Hydrolyzabilities of Different Corn Stover Fractions After Aqueous Ammonia Pretreatment

The effect of aqueous ammonia pretreatment on the hydrolysis of different corn stover fractions (rind, husk, leaf, and pith) by xylanase (XYL) with cellulases (CELs) was evaluated. The aqueous ammonia pretreatment had excellent delignification ability (above 66 %) for different corn stover fractions. The corn rind exhibited the lowest susceptibility to aqueous ammonia pretreatment. The pretreated rind showed the lowest hydrolyzability by CEL and XYL, which was supported by a high content of crystalline cellulose in the hydrolyzed residues of rind, as confirmed by X-ray diffraction (XRD). With the addition of 1 mg XYL/g dry matter, a high glucose yield (above 90 %) could be obtained from the pretreated rind by CEL. The results revealed that a high hydrolyzate yield of corn rind after aqueous ammonia pretreatment could be obtained with 1 mg xylanase/g dry matter, showing that aqueous ammonia pretreatment and xylanase addition to cellulases have great potential for the efficient hydrolysis of corn stover without previous fractionation.     

Influence of ionic-liquid incubation temperature on changes in cellulose structure,biomass composition,and enzymatic digestibility

Varying ionic liquid, 1-ethyl 3-methyl imidazolium acetate, pretreatment incubation temperature on lignocellulosic biomass substrates, corn stover, switchgrass and poplar, can have dramatic effects on the enzymatic digestibility of the resultant regenerated biomass. In order to delineate the chemical and physical changes resulting from the pretreatment process and correlate changes with enzymatic digestibility, X-ray powder and fiber diffraction, ¹³C cross polarization/magic angle spinning nuclear magnetic resonance spectroscopy, and compositional analysis was completed on poplar, corn stover and switchgrass samples. Optimal pretreatment incubation temperatures were most closely associated with the retention of amorphous substrates upon drying of regenerated biomass. Maximal glucan to glucose conversion for 24 h enzyme hydrolysis was observed for corn stover, switchgrass and poplar at ionic liquid incubation temperatures of 100, 110 and 120 °C, respectively. We hypothesize that effective pretreatment temperatures must attain lignin redistribution and retention of xylan for optimal enzyme digestibility.     

Pretreatment of corn stover by soaking in aqueous ammonia at moderate temperatures

Soaking in aqueous ammonia at moderate temperatures was investigated as a method of pretreatment for enzymatic hydrolysis as well as simultaneous saccharification and cofermentation (SSCF) of corn stover. The method involves batch treatment of the feedstock with aqueous ammonia (15-30 wt%) at 40-90 degrees C for 6-24 h. The optimum treatment conditions were found to be 15 wt% of NH(3), 60 degrees C, 1:6 of solid-to-liquid ratio, and 12 h of treatment time. The treated corn stover retained 100% glucan and 85% of xylan, but removed 62% of lignin. The enzymatic digestibility of the glucan content increased from 17 to 85% with 15 FPU/g-glucan enzyme loading, whereas the digestibility of the xylan content increased to 78%. The treated corn stover was also subjected to SSCF test using Spezyme-CP and recombinant Escherichia coli (KO11). The SSCF of the soaking in aqueous ammonia treated corn stover resulted in an ethanol concentration of 19.2 g/L from 3% (w/v) glucan loading, which corresponds to 77% of the maximum theoretical yield based on glucan and xylan.     

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.     

