Pretreatment of switchgrass by ammonia fiber explosion (AFEX)

The effects of ammonia fiber explosion (AFEX) pretreatment of switch grass using its major process variables are reported. The optimal pretreatment conditions for switchgrass were found to be near 100°C reactor temperature, and ammonia loading of 1:1 kg of ammonia: kg of dry matter with 80% moisture content (dry weight basis [dwb]) at 5 min residence time. Hydrolysis results of AFEX-treated and untreated samples showed 93% vs 16% glucan conversion, respectively. The ethanol yield of optimized AFEX-treated switchgrass was measured to be about 0.2 g ethanol/g dry biomass, which is 2.5 times more than that of the untreated sample.     

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.     

Ammonia fiber expansion pretreatment and enzymatic hydrolysis on two different growth stages of reed canarygrass

Plant materials from the vegetative growth stage of reed canarygrass and the seed stage of reed canarygrass are pretreated by ammonia fiber expansion (AFEX) and enzymatically hydrolyzed using 15 filter paper units (FPU) cellulase/g glucan to evaluate glucose and xylose yields. Percent conversions of glucose and xylose, effects of temperature and ammonia loading, and hydrolysis profiles are analyzed to determine the most effective AFEX treatment condition for each of the selected materials. The controls used in this study were untreated samples of each biomass material. All pretreatment conditions tested enhanced enzyme digestibility and improved sugar conversions for reed canarygrass compared with their untreated counterparts. Based on 168 h hydrolysis results using 15 FPU Spezyme CP cellulase/g glucan the most effective AFEX treatment conditions were determined as: vegetative growth stage of reed canarygrass--100 degrees C, 60% moisture content, 1.2:1 kg ammonia/kg of dry matter (86% glucose and 78% xylose) and seed stage of reed canarygrass--100 degrees C, 60% moisture content, 0.8:1 kg ammonia/kg of dry matter (89% glucose and 81% xylose). Supplementation by commercial Multifect 720 xylanase along with cellulase further increased both glucose and xylose yields by 10-12% at the most effective AFEX conditions.     

Ammonia Fiber Expansion (AFEX) Pretreatment,Enzymatic Hydrolysis,and Fermentation on Empty Palm Fruit Bunch Fiber (EPFBF) for Cellulosic Ethanol Production

Empty palm fruit bunch fiber (EPFBF), a readily available cellulosic biomass from palm processing facilities, is investigated as a potential carbohydrate source for cellulosic ethanol production. This feedstock was pretreated using ammonia fiber expansion (AFEX) and enzymatically hydrolyzed. The best tested AFEX conditions were at 135 °C, 45 min retention time, water to dry biomass loading of 1:1 (weight ratio), and ammonia to dry biomass loading of 1:1 (weight ratio). The particle size of the pretreated biomass was reduced post-AFEX. The optimized enzyme formulation consists of Accellerase (84 μL/g biomass), Multifect Xylanase (31 μL/g biomass), and Multifect Pectinase (24 μL/g biomass). This mixture achieved close to 90% of the total maximum yield within 72 h of enzymatic hydrolysis. Fermentation on the water extract of this biomass affirms that nutrients solely from the pretreated EPFBF can support yeast growth for complete glucose fermentation. These results suggest that AFEX-treated EPFBF can be used for cellulosic biofuels production because biomass recalcitrance has been overcome without reducing the fermentability of the pretreated materials.     

Extrusion processing for ammonia fiber explosion (AFEX)

The ammonia fiber explosion (AFEX) process, previously run only in a batch reactor, has been adapted to run on a twin-screw extruder. The sugar yield of AFEX material after enzymatic hydrolysis has been increased up to 3.5 times over that of completely untreated material. The ruminant digestibility of corn fodder has been increased up to 32% (from 54–71%) over completely untreated material, and 23% (from 63–77%) over material extruded with no ammonia. Extrusion-treated material proved more digestible by the ruminant at 48 h than material treated in the batch reactor.     

Pretreatment Methods for Bioethanol Production

Lignocellulosic biomass, such as wood, grass, agricultural, and forest residues, are potential resources for the production of bioethanol. The current biochemical process of converting biomass to bioethanol typically consists of three main steps: pretreatment, enzymatic hydrolysis, and fermentation. For this process, pretreatment is probably the most crucial step since it has a large impact on the efficiency of the overall bioconversion. The aim of pretreatment is to disrupt recalcitrant structures of cellulosic biomass to make cellulose more accessible to the enzymes that convert carbohydrate polymers into fermentable sugars. This paper reviews several leading acidic, neutral, and alkaline pretreatments technologies. Different pretreatment methods, including dilute acid pretreatment (DAP), steam explosion pretreatment (SEP), organosolv, liquid hot water (LHW), ammonia fiber expansion (AFEX), soaking in aqueous ammonia (SAA), sodium hydroxide/lime pretreatments, and ozonolysis are intensively introduced and discussed. In this minireview, the key points are focused on the structural changes primarily in cellulose, hemicellulose, and lignin during the above leading pretreatment technologies.     

