Low-moisture Anhydrous Ammonia Research Articles

Environmental and economic analysis of low-moisture anhydrous ammonia (LMAA) as a pretreatment for cellulosic ethanol production

In the context of global energy supply, cellulosic ethanol plays an essential role due to being considered a renewable fuel and minimize environmental emissions when compared to the exploration and utilization of fossil fuels. However, the cellulosic ethanol production requires pretreatment before the fermentation process, which is considered an expensive procedure that makes the cellulosic ethanol production costly. Therefore, the main goal of this research was to evaluate the economic and environmental impacts of using low-moisture anhydrous ammonia (LMAA) pretreatment for five different scenarios, which were 1000 tonne (t) of feedstock per day (Scenario I), 2000 t/d (Scenario II), 3000 t/d (Scenario III), 5000 t/d (Scenario IV), and 10,000 t/d (Scenario V), for ethanol production from sugarcane bagasse by using SuperPro software. Life Cycle Assessment (LCA) and Techno-Economic Analysis (TEA) were used to evaluate the feasibility and environmental impacts of the scenarios simulated. From the results, the capital costs increased according to the increase of the feedstock amount to be processed, having the most significant investment to the largest facility simulated (10,000 t/d). Mostly all the scenarios presented material costs (sugarcane bagasse, enzyme, yeast, and ammonia costs) as the expenses that impacted the most the overall annual operating costs, where sugarcane bagasse and ammonia were the responsible for around 58% and 40% of the total investment for material costs. In addition, from utility costs, it was observed that electricity represented around 45% of the annual utility costs for almost all the scenarios simulated. All the scenarios resulted in negative profit, and sensitivity analysis showed that ethanol sale price was the parameter that presented the greatest sensitivity for sugarcane bagasse ethanol production under LMAA pretreatment conditions evaluated in this study. Related to environmental aspects, the LCA results showed that the 2000 t/d facility simulated (Scenario II) presented the lowest Global Warming Potential (GWP) for several electricity supply technologies analyzed and presented a significant reduction of CO2 equivalent emissions, representing more than 80% lower greenhouse gasses emissions (GHG) when compared to the 10,000 t/d facility simulated (Scenario V).

Low Moisture Anhydrous Ammonia Pretreatment of Four Lignocellulosic Materials—Distillers Dried Grains With Solubles, Corn Gluten Feed, Corn Fiber, and Oil Palm Frond

Lignin and hemicellulose structures in cellulosic materials serve as a barrier for enzyme reactions. A pretreatment step is often needed to break these components to allow the biomass to be utilized as a source of value-added products. Various available pretreatment methods possess common drawbacks of the high amount of liquid and chemical requirements, harsh process conditions, and the high amount of waste produced, which driving up the production costs of bioproducts. Low moisture anhydrous ammonia (LMAA) pretreatment capable of eliminating those drawbacks. In this study, Distillers Dried Grains with Solubles (DDGS), corn gluten feed (CGF), corn fiber (CF), and oil palm frond (OPF) with different moisture contents were subjected to LMAA pretreatment at the specific ammonia loading rate, 1 h ammoniation, and 75°C incubation temperature. This pretreatment successfully decreased the lignin content of the materials, increased their percentage of α-cellulose, and improved enzymatic digestibility for most of the materials tested. The effect of moisture content (30 and 50% db) was found to be more significant than that of incubation time (24 and 72 h).

Butyric Acid Generation by Clostridium tyrobutyricum from Low-Moisture Anhydrous Ammonia (LMAA) Pretreated Sweet Sorghum Bagasse.

