Summary of findings from the Biomass Refining Consortium for Applied Fundamentals and Innovation (CAFI): corn stover pretreatment

The Biomass Refining Consortium for Applied Fundamentals and Innovation, with members from Auburn University, Dartmouth College, Michigan State University, the National Renewable Energy Laboratory, Purdue University, Texas A&M University, the University of British Columbia, and the University of California at Riverside, has developed comparative data on the conversion of corn stover to sugars by several leading pretreatment technologies. These technologies include ammonia fiber expansion pretreatment, ammonia recycle percolation pretreatment, dilute sulfuric acid pretreatment, flowthrough pretreatment (hot water or dilute acid), lime pretreatment, controlled pH hot water pretreatment, and sulfur dioxide steam explosion pretreatment. Over the course of two separate USDA- and DOE-funded projects, these pretreatment technologies were applied to two different corn stover batches, followed by enzymatic hydrolysis of the remaining solids from each pretreatment technology using identical enzyme preparations, enzyme loadings, and enzymatic hydrolysis assays. Identical analytical methods and a consistent material balance methodology were employed to develop comparative sugar yield data for each pretreatment and subsequent enzymatic hydrolysis. Although there were differences in the profiles of sugar release, with the more acidic pretreatments releasing more xylose directly in the pretreatment step than the alkaline pretreatments, the overall glucose and xylose yields (monomers + oligomers) from combined pretreatment and enzymatic hydrolysis process steps were very similar for all of these leading pretreatment technologies. Some of the water-only and alkaline pretreatment technologies resulted in significant amounts of residual xylose oligomers still remaining after enzymatic hydrolysis that may require specialized enzyme preparations to fully convert xylose oligomers to monomers.     

Microbial Lipid Production from Corn Stover via Mortierella isabellina

Microbial lipid is a promising source of oil to produce biofuel if it can be generated from lignocellulosic materials. Mortierella isabellina is a filamentous fungal species featuring high content of oil in its cell biomass. In this work, M. isabellina was studied for lipid production from corn stover. The experimental results showed that M. isabellina could grow on different kinds of carbon sources including xylose and acetate, and the lipid content reached to 35 % at C/N ratio of 20. With dilution, M. isabellina could endure inhibition effects by dilute acid pretreatment of corn stover (0.3 g/L furfural, 1.2 g/L HMF, and 1 g/L 4-hydroxybenozic acid) and the strain formed pellets in the cell cultivations. An integrated process was developed combining the dilute acid pretreatment, cellulase hydrolysis, and cell cultivation for M. isabellina to convert corn stover to oil containing fungal biomass. With 7.5 % pretreated biomass solid loading ratio, the final lipid yield from sugar in pretreated biomass was 40 % and the final lipid concentration of the culture reached to 6.46 g/L.     

Enzymatic production of xylooligosaccharides from corn stover and corn cobs treated with aqueous ammonia

A novel method of producing food-grade xylooligosaccharides from corn stover and corn cobs was investigated. The process starts with pretreatment of feedstock in aqueous ammonia, which results delignified and xylan-rich substrate. The pretreated substrates are subjected to enzymatic hydrolysis of xylan using endoxylanase for production of xylooligosaccharides. The conventional enzyme-based method involves extraction of xylan with a strong alkaline solution to form a liquid intermediate containing soluble xylan. This intermediate is heavily contaminated with various extraneous components. A costly purification step is therefore required before enzymatic hydrolysis. In the present method, xylan is obtained in solid form after pretreatment. Water-washing is all that is required for enzymatic hydrolysis of this material. The complex step of purifying soluble xylan from contaminant is essentially eliminated. Refining of xylooligosaccharides to food-grade is accomplished by charcoal adsorption followed by ethanol elution. Xylanlytic hydrolysis of the pretreated corn stover yielded glucan-rich residue that is easily digestible by cellulase enzyme. The digestibility of the residue reached 86% with enzyme loading of 10 filter paper units/g-glucan. As a feedstock for xylooligosaccharides production, corn cobs are superior to corn stover because of high xylan content and high packing density. The high packing density of corn cobs reduces water input and eventually raises the product concentration.     

