover pretreated in such a manner were studied. The Power Law parameters were sensitive to corn stover suspension concentration becoming more non-Newtonian with slope n, ranging from 0.92 to 0.05 between 5 and 30% solids. The Casson and the Power Law models described the experimental data with correlation coefficients ranging from 0.90 to 0.99 and 0.85 to 0.99, respectively. The yield stress predicted by direct data extrapolation and by the Herschel-Bulkley model was similar for each concentration of corn stover tested.     

Fermentation of Acid-pretreated Corn Stover to Ethanol Without Detoxification Using Pichia stipitis

In this work, the effect of adaptation on P. stipitis fermentation using acid-pretreated corn stover hydrolyzates without detoxification was examined. Two different types of adaptation were employed, liquid hydrolyzate and solid state agar adaptation. Fermentation of 12.5% total solids undetoxified acid-pretreated corn stover was performed in shake flasks at different rotation speeds. At low rotation speed (100 rpm), both liquid hydrolyzate and solid agar adaptation highly improved the sugar consumption rate as well as ethanol production rate compared to the wild-type strains. The fermentation rate was higher for solid agar-adapted strains compared to liquid hydrolyzate-adapted strains. At a higher rotation speed (150 rpm), there was a faster sugar consumption and ethanol production for both the liquid-adapted and the wild-type strains. However, improvements in the fermentation rate between the liquid-adapted and wild strains were less pronounced at the high rotation speed.     

Effects of temperature and moisture on dilute-acid steam explosion pretreatment of corn stover and cellulase enzyme digestibility

Corn stover is emerging as a viable feedstock for producing bioethanol from renewable resources. Dilute-acid pretreatment of corn stover can solubilize a significant portion of the hemicellulosic component and enhance the enzymatic digestibility of the remaining cellulose for fermentation into ethanol. In this study, dilute H₂SO₄ pretreatment of corn stover was performed in a steam explosion reactor at 160°C, 180°C, and 190°C, approx 1 wt% H₂SO₄, and 70-s to 840-s residence times. The combined severity (Log₁₀ [R _o ] - pH), an expression relating pH, temperature, and residence time of pretreatment, ranged from 1.8 to 2.4. Soluble xylose yields varied from 63 to 77% of theoretical from pretreatments of corn stover at 160 and 180°C. However, yields >90% of theoretical were found with dilute-acid pretreatments at 190°C. A narrower range of higher combined severities was required for pretreatment to obtain high soluble xylose yields when the moisture content of the acid-impregnated feedstock was increased from 55 to 63 wt%. Simultaneous saccharification and fermentation (SSF) of washed solids from corn stover pretreated at 190°C, using an enzyme loading of 15 filter paper units (FPU)/g of cellulose, gave ethanol yields in excess of 85%. Similar SSF ethanol yields were found using washed solid residues from 160 and 180°C pretreatments at similar combined severities but required a higher enzyme loading of approx 25 FPU/g of cellulose.     

Improved multivariate calibration models for corn stover feedstock and dilute-acid pretreated corn stover

We have studied rapid calibration models to predict the composition of a variety of biomass feedstocks by correlating near-infrared (NIR) spectroscopic data to compositional data produced using traditional wet chemical analysis techniques. The rapid calibration models are developed using multivariate statistical analysis of the spectroscopic and wet chemical data. This work discusses the latest versions of the NIR calibration models for corn stover feedstock and dilute-acid pretreated corn stover. Measures of the calibration precision and uncertainty are presented. No statistically significant differences (p = 0.05) are seen between NIR calibration models built using different mathematical pretreatments. Finally, two common algorithms for building NIR calibration models are compared; no statistically significant differences (p = 0.05) are seen for the major constituents glucan, xylan, and lignin, but the algorithms did produce different predictions for total extractives. A single calibration model combining the corn stover feedstock and dilute-acid pretreated corn stover samples gave less satisfactory predictions than the separate models.     

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.     