Sweet sorghum bagasse (SSB) is an under-utilized feedstock for biochemical conversion to biofuels or high value chemicals. One such chemical that can be generated biochemically and applied to a wide array of industries from pharmaceuticals to the production of liquid transportation fuels is butyric acid. This work investigated cultivating the butyric acid producing strain Clostridium tyrobutyricum ATCC 25755 on low-moisture anhydrous ammonia (LMAA) pretreated SSB. Pretreated SSB hydrolysate was detoxified and supplemented with urea for shake flask batch fermentation to show that up to 11.4g/L butyric acid could be produced with a selectivity of 87% compared to other organic acids. Bioreactor fermentation with pH control showed high biomass growth, but a similar output of 11.3g/L butyric acid was achieved. However, the butyric acid productivity increased to 0.251g/L∙hr with a butyric acid yield of 0.29g/g sugar consumed. This butyric acid output represented an 83% theoretical yield. Further improvements in butyric acid titer and yield can be achieved by optimizing nutrient supplementation and incorporating fed-batch fermentation processing of pretreated SSB hydrolysate. Construction of ZGO:Sr NR- and ZGC@PDA NP-driven ratiometric aptasensor for CEA detection.

Effects of Additional Xylanase on Saccharification and Ethanol Fermentation of Ammonia-Pretreated Corn Stover and Rice Straw

Synergistic effect of cellulase and hemicellulase (xylanase) was evaluated because lignocellulosic material is a heterogeneous complex of cellulose and hemicellulose. Various effects of HTec2 addition on enzymatic saccharification and fermentation were evaluated using two different substrates such as corn stover and rice straw. Corn stover and rice straw were pretreated by the LMAA (low-moisture anhydrous ammonia) method at the preselected same conditions (90 °C, 120 h, moisture content = 50%, NH3 loading = 0.1 g NH3/g). It was observed that the enzymatic saccharification yield of pretreated corn stover (76.4% for glucan digestibility) was higher than that of pretreated rice straw (70.9% for glucan) using CTec2 cellulase without HTec2 addition. Glucan digestibility of pretreated corn stover was significantly increased from 76.4% to 91.1% when the HTec2/CTec2 (v/v) increased from 0 to 10. However, it was interesting that the ethanol production was decreased from 89.9% to 76.3% for SSF and 118.0% to 87.9% for SSCF at higher HTec2/CTec2. As the glucan loading increased from 2.0% to 7.0%, the ethanol yields of both SSF and SSCF were decreased from 96.3% to 88.9% and from 116.6% to 92.4%, respectively. In addition, the smallest inoculum size (optical density of 0.25) resulted in the highest ethanol production (20.5 g/L).

Techno-Economic Analysis (TEA) of Different Pretreatment and Product Separation Technologies for Cellulosic Butanol Production from Oil Palm Frond

Among the driving factors for the high production cost of cellulosic butanol lies the pretreatment and product separation sections, which often demand high amounts of energy, chemicals, and water. In this study, techno-economic analysis of several pretreatments and product separation technologies were conducted and compared. Among the pretreatment technologies evaluated, low-moisture anhydrous ammonia (LMAA) pretreatment has shown notable potential with a pretreatment cost of $0.16/L butanol. Other pretreatment technologies evaluated were autohydrolysis, soaking in aqueous ammonia (SAA), and soaking in sodium hydroxide solution (NaOH) with pretreatment costs of $1.98/L, $3.77/L, and $0.61/L, respectively. Evaluation of different product separation technologies for acetone-butanol-ethanol (ABE) fermentation process have shown that in situ stripping has the lowest separation cost, which was $0.21/L. Other product separation technologies tested were dual extraction, adsorption, and membrane pervaporation, with the separation costs of $0.38/L, $2.25/L, and $0.45/L, respectively. The evaluations have shown that production of cellulosic butanol using combined LMAA pretreatment and in situ stripping or with dual extraction recorded among the lowest butanol production cost. However, dual extraction model has a total solvent productivity of approximately 6% higher than those of in situ stripping model.