Effects of ammonia fiber explosion treatment on activity of endoglucanase from Acidothermus cellulolyticus in transgenic plant

A critical parameter affecting the economic feasibility of lignocellulosic bioconversion is the production of inexpensive and highly active cellulase enzymes in bulk quantity. A promising approach to reduce enzyme costs is to genetically transform plants with the genes of these enzymes, thereby producing the desired cellulases in the plants themselves. Extraction and recovery of active proteins or release of active cellulase from the plants during bioconversion could have a significant positive impact on overall lignocellulose conversion economics. The effects of ammonia fiber explosion (AFEX) pretreatment variables (treatment temperature, moisture content, and ammonia loading) on the activity of plant-produced heterologous cellulase enzyme were individually investigated via heat treatmett or ammonia treatment. Finally, we studied the effects of all these variables in concert through the AFEX process. The plant materials included transgenic tobacco plants expressing E1 (endoglucanase from Acidothermus cellulolyticus). The E1 activity was measured in untreated and AFEX-treated tobacco leaves to investigate the effects of the treatment on the activity of this enzyme. The maximum observed activity retention in AFEX-treated transgenic tobacco samples compared with untreated samples was approx 35% (at 60°C, 0.5∶1 ammonia loading, and 40% moisture). Based on these findings, it is our opinion that AFEX pretreatment is not a suitable option for releasing cellulase enzyme from transgenic plants.     

Cost estimates and sensitivity analyses for the ammonia fiber explosion process

Process designs were conducted for each unit of the conceptual ammonia fiber explosion (AFEX) process, and fixed capital investment and operating costs were estimated. AFEX costs about $20-40/t of dry bio-mass treated. Several promising areas for reducing process costs exist. Return on investment (ROI) calculations were also done for AFEX-treated materials (as digestibility-enhanced animal feeds), in conjunction with sensitivity analyses on the overall processing costs. Estimated ROIs range from over 100%/y to negative, depending on the system variables. The most important variables are the cost of corn and corn fiber, ammonia loading, and whether or not drying is required.     

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.     

Understanding the Impacts of AFEX™ Pretreatment and Densification on the Fast Pyrolysis of Corn Stover,Prairie Cord Grass,and Switchgrass

Lignocellulosic feedstocks corn stover, prairie cord grass, and switchgrass were subjected to ammonia fiber expansion (AFEX?) pretreatment and densified using extrusion pelleting and ComPAKco densification technique. The effects of AFEX? pretreatment and densification were studied on the fast pyrolysis product yields. Feedstocks were milled in a hammer mill using three different screen sizes (2, 4, and 8 mm) and were subjected to AFEX? pretreatment. The untreated and AFEX?-pretreated feedstocks were moisture adjusted at three levels (5, 10, and 15 % wb) and were extruded using a lab-scale single screw extruder. The barrel temperature of the extruder was maintained at 75, 100, and 125 °C. Durability of the extruded pellets made from AFEX?-pretreated corn stover, prairie cord grass, and switchgrass varied from 94.5 to 99.2, 94.3 to 98.7, and 90.1 to 97.5 %, respectively. Results of the thermogravimetric analysis showed the decrease in the decomposition temperature of the all the feedstocks after AFEX? pretreatment indicating the increase in thermal stability. Loose and densified feedstocks were subjected to fast pyrolysis in a lab-scale reactor, and the yields (bio-oil and bio-char) were measured. Bio-char obtained from the AFEX?-pretreated feedstocks exhibited increased bulk and particle density compared to the untreated feedstocks. The properties of the bio-oil were statistically similar for the untreated, AFEX?-pretreated, and AFEX?-pretreated densified feedstocks. Based on the bio-char and bio-oil yields, the AFEX?-pretreated feedstocks and the densified AFEX?-pretreated feedstocks (pellets and PAKs) exhibited similar behavior. Hence, it can be concluded that densifying the AFEX?-pretreated feedstocks could be a viable option in the biomass-processing depots to reduce the transportation costs and the logistical impediments without affecting the product yields.     

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.     

Optimization of SO2-catalyzed steam pretreatment of corn fiber for ethanol production

A batch reactor was employed to steam explode corn fiber at various degrees of severity to evaluate the potential of using this feedstock as part of an enzymatically mediated cellulose-to-ethanol process. Severity was controlled by altering temperature (150–230°C), residence time (1–9 min), and SO₂ concentration (0–6% [w/w] dry matter). The effects of varying the different parameters were assessed by response surface modeling. The results indicated that maximum sugar yields (hemicellulose-derived water soluble, and cellulose-derived following enzymatic hydrolysis) were recovered from corn fiber pretreated at 190°C for 5 minutes after exposure to 3% SO₂. Sequential SO₂-catalyzed steam explosion and enzymatic hydrolysis resulted in a conversion efficiency of 81% of the combined original hemicellulose and cellulose in the corn fiber to monomeric sugars. An additional posthydrolysis step performed on water soluble hemicellulose stream increased the concentration of sugars available for fermentation by 10%, resulting in the high conversion efficiency of 91%. Saccharomyces cerevisiae was able to ferment the resultant corn fiber hydrolysates, perhydrolysate, and liquid fraction from the posthydrolysis steps to 89, 94, and 85% of theoretical ethanol conversion, respectively. It was apparent that all of the parameters investigated during the steam explosion pretreatment had a significant effect on sugar recovery, inhibitory formation, enzymatic conversion efficiency, and fermentation capacity of the yeast.     