Cellulase retention and sugar removal by membrane ultrafiltration during lignocellulosic biomass hydrolysis

Technologies suitable for the separation and reuse of cellulase enzymes during the enzymatic saccharification of pretreated corn stover are investigated to examine the economic and technical viability of processes that promote cellulase reuse while removing inhibitory reaction products such as glucose and cellobiose. The simplest and most suitable separation is a filter with relatively large pores on the order of 20–25 mm that retains residual corn stover solids while passing reaction products such as glucose and cellobiose to form a sugar stream for a variety of end uses. Such a simple separation is effective because cellulase remains bound to the residual solids. Ultrafiltration using 50-kDa polyethersulfone membranes to recover cellulase enzymes in solution was shown not to enhance further the saccharification rate or overall conversion. Instead, it appears that the necessary cellulase enzymes, including β-glucosidase, are tightly bound to the substrate; when fresh corn stover is contacted with highly washed residual solids, without the addition of fresh enzymes, glucose is generated at a high rate. When filtration was applied multiple times, the concentration of inhibitory reaction products such as glucose and cellobiose was reduced from 70 to 10 g/L. However, an enhanced saccharification performance was not observed, most likely because the concentration of the inhibitory products remained too high. Further reduction in the product concentration was not investigated, because it would make the reaction unnecessarily complex and result in a product stream that is much too dilute to be useful. Finally, an economic analysis shows that reuse of cellulase can reduce glucose production costs, especially when the enzyme price is high. The most economic performance is shown to occur when the cellulase enzyme is reused and a small amount of fresh enzyme is added after each separation step to replace lost or deactivated enzyme.     

Pretreatment of corn stover by soaking in aqueous ammonia

Soaking in aqueous ammonia (SAA) was investigated as a pretreatment method for corn stover. In this method, the feedstock was soaked in aqueous ammonia over an extended period (10–60 d) at room temperature. It was done without agitation at atmospheric pressure. SAA treatment removed 55–74% of the lignin, but retained nearly 100% of the glucan and 85% of the xylan. The xylan remaining in the corn stover after SAA treatment was hydrolyzed along with the glucan by xylanase present in the Spezyme CP enzyme. In the simultaneous saccharification and fermentation (SSF) test of SAA-treated corn stover, using S. cerevisiae (D₅A), an ethanol yield of 73% of theoretical maximum was obtained on the basis of the glucan content in the treated corn stover. The accumulation of xylose in the SSF appears to inhibit the cellulase activity on glucan hydrolysis, which limits the yield of ethanol. In the simultaneous saccharification and co-fermentation (SSCF) test, using recombinant E. coli (KO11), both the glucan and xylose were effectively utilized, resulting in on overall ethanol yield of 77% based on the glucan and xylan content of the substrate. When the SSCF process is used, the fact that the xylan fraction is retained during pretreatment is a desirable feature since the overall bioconversion can be carried out in a single step without separate recovery of xylose from the pretreatment liquid.     

Can delignification decrease cellulose digestibility in acid pretreated corn stover?

It has previously been shown that the improved digestibility of dilute acid pretreated corn stover is at least partially due to the removal of xylan and the consequent increase in accessibility of the cellulose to cellobiohydrolase enzymes. We now report on the impact that lignin removal has on the accessibility and digestibility of dilute acid pretreated corn stover. Samples of corn stover were subjected to dilute sulfuric acid pretreatment with and without simultaneous (partial) lignin removal. In addition, some samples were completely delignified after the pretreatment step using acidified sodium chlorite. The accessibility and digestibility of the samples were tested using a fluorescence-labeled cellobiohydrolase (Trichoderma reesei Cel7A) purified from a commercial cellulase preparation. Partial delignification of corn stover during dilute acid pretreatment was shown to improve cellulose digestibility by T. reesei Cel7A; however, decreasing the lignin content below 5% (g g⁻¹) by treatment with acidified sodium chlorite resulted in a dramatic reduction in cellulose digestibility. Importantly, this effect was found to be enhanced in samples with lower xylan contents suggesting that the near complete removal of xylan and lignin may cause aggregation of the cellulose microfibrils resulting in decreased cellulase accessibility.     