Pretreatment and Fractionation of Corn Stover by Soaking In Ethanol and Aqueous Ammonia

A new process for pretreatment of lignocellulosic biomass, designated the soaking in ethanol and aqueous ammonia (SEAA) process, was developed to improve hemicellulose preservation in solid form. In the SEAA process, an aqueous ammonia solution containing ethanol is used. Corn stover was treated with 15 wt.% ammonia at 1:9 solid–liquid ratio (by weight) at 60 °C for 24 h with ethanol added at 1, 5, 20, and 49 wt.% (balance was water). The extents by which xylan was solubilized with no ethanol and with ethanol added at 1, 5, 20, and 49 wt.% of the total liquid were 17.2%, 16.7%, 14.5%, 10.4%, and 6.3% of the original xylan, respectively. Thus, at the highest ethanol concentration used the loss of hemicellulose to the liquid phase was reduced by 63%. The digestibility of glucan and xylan in the pretreated corn stover samples by cellulase was not affected by ethanol addition of up to 20 wt.%. The enzymatic digestibility of the corn stover treated with 49 wt.% ethanol added was lower than the digestibility of the sample treated with no ethanol addition. Thus, based on these results, 20 wt.% was found to be the optimum ethanol concentration for use in the SEAA process for pretreatment of corn stover.     

Enhancing the Enzymatic Hydrolysis of Corn Stover by an Integrated Wet-milling and Alkali Pretreatment

An integrated wet-milling and alkali pretreatment was applied to corn stover prior to enzymatic hydrolysis. The effects of NaOH concentration in the pretreatment on crystalline structure, chemical composition, and reducing-sugar yield of corn stover were investigated, and the mechanism of increasing reducing-sugar yield by the pretreatment was discussed. The experimental results showed that the crystalline structure of corn stover was disrupted, and lignin was removed, while cellulose and hemicellulose were retained in corn stover by the pretreatment with 1% NaOH in 1 h. The reducing-sugar yield from the pretreated corn stovers increased from 20.2% to 46.7% when the NaOH concentration increased from 0% to 1%. The 1% NaOH pretreated corn stover had a holocellulose conversion of 55.1%. The increase in reducing-sugar yield was related to the crystalline structure disruption and delignification of corn stover. It was clarified that the pretreatment significantly enhanced the conversion of cellulose and hemicellulose in the corn stover to sugars.     

Microbial Pretreatment of Corn Stovers by Solid-State Cultivation of Phanerochaete chrysosporium for Biogas Production

The microbial pretreatment of corn stover and corn stover silage was achieved via the solid-state cultivation of Phanerochaete chrysosporium; pretreatment effects on the biodegradability and subsequent anaerobic production of biogas were investigated. The peak levels of daily biogas production and CH₄ yield from corn stover silage were approximately twice that of corn stover. Results suggested that ensiling was a potential pretreatment method to stimulate biogas production from corn stover. Surface morphology and Fourier-transform infrared spectroscopy analyses demonstrated that the microbial pretreatment of corn stover silage improved biogas production by 10.5 to 19.7 % and CH₄ yield by 11.7 to 21.2 % because pretreatment could decrease dry mass loss (14.2 %) and increase substrate biodegradability (19.9 % cellulose, 32.4 % hemicellulose, and 22.6 % lignin). By contrast, the higher dry mass loss in corn stover (55.3 %) after microbial pretreatment was accompanied by 54.7 % cellulose, 64.0 % hemicellulose, and 61.1 % lignin degradation but did not significantly influence biogas production.     

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.     

Pretreatment of corn stover by low-liquid ammonia recycle percolation process

A pretreatment method using aqueous ammonia was investigated with the intent of minimizing the liquid throughput. This process uses a flow-through packed column reactor (or percolation reactor). In comparison to the ammonia recycle percolation (ARP) process developed previously in our laboratory, this process significantly reduces the liquid throughput to one reactor void volume in packed bed (2.0–4.7 mL of liquid/g of corn stover) and, thus, is termed low-liquid ARP (LLARP). In addition to attaining short residence time and reduced energy input, this process achieves 59–70% of lignin removal and 48–57% of xylan retention. With optimum operation of the LLARP to corn stover, enzymatic digestibilities of 95, 90 and 86% were achieved with 60, 15, and 7.5 filter paper units/g of glucan, respectively. In the simultaneous saccharification and fermentation test of the LLARP samples using Saccharomyces cerevisiae (NREL-D₅A), an ethanol yield of 84% of the theoretical maximum was achieved with 6% (w/v) glucan loading. In the simultaneous saccharification and cofermentation (SSCF) test using recombinant Escherichia coli (KO11), both the glucan and xylan in the solid were effectively utilized, giving an overall ethanol yield of 109% of the theoretical maximum based on glucan, a clear indication that the xylan content was converted into ethanol. The xylooligomers existing in the LLARP effluent were not effectively hydrolyzed by cellulase enzyme, achieving only 60% of digestibility. SSCF of the treated corn stover was severely hampered when the substrate was supplemented with the LLARP effluent, giving only 56% the overall yield of ethanol. The effluent appears to significantly inhibit cellulase and microbial activities.     