Life-Cycle Assessment (LCA) of Different Pretreatment and Product Separation Technologies for Butanol Bioprocessing from Oil Palm Frond

Environmental impact assessment is a crucial aspect of biofuels production to ensure that the process generates emissions within the designated limits. In typical cellulosic biofuel production process, the pretreatment and downstream processing stages were reported to require a high amount of chemicals and energy, thus generating high emissions. Cellulosic butanol production while using low moisture anhydrous ammonia (LMAA) pretreatment was expected to have a low chemical, water, and energy footprint, especially when the process was combined with more efficient downstream processing technologies. In this study, the quantification of environmental impact potentials from cellulosic butanol production plants was conducted with modeled different pretreatment and product separation approaches. The results have shown that LMAA pretreatment possessed a potential for commercialization by having low energy requirements when compared to the other modeled pretreatments. With high safety measures that reduce the possibility of anhydrous ammonia leaking to the air, LMAA pretreatment resulted in GWP of 5.72 kg CO2 eq./L butanol, ecotoxicity potential of 2.84 × 10−6 CTU eco/L butanol, and eutrophication potential of 0.011 kg N eq./L butanol. The lowest energy requirement in biobutanol production (19.43 MJ/L), as well as better life-cycle energy metrics performances (NEV of 24.69 MJ/L and NER of 2.27) and environmental impacts potentials (GWP of 3.92 kg N eq./L butanol and ecotoxicity potential of 2.14 × 10−4 CTU eco/L butanol), were recorded when the LMAA pretreatment was combined with the membrane pervaporation process in the product separation stage.

Techno-economic analysis of low moisture anhydrous ammonia (LMAA) pretreatment for butanol production from oil palm frond

Cellulosic butanol production has been of interest because of the attractive properties of butanol as a fuel and its ability in environmental preservation. Biological pathway for butanol production is currently a subject of development efforts from various processing aspects. One crucial stage is the pretreatment process, which has considerably affected the production cost of butanol. There are some concerns regarding the overall effectiveness of the process, including high total energy and liquid requirement, and production of inhibitory compounds from the pretreatment process. Low-moisture anhydrous ammonia (LMAA) pretreatment, which requires less water, work at atmospheric conditions, and gives milder pretreatment intensity, was developed to solve these problems. In this study, the techno-economic analysis (TEA) of butanol production from oil palm fronds (OPF) was conducted. The simulation employed LMAA pretreatment, followed by ABE fermentation and downstream processing. The lowest butanol production cost was $ 2.11/L, which was recorded from 95.34 × 106 L butanol/y plant capacity. Detail analysis of each cost component revealed that operating costs contributing nearly 63% of the production cost with 24% and 17% contribution from utility and material costs, respectively. Optimization of process parameters estimated the potential of production cost reduction to lower than $ 1.65/L by an increase in the enzyme hydrolysis yield. The study aimed to provide data towards utilization effort of OPF, especially for butanol production.

Two-stage processing of Miscanthus giganteus using anhydrous ammonia and hot water for effective xylan recovery and improved enzymatic saccharification

A two-stage method using gaseous ammonia and hot water was proposed to recover xylan and lignin from Miscanthus. In this method, Miscanthus was treated with gaseous ammonia at elevated temperatures (60–150 °C) for various reaction times (1–48 h) in the first stage, termed as LMAA (low-moisture anhydrous ammonia) treatment. In the following stage, the LMAA-treated solid was subjected to hot-water treatment in a flow-through column reactor under various reaction conditions (170–220 °C, 30–90 min). After two-stage processing, the remaining solid contained mostly glucan (∼80% cellulose), which became highly digestible by enzymes. The optimal treatment conditions for sugar recovery using two-stage process were 120 °C and 12 h for the 1st stage and 190 °C, 90 min, and 5 mL/min for the 2nd stage, which resulted in 84.2% xylan recovery in liquid phase and 95.3% glucan digestibility of the treated solid, using 15 FPU/g-glucan enzyme loading after the two-stage treatment.