Pretreatment and enzymatic saccharification of corn fiber

Corn fiber consists of about 20% starch, 14% cellulose, and 35% hemicellulose, and has the potential to serve as a low-cost feedstock for production of fuel ethanol. Several pretreatments (hot water, alkali, and dilute, acid) and enzymatic saccharification procedures were evaluated for the conversion of corn fiber starch, cellulose, and hemicellulose to monomeric sugars. Hot water pretreatment (121°C, 1 h) facilitated the enzymatic sacch arification of starch and cellulose but not hemicellulose. Hydrolysis of corn fiber pretreated with alkali un dersimilar conditions by enzymatic means gave similar results. Hemicellulose and starch components were converted to monomeric sugars by dilute H₂SO₄ pretreatment (0.5–1.0%, v/v) at 121°C. Based on these findings, a method for pretreatment and enzymatic saccharification of corn fiber is presented. It in volves the pretreatment of corn fiber (15% solid, w/v) with dilute acid (0.5% H₂SO₄, v/v) at 121°C for 1 h, neutralization to pH 5.0, then saccharification of the pretreated corn fiber material with commercial cellulase and β-glucosidase preparations The yield of monomeric sugars from corn fiber was typically 85–100% of the theoretical yield. Names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by USDA implies no approval of the product to the exclusion of others that may also be suitable.     

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.     

Hydrolytic Enzyme of Cellulose for Complex Formulation Applied Research

To improve the enzymatic hydrolytic efficiency and reduce the supplementation of enzymes, the mixture designed experimental approach was used to optimize the composition of enzyme mixture and promote the hydrolysis of ball-milled corn stover. From the experimental results, a synergistic effect was found when combinations of the three enzymes, two kinds of cellulases and a kind of xylanase, were used. The optimal hydrolysis of pretreated corn stover accorded with enzymes activity ration of FPU/CMCase/β-glucosidase/xylanase = 4.4:1:75:829, and the hydrolysis efficiency of corn stover increased significantly compared with using individual enzyme. The results indicated that the mixture design experiment could be an effective tool for optimized enzyme mixture for lignocellulose hydrolysis.     

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.     

秸秆稀酸水解液的气相色谱／质谱法研究

在秸秆两步稀酸水解工艺中,用气相色谱/质谱(GC/MS)法对其水解液中的单糖成分进行测定,采用2%硼氢化钠的氨溶液将稀酸水解液中的单糖还原成糖醇,然后在甲基咪唑催化剂的作用下和乙酸酐在水相中直接反应生成乙酰化的糖醇,用二氯甲烷萃取后进行GC/MS测定.研究结果表明:秸秆稀酸水解液中有五种单糖,主要是木糖和葡萄糖,其次是阿拉伯糖、半乳糖和少量的甘露糖;利用此方法测定了一批秸秆稀酸水解液,得到了该秸秆稀酸水解过程的最佳的反应时间.该方法可快速、准确测定秸秆稀酸水解液中单糖的浓度,为水解工艺的研究提供一种有效的分析方法.     

Lipid Accumulation by Pelletized Culture of Mucor circinelloides on Corn Stover Hydrolysate

Microbial oil accumulated by fungal cells is a potential feedstock for biodiesel production, and lignocellulosic materials can serve as the carbon source to support the fungal growth. The dilute acid pretreatment of corn stover can effectively break down its lignin structure, and this process generates a hydrolysate containing mostly xylose at very dilute concentration and numerous by-products that may significantly inhibit the cell growth. This study utilized corn stover hydrolysate as the culture media for the growth of Mucor circinelloides. The results showed that Mucor cells formed pellets during the cell growth, which facilitates the cell harvest from dilute solution. The results also showed that the inhibitory effect of furfural, 5-hydroxymethylfurfural (HMF), and acetic acid could be avoided if their concentration was low. In fact, all these by-products may be assimilated as carbon sources for the fungal growth. The results proved the feasibility to reuse the cultural broth water for acid pretreatment and then use for subsequent cell cultivation. The results will have a direct impact on the overall water usage of the process.     