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.     

Aqueous Ammonia Soaking of Switchgrass Followed by Simultaneous Saccharification and Fermentation

Simultaneous saccharification and fermentation (SSF) of switchgrass was performed following aqueous ammonia pretreatment. Switchgrass was soaked in aqueous ammonium hydroxide (30%) with different liquid–solid ratios (5 and 10 ml/g) for either 5 or 10 days. The pretreatment was carried out at atmospheric conditions without agitation. A 40–50% delignification (Klason lignin basis) was achieved, whereas cellulose content remained unchanged and hemicellulose content decreased by approximately 50%. The Sacccharomyces cerevisiae (D₅A)-mediated SSF of ammonia-treated switchgrass was investigated at two glucan loadings (3 and 6%) and three enzyme loadings (26, 38.5, and 77 FPU/g cellulose), using Spezyme CP. The percentage of maximum theoretical ethanol yield achieved was 72. Liquid–solid ratio and steeping time affected lignin removal slightly, but did not cause a significant change in overall ethanol conversion yields at sufficiently high enzyme loadings. These results suggest that ammonia steeping may be an effective method of pretreatment for lignocellulosic feedstocks.     

Optimization of dilute-acid pretreatment of corn stover using a high-solids percolation reactor

We have previously demonstrated that pretreatment of corn stover with dilute sulfuric acid can achieve high digestibility and efficient recovery of hemicellulose sugars with high yield and concentration. Further improvement of this process was sought in this work. A modification was made in the operation of the percolation reactor that the reactor is preheated under atmospheric pressure to remove moisture that causes autohydrolysis. This eliminated sugar decomposition during the preheating stage and led to a considerable improvement in overall sugar yield. In addition, liquid throughput was minimized to the extent that only one reactor void volume of liquid was collected. This was done to attain a high xylose concentration in the hydrolyzate. The optimum reaction and operating conditions were identified wherein near quantitative enzymatic digestibilities are obtained with enzyme loading of 15 FPU/g glucan. With a reduced enzyme loading of 5 FPU/g glucan, the enzymatic digestibility was decreased, but still reached a level of 92%. Decomposition of carbohydrates was extremely low as indicated by the measured glucan and xylan mass closures (recovered sugar plus unreacted) which were 98% and 94%, respectively. The data obtained in this work indicate that the digestibility is related to the extent of xylan removal.     

Impact of fluid velocity on hot water only pretreatment of corn stover in a flowthrough reactor

Flowthrough pretreatment with hot water only offers many promising features for advanced pretreatment of biomass, and a better understanding of the mechanisms responsible for flowthrough behavior could allow researchers to capitalize on key attributes while overcoming limitations. In this study, the effect of fluid velocity on the fate of total mass, hemicellulose, and lignin was evaluated for hot water only pretreatment of corn stover in tubular flow through reactors. Increasing fluid velocity significantly accelerated solubilization of total mass, hemicellulose, and lignin at early times. For example, when fluid velocity was increased from 2.8 to 10.7 cm/min, xylan removal increased from 60 to 82% for hot water only pretreatment of corn stover at 200°C after 8 min. At the same time, lignin removal increased from 30 to 46%. Dissolved hemicellulose was almost all in oligomeric form, and solubilization of hemicellulose was always accompanied by lignin release. The increase in removal of xylan and lignin with velocity, especially in the early reaction stage, suggests that chemical reaction is not the only factor controlling hemicellulose hydrolysis and that mass transfer and other physical effects may also play an important trole in hemicellulose and lignin degradation and removal.     