Assessment of Antioxidant and Antimicrobial Properties of Lignin from Corn Stover Residue Pretreated with Low-Moisture Anhydrous Ammonia and Enzymatic Hydrolysis Process.

Lignin accounts for 15-35% of dry biomass materials. Therefore, developing value-added co-products from lignin residues is increasingly important to improve the economic viability of biofuel production from biomass resources. The main objective of this work was to study the lignin extracts from corn stover residue obtained from a new and improved process for bioethanol production. Extraction conditions that favored high lignin yield were optimized, and antioxidant and antimicrobial activities of the resulting lignin were investigated. Potential estrogenic toxicity of lignin extracts was also evaluated. The corn stover was pretreated by low-moisture anhydrous ammonia (LMAA) and then subjected to enzymatic hydrolysis using cellulase and hemicellulase. The residues were then added with sodium hydroxide and extracted for different temperatures and times for enhancing lignin yield and the bioactivities. The optimal extraction conditions using 4% (w/v) sodium hydroxide were determined to be 50°C, 120min, and 1:8 (w:v), the ratio between corn stover solids and extracting liquid. Under the optimal condition, 33.92g of lignin yield per 100g of corn stover residue was obtained. Furthermore, the extracts produced using these conditions showed the highest antioxidant activity by the hydrophilic oxygen radical absorbance capacity (ORAC) assay. The extracts also displayed significant antimicrobial activities against Listeria innocua. Minimal estrogenic impacts were observed for all lignin extracts when tested using the MCF-7 cell proliferation assay. Thus, the lignin extracts could be used for antioxidant and antimicrobial applications, and improve the value of the co-products from the biomass-based biorefinery.

Anhydrous Ammonia Pretreatment of Corn Stover and Enzymatic Hydrolysis of Glucan from Pretreated Corn Stover

As a promising alternative of fossil fuel, ethanol has been widely used. In recent years, much attention has been devoted to bioethanol production from lignocellulosic biomass. In previous research, it is found that the pretreatment method named low-moisture anhydrous ammonia (LMAA) has the advantage of high conversion efficiency and less washing requirements. The purpose of this study was to explore the optimal conditions by employing the LMAA pretreatment method. Corn stover was treated under three levels of moisture content: 20, 50, 80 w.b.% (wet basis), and three levels of particle size: <0.09, 0.09–2, >2 mm; it was also ammoniated with a loading rate of 0.1g NH3/g biomass (dry matter). Ammoniated corn stover was then subjected to different pretreatment times (24, 96, 168 h) and temperatures (20, 75, 130 °C). After pretreatment, compositional analysis and enzymatic digestibility were conducted to determine the highest glucose yield. As a result, the highest glucose yield was obtained under the condition of 96 h and 75 °C with 50 w.b.% and 0.09–2 mm of corn stover. The main findings of this study could improve the efficiency of bioethanol production processing in the near future.

Small-scale low-moisture anhydrous ammonia (LMAA) pretreatment of corn stover

With many economic benefits, corn-based ethanol has been widely used in recent years. Cellulosic ethanol, however, require pretreatment to break down lingo-cellulosic structures prior to fermentation. Among all the pretreatment reagents that have been used, ammonia has proven to be one of the most effective, because it can readily delignify, swell, and preserve the polysaccharides. The purpose of this study was to determine optimal conditions (i.e. highest glucose yield) using the low-moisture anhydrous ammonia (LMAA) pretreatment process. In this experiment, corn stover was prepared with different moisture contents (20% w.b., 50% w.b., 80% w.b.) and particle sizes (9–30 mesh, 30–144 mesh). Corn stover was ammoniated for 30 min. Ammoniated corn stover was then subjected to different incubation times (24 h, 75 h, and 144 h) under different temperatures (20 °C, 70 °C, 120 °C). After that, compositional analyses, including moisture content, ash content, carbohydrates, and lignin content, were conducted. Enzymatic digestibility tests were also conducted. Results from this study will be used to improve the efficiency of pretreatment processing.