Impact of Pretreatment with Dilute Sulfuric Acid Under Moderate Temperature on Hydrolysis of Corn Stover with Two Enzyme Systems

Pretreatment of corn stover with dilute sulfuric acid at moderate temperature was investigated, and glucan digestibility by Cellic CTec2 and Celluclast on the pretreated biomass was compared. Pretreatments were carried out from 60 to 180 min at the temperature from 105 to 135 °C, with acid concentrations ranging from 0.5 to 2 % (w/v). Significant portion of xylan was removed during pretreatment, and the glucan digestibility by CTec2 was significantly better than that by Celluclast in all cases. Analysis showed that glucan digestibility by both two enzymes correlated directly with the extent of xylan removal in pretreatment. Confidence interval was built to give a more precise range of glucan conversion and to test the significant difference among pretreatment conditions. Response surface model was built to obtain the optimal pretreatment condition to achieve high glucan conversion after enzymatic hydrolysis. Considering the cost and energy savings, the optimal pretreatment condition of 1.75 % acid for 160 min at 135 °C was determined, and glucan conversion can achieve the range from 72.86 to 76.69 % at 95 % confidence level after enzymatic hydrolysis, making total glucan recovery up to the range from 89.42 to 93.25 %.     

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.     

Ammonia fiber explosion treatment of corn stover

Optimizing process conditions and parameters such as ammonia loading, moisture content of biomass, temperature, and residence time is necessary for maximum effectiveness of the ammonia fiber explosion process. Approximate optimal pretreatment conditions for corn stover were found to be temperature of 90°C, ammonia: dry corn stover mass ratio of 1∶1, moisture content of corn stover of 60% (dry weight basis), and residence time (holding at target temperature), of 5 min. Approximately 98% of the theoretical glucose yield was obtained during enzymatic hydrolysis of the optimal treated corn stover using 60 filter paper units (FPU) of cellulase enzyme/g of glucan (equal to 22 FPU/g of dry corn stover). The ethanol yield from this sample was increased up to 2.2 times over that of untreated sample. Lowering enzyme loading to 15 and 7.5 FPU/g of glucan did not significantly affect the glucose yield compared with 60 FPU, and any differences between effects at different enzyme levels decreased as the treatment temperature increased.     

Pretreatment of corn stover using wet oxidation to enhance enzymatic digestibility

Corn stover is an abundant, promising raw material for fuel ethanol production. Although it has a high cellulose content, without pretreatment it resists enzymatic hydrolysis, like most lignocellulosic materials. Wet oxidation (water, oxygen, mild alkali or acid, elevated temperature and pressure) was investigated to enhance the enzymatic digestibility of corn stover. Six different combinations of reaction temperature, time, and pH were applied. The best conditions (60g/L of corn stover, 195°C, 15 min, 12 bar O₂, 2 g/L of Na₂CO₃) increased the enzymatic conversion of corn stover four times, compared to untreated material. Under these conditions 60% of hemicellulose and 30% of lignin were solubilized, whereas 90% of cellulose remained in the solid fraction. After 24-h hydrolysis at 50°C using 25 filter paper units (FPU)/g of dry matter (DM) biomass, the achieved conversion of cellulose to glucose was about 85%. Decreasing the hydrolysis temperature to 40°C increased hydrolysis time from 24 to 72 h. Decreasing the enzyme loading to 5 FPU/g of DM biomass slightly decreased the enzymatic conversion from 83.4 to 71%. Thus, enzyme loading can be reduced without significantly affecting the efficiency of hydrolysis, an important economical aspect.     

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.     

Ball Milling Pretreatment of Corn Stover for Enhancing the Efficiency of Enzymatic Hydrolysis

Ethanol can be produced from lignocellulosic biomass with the usage of ball milling pretreatment followed by enzymatic hydrolysis and fermentation. The sugar yields from lignocellulosic feed stocks are critical parameters for ethanol production process. The research results from this paper indicated that the yields of glucose and xylose were improved by adding any of the following dilute chemical reagents: H₂SO₄, HCl, HNO₃, CH₃COOH, HCOOH, H₃PO₄, and NaOH, KOH, Ca(OH)₂, NH₃·H₂O in the ball milling pretreatment of corn stover. The optimal enzymatic hydrolysis efficiencies were obtained under the conditions of ball milling in the alkali medium that was due to delignification. The data also demonstrated that ball milling pretreatment was a robust process. From the microscope image of ball milling-pretreated corn stover, it could be observed that the particle size of material was decreased and the fiber structure was more loosely organized. Meanwhile, the results indicate that the treatment effect of wet milling is better than that of dry milling. The optimum parameters for the milling process were ball speed of 350 r/min, solid/liquid ratio of 1:10, raw material particle size with 0.5 mm, and number of balls of 20 (steel ball, Φ = 10 mm), grinding for 30 min. In comparison with water milling process, alkaline milling treatment could increase the enzymatic hydrolysis efficiency of corn stover by 110%; and through the digestion process with the combination of xylanase and cellulase mixture, the hydrolysis efficiency could increase by 160%.     