Extraction and characterization of native heteroxylans from delignified corn stover and aspen

Dimethylsulfoxide-solubilized polysaccharides from delignified corn stover and aspen were characterized. The biomass was delignified by two different techniques; a standard acid chlorite and a pulp and paper QPD technique comprising chelation (Q), peroxide (P), and acid-chlorite (D). Major polysaccharides in all fractions were diversely substituted xylan. Xylan acetylation was intact after chlorite delignification and, as expected, xylan from QPD-delignified fraction was de-acetylated by the alkaline peroxide step. The study of DMSO-extractable xylans from chlorite-delignified biomass revealed major differences in native acetylation patterns between corn stover and aspen xylan. Xylan from cell walls of corn stover contains 2-O- and 3-O-mono-acetylated xylan and [MeGlcA-α-(1 → 2)][3-OAc]-xylp units. In addition, aspen xylan also contains 2,3-di-O-acetylated xylose. 1,4-β-d-xylp residues substituted with MeGlcA at O-2 position are absent in chlorite-delignified aspen xylan. Sugar composition in accord with NMR-spectroscopic data indicated that corn stover xylan is arabinosylated while aspen xylan is not. We have shown that corn stover xylan has similar structure with xylans from other plants of Poales order. No evidence was found to indicate the presence of 1,4-β-d-[MeGlcA-α-(1 → 2)][Ara-α-(1 → 3)]-xylp in corn stover xylan fractions.     

Reintroduced solids increase inhibitor levels in a pretreated corn stover hydrolysate

Following detoxification of the liquid hydrolysate produced in a corn stover pretreatment process, inhibitor levels are seen to increase with the re-addition of solids for the ensuing hydrolysis and fermentation processes. The solids that were separated from the slurry before detoxification of the liquor contain approx 60% (w/w) moisture, and contamination occurs owing to the diffusion of inhibitors from the moisture entrained in the porous structure of the corn stover solids into the bulk fluid. This evidence suggests the need for additional separation and detoxification steps to purge residual inhibitors entrained in the moisture in the solids. An overliming process to remove furans from the hydrolysate failed to reduce total organic acids concentration, so acids were removed by treatment with an activated carbon powder. Smaller carbon doses proved more efficient in removing organic acids in terms of grams of acid removed per gram of carbon powder. Sugar adsorption by the activated carbon powder was minimal.     

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.     

Model-based fed-batch for high-solids enzymatic cellulose hydrolysis

While many kinetic models have been developed for the enzymatic hydrolysis of cellulose, few have been extensively applied for process design, optimization, or control. High-solids operation of the enzymatic hydrolysis of lignocellulose is motivated by both its operation decreasing capital costs and increasing product concentration and hence separation costs. This work utilizes both insights obtained from experimental work and kinetic modeling to develop an optimization strategy for cellulose saccharification at insoluble solids levels greater than 15% (w/w), where mixing in stirred tank reactors (STRs) becomes problematic. A previously developed model for batch enzymatic hydrolysis of cellulose was modified to consider the effects of feeding in the context of fed-batch operation. By solving the set of model differential equations, a feeding profile was developed to maintain the insoluble solids concentration at a constant or manageable level throughout the course of the reaction. Using this approach, a stream of relatively concentrated solids (and cellulase enzymes) can be used to increase the final sugar concentration within the reactor without requiring the high initial levels of insoluble solids that would be required if the operation were performed in batch mode. Experimental application in bench-scale STRs using a feed stream of dilute acid-pretreated corn stover solids and cellulase enzymes resulted in similar cellulose conversion profiles to those achieved in batch shake-flask reactors where temperature control issues are mitigated. Final cellulose conversions reached approximately 80% of theoretical for fed-batch STRs fed to reach a cumulative solids level of 25% (w/w) initial insoluble solids.     

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.     