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).     

Separation of glucose and pentose sugars by selective enzyme hydrolysis of AFEX-treated corn fiber

A process was developed to fractionate corn fiber into glucose- and pentose-rich fractions. Corn fiber was ammonia fiber explosion treated at 90 degrees C, using 1 g anhydrous ammonia pergram of drybiomass, 60% moisture, and 30-min residence time. Twenty four hour hydrolysis of ammonia fiber explosion-treated corn fiber with cellulase converted 83% of available glucanto-glucose. In this hydrolysis the hemicellulose was partially broken down with 81% of the xylan and 68% of the arabinan being contained in the hydrolysate after filtration to remove lignin and other insoluble material. Addition of ethanol was used to precipitate and recover the solubilized hemicellulose from the hydrolysate, followed by hydrolysis with 2% (v/v) sulfuric acid to convert the recovered xylan and arabinan to monomeric sugars. Using this method, 57% of xylose and 54% of arabinose available in corn fiber were recovered in a pentose-rich stream. The carbohydrate composition of the pentose-enriched stream was 5% glucose, 57% xylose, 27% arabinose, and 11% galactose. The carbohydrate composition of the glucose-enriched stream was 87% glucose, 5% xylose, 6% arabinose, and 1% galactose, and contained 83% of glucose available from the corn fiber.     

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.     

Fungal Cellulase/Xylanase Production and Corresponding Hydrolysis Using Pretreated Corn Stover as Substrates

Three pretreated corn stover (ammonia fiber expansion, dilute acid, and dilute alkali) were used as carbon source to culture Trichoderma reesei Rut C-30 for cellulase and xylanase production. The results indicated that the cultures on ammonia fiber expansion and alkali pretreated corn stover had better enzyme production than the acid pretreated ones. The consequent enzymatic hydrolysis was performed applying fungal enzymes on pretreated corn stover samples. Tukey’s statistical comparisons exhibited that there were significant differences on enzymatic hydrolysis among different combination of fungal enzymes and pretreated corn stover. The higher sugar yields were achieved by the enzymatic hydrolysis of dilute alkali pretreated corn stover.     

Predicting digestibility of ammonia fiber explosion (AFEX)-treated rice straw

The enzymatic digestibility of ammonia fiber explosion (AFEX)-treated rice straw was modeled by statistically correlating the variability of samples to differences in treatment using several different analytical techniques. Lignin content and crystallinity index of cellulose affect enzymatic hydrolysis the most. X-ray diffraction was used to measure the crystallinity index (CrI), while fluorescence and diffuse reflectance infrared (DRIFT) spectroscopy measured the lignin content of the samples. Multivariate analysis was applied to correlate the enzymatic hydrolysis results of the various samples with X-ray diffraction and spectroscopic data. Principal component analysis (PCA) and multilinear regression (MLR) techniques did not accurately predict the digestibility of the rice straw samples. The best correlation (R value of 0.775) was found between the treatment conditions of the AFEX process and the concentration of xylose at 24 h after enzymatic hydrolysis.     

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

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

Deficiency of Cellulase Activity Measurements for Enzyme Evaluation

Switchgrass was used as a model feedstock to determine the influence of pretreatment conditions and biomass quality on enzymatic hydrolysis using different enzyme products. Dilute sulfuric acid and soaking in aqueous ammonia pretreatments were used to produce biomass with varied levels of hemicellulose and lignin sheathing. Pretreated switchgrass solids were tested with simple enzymatic hydrolysis and simultaneous saccharification and fermentation (SSF) with three commercial enzyme products: Accellerase 1000 (Genencor), Spezyme CP (Genencor)/Novozyme 188 (Novozymes), and Celluclast/Novozyme 188 (Novozymes). Enzymes were loaded on a common activity basis (FPU/g cellulose and CBU/g cellulose). Despite identical enzyme loadings, glucose yields were significantly different for both acid and alkaline pretreatments but differences diminished as hydrolysis progressed for acid-pretreated biomass. Cellobiose concentrations in Accellerase treatments indicated an initial β-glucosidase limitation that became less significant over time. SSF experiments showed that differences in glucose and ethanol yields could not be attributed to enzyme product inhibition. Yield discrepancies of glucose or ethanol in acid pretreatment, alkaline pretreatment, and acid pretreatment/SSF were as much as 15%, 19%, and 5%. These results indicate that standardized protocols for measuring enzyme activity may not be adequate for assessing activity using pretreated biomass substrates.