Determination of glucose and ethanol after enzymatic hydrolysis and fermentation of biomass using Raman spectroscopy

Raman spectroscopy has been used for the quantitative determination of the conversion efficiency at each step in the production of ethanol from biomass. The method requires little sample preparation; therefore, it is suitable for screening large numbers of biomass samples and reaction conditions in a complex sample matrix. Dilute acid or ammonia-pretreated corn stover was used as a model biomass for these studies. Ammonia pretreatment was suitable for subsequent measurements with Raman spectroscopy, but dilute acid-pretreated corn stover generated a large background signal that surpassed the Raman signal. The background signal is attributed to lignin, which remains in the plant tissue after dilute acid pretreatment. A commercial enzyme mixture was used for the enzymatic hydrolysis of corn stover, and glucose levels were measured with a dispersive 785 nm Raman spectrometer. The glucose detection limit in hydrolysis liquor by Raman spectroscopy was 8 g L⁻¹. The mean hydrolysis efficiency for three replicate measurements obtained with Raman spectroscopy (86 ± 4%) was compared to the result obtained using an enzymatic reaction with UV-vis spectrophotometry detection (78 ± 8%). The results indicate good accuracy, as determined using a Student's t-test, and better precision for the Raman spectroscopy measurement relative to the enzymatic detection assay. The detection of glucose in hydrolysis broth by Raman spectroscopy showed no spectral interference, provided the sample was filtered to remove insoluble cellulose prior to analysis. The hydrolysate was further subjected to fermentation to yield ethanol. The detection limit for ethanol in fermentation broth by Raman spectroscopy was found to be 6 g L⁻¹. Comparison of the fermentation efficiencies measured by Raman spectroscopy (80 ± 10%) and gas chromatrography-mass spectrometry (87 ± 9%) were statistically the same. The work demonstrates the utility of Raman spectroscopy for screening the entire conversion process to generate lignocellulosic ethanol.     

Effect of Initial Particle Size and Densification on AFEX-Pretreated Biomass for Ethanol Production

Switchgrass (SG), corn stover (CS), and prairie cordgrass (PCG) pretreated with ammonia fiber expansion (AFEX) were densified using a novel low-temperature, low-pressure densification method. Simultaneous saccharification and fermentation (SSF) and separate hydrolysis and fermentation (SHF) were performed with loose and densified AFEX-treated biomass to determine the effect of post-AFEX densification. Biomass particle size reduction before pretreatment increased 144-h SSF ethanol yields from densified material by 8–9 % although no significant differences were seen in the first 72 h. Grinding material after densification had no impact on final ethanol yields but increased production rates in the first 24–48 h. Low-pressure, post-AFEX densification had no adverse effects on SSF ethanol yields from SG or CS but reduced yields from densified PCG by 16 %. Glucose concentrations after hydrolysis (SHF) showed similar trends. Ethanol yields after SHF, however, showed that densification had no significant impact on CS or PCG but reduced final ethanol yields from SG.     

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.     

Chemical pretreatments of corn stover for enhancing enzymatic digestibility

Corn stover, the most abundant agricultural residue in Hungary, is a potential raw material for the production of fuel ethanol as a result of its high content of carbohydrates, but a pretreatment is required for its efficient hydrolysis. In this article, we describe the results using various chemicals such as dilute H₂SO₄, HCl, and NaOH separately as well as consecutively under relative mild conditions (120°C, 1h). Pretreatment with 5% H₂SO₄ or 5% HCl solubilized 85% of the hemicellulose fraction, but the enzymatic conversion of pretreated materials increased only two times compared to the untreated corn stover. Applying acidic pretreatment following a 1-d soaking in base achieved enzymatic conversion that was nearly the theoretical maximum (95.7%). Pretreatment with 10% NaOH decreased the lignin fraction >95%, increased the enzymatic conversion more than four times, and gave a 79.4% enzymatic conversion. However, by increasing the reaction time, the enzymatic degradability could also be increased significantly, using a less concentrated base. When the time of pretreatment was increased three times (0.5% NaOH at 120°C), the amount of total released sugars was 47.9 g from 100 g (dry matter) of untreated corn stover.     

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.