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.     

Fractionation of Corn Fiber Treated by Soaking in Aqueous Ammonia (SAA) for Isolation of Hemicellulose B and Production of C5 Sugars by Enzyme Hydrolysis

A process was developed to fractionate and isolate the hemicellulose B component of corn fiber generated by corn wet milling. The process consisted of pretreatment by soaking in aqueous ammonia followed by enzymatic cellulose hydrolysis, during which the hemicellulose B was solubilized by cleavage into xylo-oligosaccharides and subsequently recovered by precipitation with ethanol. The pretreatment step resulted in high retention of major sugars and improvement of subsequent enzymatic hydrolysis. The recovered hemicellulose B was hydrolyzed by a cocktail of enzymes that consisted of β-glucosidase, pectinase, xylanase, and ferulic acid esterase (FAE). Xylanase alone was ineffective, demonstrating yields of less than 2% of xylose and arabinose. The greatest xylose and arabinose yields, 44% and 53%, respectively, were obtained by the combination of pectinase and FAE. A mass balance accounted for 87% of the initially present glucan, 91% of the xylan, and 90% of the arabinan. The developed process offered a means for production of corn fiber gum as a value-added co-product and C5 sugars, which could be converted to other valuable co-products through fermentation in a corn wet-milling biorefinery.     

Ethanol fermentation of various pretreated and hydrolyzed substrates at low initial pH

Lignocellulosic materials represent an abundant feedstock for bioethanol production. Because of their complex structure pretreatment is necessary to make it accessible for enzymatic attack. Steam pretreatment with or without acid catalysts seems to be one of the most promising techniques, which has already been applied for large variety of lignocellulosics in order to improve enzymatic digestibility. During this process a range of toxic compounds (lignin and sugar degradation products) are formed which inhibit ethanol fermentation. In this study, the toxicity of hemicellulose hydrolysates obtained in the steam pretreatment of spruce, willow, and corn stover were investigated in ethanol fermentation tests using a yeast strain, which has been previously reported to have a resistance to inhibitory compounds generated during steam pretreatment. To overcome bacterial contamination, fermentations were carried out at low initial pH. The fermentability of hemicellulose hydrolysates of pretreated lignocellulosic substrates at low pH gave promising results with the economically profitable final 5 vol% ethanol concentration corresponding to 85% of theoretical. Adaptation experiments have shown that inhibitor tolerance of yeast strain can be improved by subsequent transfer of the yeast to inhibitory medium.     

Two-Stage Fractionation of Corn Stover Using Aqueous Ammonia and Hot Water

Hot water and aqueous ammonia fractionation of corn stover were used to separate hemicellulose and lignin and improve enzymatic digestibility of cellulose. A two-stage approach was used: The first stage was designed to recover soluble lignin using aqueous ammonia at low temperature, while the second stage was designed to recover xylan using hot water at high temperature. Specifically, the first stage employed a batch reaction using 15 wt.% ammonia at 60 °C, in a 1:10 solid:liquid ratio for 8 h, while the second stage employed a percolation reaction using hot water, 190–210 °C, at a 20 ml/min flow rate for 10 min. After fractionation, the remaining solids were nearly pure cellulose. The two-stage fractionation process achieved 68% lignin purity with 47% lignin recovery in the first stage, and 78% xylan purity, with 65% xylan recovery in the second stage. Two-stage treatment enhanced the enzymatic hydrolysis of remaining cellulose to 96% with 15 FPU/g of glucan using commercial cellulase enzymes. Enzyme hydrolyses were nearly completed within 12–24 h with the remaining solids fraction.     