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 (0.1g 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 72-144h. 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 20min, followed by pretreatment at 90°C for 48h).

Comparison of sealing and open conditions for long term storage of corn stover using low-moisture anhydrous ammonia pretreatment method

As a promising material for bioethanol production, corn stover has been studied under various pretreatment methods prior to production of bioethanol. However, the storage of pretreated corn stover is still challenged by both weather conditions and the physical properties of its own. The main objective of this experiment is to evaluate the effect of low-moisture anhydrous ammonia (LMAA) pretreatment method on biomass quality during long periods of storage. In this study, corn stover was contacted with various ammonia loadings (0, 0.1, and 0.2g/g DM biomass) and moisture content (20wt.%, 40wt.%, and 60wt.%) from 1 day to 90 days both in sealed and open containers. As a result, the mass loss in sealed container increased with time; however, the mass loss in open container was affected by the conditions of the environment. In terms of the carbohydrate, no significant reduction was observed in either sealed or open containers.

Pretreatment of Corn Stover by Low Moisture Anhydrous Ammonia (LMAA) in a Pilot-Scale Reactor and Bioconversion to Fuel Ethanol and Industrial Chemicals.

Corn stover (CS) adjusted to 50, 66, and 70% moisture was pretreated by the low moisture anhydrous ammonia (LMAA) process in a pilot-scale ammoniation reactor. After ammoniation, the 70% moisture CS was treated at 90 and 100°C whereas the others were treated at 90°C only. The 70% moisture pretreated CS then was subjected to a storage study under non-sterile conditions for 3months. It was found that storage time did not have significant effects on the compositions of the pretreated materials and their hydrolysis by commercial enzymes. The 70% moisture CS treated at 90°C was used for preparation of a mix sugar hydrolysate (MSH) using combination of cellulase and xylanase. The MSH was used to prepare a corn mash at 9.5wt% solid then subjected to ethanol fermentation by Escherichia coli KO11. The 66% moisture CS treated at 90°C was hydrolyzed with xylanase to make a xylose-rich hydrolysate (XRH), which was subsequently used for butyric acid fermentation by Clostridium tyrobutyricum. The resultant cellulose-enriched residue was hydrolyzed with cellulase to make a glucose-rich hydrolysate (GRH), which was subsequently used for succinic acid fermentation by E. coli AFP184.

Understanding the Physicochemical Characteristics and the Improved Enzymatic Saccharification of Corn Stover Pretreated with Aqueous and Gaseous Ammonia

AbstractPhysicochemical characteristics of corn stover pretreated by soaking in aqueous ammonia (SAA) and low-moisture anhydrous ammonia (LMAA) were compared and investigated. The glucan digestibility of the treated biomass reached 90 % (SAA) and 84 % (LMAA). The LMAA pretreatment enhanced the digestibility by cleaving cross-linkages between cell wall components, whereas the SAA pretreatment additionally improved the digestibility by efficiently removing a major portion of the lignin under mild reaction conditions without significant loss of carbohydrates. Fourier transform infrared spectroscopy (FTIR), nuclear magnetic resonance (NMR), and gel permeation chromatography (GPC) revealed the structural and chemical transformations of lignin during the pretreatments. Both pretreatments effectively cleaved ferulate cell wall cross-linking that is associated with the recalcitrance of grass lignocellulosics toward enzymatic saccharification. Extracted lignin from SAA pretreatment was extensively depolymerized but retained “native” character, as evidenced by the retention of β-ether linkages.