Heterologous Expression of Two Ferulic Acid Esterases from Penicillium funiculosum

Two recombinant ferulic acid esterases from Penicillium funiculosum produced in Aspergillus awamori were evaluated for their ability to improve the digestibility of pretreated corn stover. The genes, faeA and faeB, were cloned from P. funiculosum and expressed in A. awamori using their native signal sequences. Both enzymes contain a catalytic domain connected to a family 1 carbohydrate-binding module by a threonine-rich linker peptide. Interestingly, the carbohydrate binding-module is N-terminal in FaeA and C-terminal in FaeB. The enzymes were purified to homogeneity using column chromatography, and their thermal stability was characterized by differential scanning microcalorimetry. We evaluated both enzymes for their potential to enhance the cellulolytic activity of purified Trichoderma reesei Cel7A on pretreated corn stover.     

Conversion of aqueous ammonia-treated corn stover to lactic acid by simultaneous saccharification and cofermentation

Treatment of corn stover with aqueous ammonia removes most of the structural lignin, whereas retaining the majority of the carbohydrates in the solids. After treatment, both the cellulose and hemicellulose in corn stover become highly susceptible to enzymatic digestion. In this study, corn stover treated by aqueous ammonia was investigated as the substrate for lactic acid production by simultaneous saccharification and cofermentation (SSCF). A commercial cellulase (Spezyme-CP) and Lactobacillus pentosus American Type Culture Collection (ATCC) 8041 (Spanish Type Culture Collection [CECT]-4023) were used for hydrolysis and fermentation, respectively. In batch SSCF operation, the carbohydrates in the treated corn stover were converted to lactic acid with high yields, the maximum lactic acid yield reaching 92% of the stoichiometric maximum based on total fermentable carbohydrates (glucose, xylose, and arabinose). A small amount of acetic acid was also produced from pentoses through the phosphoketolase pathway. Among the major process variables for batch SSCF, enzyme loading and the amount of yeast extract were found to be the key factors affecting lactic acid production. Further tests on nutrients indicated that corn steep liquor could be substituted for yeast extract as a nitrogen source to achieve the same lactic acid yield. Fed-batch operation of the SSCF was beneficial in raising the concentration of lactic acid to a maximum value of 75.0 g/L.     

Retrofit of CD-6 (smith) impeller in fermentation vessels

We extended prior studies on the influence of impeller type on fermentation performance to include a novel low-power-number, high-efficiency radial flow impeller, the CD-6, possessing six curved blades on a disk turbine. Dual impeller combinations of CD-6/CD-6, CD-6/Maxflo T, and CD-6/HE-3 were compared with Rushton/Rushton and Maxflo T/Maxflo T base cases. Qualitative comparisons of unaerated and aerated power draw in both water and glycerol were conducted. These suggested minimal power drops with aeration for dual CD-6 impellers and hybrids containing the CD-6 impeller design. We also examined fermentation performance for Streptomyces and Glarea secondary metabolite fermentations. A qualitative comparison of the data suggested that dual CD-6 impellers and hybrids containing the CD-6 impeller design resulted in reasonable power draws, improved mass transfer rates with airflow increases, and acceptable peak titers. These arrangements may warrant further study under a wider range of production conditions.     

Optimization of steam pretreatment of corn stover to enhance enzymatic digestibility

Among the available agricultural byproducts, corn stover, with its yearly production of 10 million t (dry basis), is the most abundant promising raw material for fuel ethanol production in Hungary. In the United States, more than 216 million to fcorn stover is produced annually, of which a portion also could possibly be collected for conversion to ethanol. However, a network of lignin and hemicellulose protects cellulose, which is the major source of fermentable sugars in corn stover (approx 40% of the dry matter [DM]). Steam pretreatment removes the major part of the hemicellulose from the solid material and makes the cellulose more susceptible to enzymatic digestion. We studied 12 different combinations of reaction temperature, time, and pH during steam pretreatment. The best conditions (200°C, 5 min, 2% H₂SO₄) increased the enzymatic conversion (from cellulose to glucose) of corn stover more then four times, compared to untreated material. However, steam pretreatment at 190°C for 5 min with 2% sulfuric acid resulted in the highest overall yield of sugars, 56.1 g from 100 g of untreated material (DM), corresponding to 73% of the theoretical. The liquor following steam explosion was fermented using Saccharomyces cerevisiae to investigate the inhibitory effect of the pretreatment. The achieved ethanol yield was slightly higher than that obtained with a reference sugar solution. This demonstrates that baker's yeast could adapt to the pretreated liquor and ferment the glucose to ethanol efficiently.     