Effect of sulfuric and phosphoric acid pretreatments on enzymatic hydrolysis of corn stover

The pretreatment of corn stover with H₂SO₄ and H₃PO₄ was investigated. Pretreatments were carried out from 30 to 120 min in a batch reactor at 121°C, with acid concentrations ranging from 0 to 2% (w/v) at a solid concentration of 5% (w/v). Pretreated corn stover was washed with distilled water until the filtrate was adjusted to pH 7.0, followed by surfactant swelling of the cellulosic fraction in a 0–10% (w/v) solution of Tween-80 at room temperature for 12 h. The dilute acid treatment proved to be a very effective method in terms of hemicellulose recovery and cellulose digetibility. Hemicellulose recovery was 62–90%, and enzymatic digestibility of the cellulose that remained in the solid was >80% with 2% (w/v) acid. In all cases studied, the performance of H₂SO₄ pretreatment (hemicellulose recovery and cellulose digestibility) was significantly better than obtained with H₃PO₄. Enzymatic hydrolysis was more effective using surfactant than without it, producing 10–20% more sugar. Furthermore, digestibility was investigated as a function of hemicellulose removal. It was found that digestibility was more directly related to hemicellulose removal than to delignification.     

Effects of Low Moisture Anhydrous Ammonia (LMAA) Pretreatment at Controlled Ammoniation Temperatures on Enzymatic Hydrolysis of Corn Stover

Corn stover was treated using low-moisture anhydrous ammonia (LMAA) at controlled ammoniation temperature. Moisturized corn stover (50 % moisture) was contacted with anhydrous ammonia (0.1 g NH₃/g-biomass) in a batch reactor at various temperatures (ambient to 150 °C). After ammoniation at elevated and controlled temperature, ammoniated corn stover was pretreated at various temperatures (60–150 °C) for 72–144 h. Change in composition was marginal at low pretreatment temperature but was relatively severe with pretreatment at high temperature (130–150 °C). The latter resulted in low enzymatic digestibility. It was also observed that extreme levels (either high or low) of residual ammonia affected enzymatic digestibility, while residual ammonia improved by 1.0–1.5 %. The LMAA method enhanced enzymatic digestibility compared to untreated corn stover (29.8 %). The highest glucan and xylan digestibility (84.1 and 73.6 %, respectively) was obtained under the optimal LMAA conditions (i.e., ammoniation at 70 °C for 20 min, followed by pretreatment at 90 °C for 48 h).     

Autohydrolysis of Tropical Agricultural Residues by Compressed Liquid Hot Water Pretreatment

Pretreatment is an essential step in biorefineries for improving digestibility of recalcitrant agricultural feedstocks prior to enzymatic hydrolysis to composite sugars, which can be further converted to fuels and chemicals. In this study, autohydrolysis by compressed liquid hot water (LHW) pretreatment of various tropical agricultural residues including sugarcane bagasse (BG), rice straw (RS), corn stover (CS), and empty palm fruit bunch (EPFB) was investigated. It was found that LHW pretreatment at 200 °C for 5–20 min resulted in high levels of hemicellulose solubilization into the liquid phase and marked improvement on enzymatic digestibility of the solid cellulose-enriched residues. The maximal yields of glucose and pentose were 409.8–482.7 mg/g and 81.1–174.0 mg/g of pretreated substrates, respectively. Comparative analysis based on severity factor showed varying susceptibility of biomass to LHW in the order of BG> RS> CS> EPFB. Structural analysis revealed surface modification of the pretreated biomass along with an increase in crystallinity index. Overall, 75.7–82.3 % yield of glucose and 27.4–42.4 % yield of pentose from the dried native biomass was recovered in the pretreated solid residues, while 18.3–29.7 % of pentoses were recovered in the liquid phase with dehydration by-product concentration under the threshold for ethanologens. The results suggest the potential of LHW as an efficient pretreatment strategy for implementation in biorefineries operated using various seasonal agricultural feedstocks.