Relative fermentability of lignocellulosic diluteacid prehydrolysates

The relative toxicity of the combined nonxylose components in prehydrolysates derived from three different lignocellulosic biomass feedstocks was determined. One woody (poplar) and two herbaceous (corn stover and switchgrass) feedstocks were dilute-acid pretreated under temperature and acid conditions chosen to optimize xylose recovery in the liquid prehydrolysate; xylose yields averaged 96,89,and 87% of theoretical for switchgrass,corn stover,and poplar,respectively. Prehydrolysates from each feedstock were neutralized,adjusted to equivalent xylose concentrations,and bioassayed for toxicity,using a standardized fermentation protocol withPichia stipitis NRRL 11545. Full time-courses for ethanol production (30-60 h) clearly illustrate the distinct inhibitory effects of prehydrolysates from different feedstocks. The relative toxicity of the prehydrolysates,ranked in order of decreasing toxicity,is poplar-derived prehydrolysates > switchgrass-derived prehydrolysates > corn stover-derived prehydrolysates. The inhibition of ethanol production appeared to be the result of a general slowdown of yeast metabolism,rather than the result of the production of alternative, nonethanol end products. Ethanol yields averaged 74,83,and 88% of control values for poplar,switchgrass,and corn stover prehydrolysates, respectively. Volumetric ethanol productivities (g ethanol L/h) averaged 32,70,and 102% of control values for poplar,switchgrass,and corn stover prehydrolysates,respectively. Ethanol productivities correlated closely with acetate concentrations in the prehydrolysates; however, regression lines correlating acetate concentrations and ethanol productivities were found to be feedstock-dependent. Oregon State University Agricultural Experiment Station Technical Publication Number 11114     

The Role of Product Inhibition as a Yield-Determining Factor in Enzymatic High-Solid Hydrolysis of Pretreated Corn Stover

Industrially, enzymatic hydrolysis of lignocellulose at high solid content is preferable over low solids due to a reduction in processing costs. Unfortunately, the economic benefits are counteracted by a linear decrease in yield with solid content, referred to as the “solid effect” in the literature. In the current study, we investigate the contribution of product inhibition to the solid effect (7–33 % solids). Product inhibition was measured directly by adding glucose to high-solid hydrolysis samples and indirectly through variation of water content and beta-glucosidase concentration. The results suggest that the solid effect is mainly controlled by product inhibition under the given experimental conditions (washed pretreated corn stover as substrate). Cellobiose was found to be approximately 15 times more inhibitory than glucose on a molar scale. However, considering that glucose concentrations are at least 100 times higher than cellobiose concentrations under industrial conditions, glucose inhibition of cellulases is suggested to be the main cause of the solid effect.     

Reaction kinetics of stover liquefaction in recycled stover polyol

The purpose of this research was to study the kinetics of liquefaction of crop residues. The liquefaction of corn stover in the presence of ethylene glycol and ethylene carbonate using sulfuric acid as a catalyst was studied. It was found that the liquefaction yield was a function of ratio of solvent to corn stover, temperature, residence time, and amount of catalyst. Liquefaction of corn stover was conducted over a range of conditions encompassing residence times of 0–2.5 h, temperatures of 150–170°C, sulfuric acid concentrations of 2–4% (w/w), and liquefaction reagent/corn stover ratio of 1–3. The liquefaction rate constants for individual sets of conditions were examined using a first-order reaction model. Rate constant increased with the increasing of liquefaction temperature, catalyst content, and liquefaction reagent/corn stover ratio. Reuse of liquefied biomass as liquefying agent was also evaluated. When using recycled liquefied biomass instead of fresh liquefaction reagent, the conversion is reduced. It appeared that 82% of liquefaction yield was achieved after two times of reuse.