Techno-economic analysis (TEA) of low-moisture anhydrous ammonia (LMAA) pretreatment method for corn stover

Techno-economic analysis (TEA) plays an important role in assessing economic performance and potential market acceptance for new technologies. Previous work has shown that the construction and operation of a cellulosic bioethanol plant can be very expensive. One of the largest cost categories is pretreatment processing. The purpose of this study was to conduct a detailed cost analysis to assess low moisture anhydrous ammonia (LMAA) pretreatment process at the commercial-scale, and to estimate the breakeven point in large-scale production. In this study, capital expenses, including annualized purchase and installation fees, and annual operating costs associated with each unit operation were determined. The lowest unit cost obtained from this study was $3.86/gal, but it was still high compared with current gasoline price.

Italian ryegrass (Lolium multiflorum Lam) as a High-Potential Bio-Ethanol Resource

Cellulosic bioethanol production from lignocellulosic biomass has been recognized as one of the promising sustainable energy source alternatives to petroleum-based fuels, since the lignocelluloses are not directly in competition with food sources. In order to develop a lignocellulosic raw material with high energy crops for bio-ethanol production, we selected Italian ryegrass (Lolium multiflorum Lam). Bio-ethanol production from Italian ryegrass through a low-moisture anhydrous ammonia (LMAA) pretreatment and simultaneous saccharification and co-fermentation (SSCF) was examined. At first, Italian ryegrass was subjected to LMAA pretreatment, which was performed by keeping the moist powdered Italian ryegrass under NH3 gas atmosphere at room temperature for 4 weeks. The effect of LMAA pretreatment was examined for bio-ethanol production through simultaneous saccharification and fermentation (SSF) which was performed at 34 °C using Saccharomyces cerevisiae, cellulase, and xylanase. The yields of ethanol and xylose in SSF were enhanced by LMAA-pretreatment. The SSCF of the LMAA-retreated Italian ryegrass was performed at 36 °C using Escherichia coli KO11, S. cerevisiae, cellulase, and xylanase. The SSCF of LMAA-pretreated Italian ryegrass produced ethanol in higher yield (84.6 %) compared with those of LMAA-pretreated bamboo, rice straw, Napier grass, and silvergrass.

Fuelization of Italian Ryegrass and Napier Grass through a Biological Treatment and Photocatalytic Reforming

Fuelization of Italian ryegrass and Napier grass was examined by the combination of biological treatments and photocatalytic reforming (photo-Reform). The alkali-pretreated Italian ryegrass and Napier grass were subjected to the enzymatic saccharification using cellulase and xylanase. Xylose and glucose were produced in 56.6% and 71.1% from Italian ryegrass and in 49.5% and 67.3% from Napier grass, respectively. Xylose and glucose were converted to hydrogen by the photo-Reform using a Pt-loaded titanium oxide (Pt/TiO2) under UV irradiation. Moreover, a low-moisture anhydrous ammonia (LMAA) pretreatment was performed for Italian ryegrass and Napier grass by keeping moist powdered biomass under NH3 gas atmosphere at room temperature for four weeks. The Italian ryegrass and Napier grass which were pretreated by LMAA method were subjected to simultaneous saccharification and fermentation (SSF) using a mixture of cellulase and xylanase as well as Saccharomyces cerevisiae in acetate buffer (pH 5.0). Ethanol and xylose were produced in 91.6% and 51.6% from LMAA-pretreated Italian ryegrass and 78.6% and 68.8% from Napier grass, respectively. After the evaporation of ethanol, xylose was converted to hydrogen by the photo-Reform. In the case of saccharification→photo-Reform, energy was recovered as hydrogen from the alkali-pretreated Italian ryegrass and Napier grass in 71.9% and 77.0% of energy recovery efficiency, respectively. In the case of SSF→photo-Reform, the energy was recovered in 82.7% and 77.2% as ethanol and hydrogen from the LMAA-pretreated Italian ryegrass and Napier grass, respectively.

Napier grass (Pennisetum purpureum Schumach) as raw material for bioethanol production: Pretreatment, saccharification, and fermentation

Recently Napier grass (Pennisetum purpureum Schumach) has been recognized to meet the requirement of lignocellulosic bioethanol production, because it has low lignin-content and a relatively high herbage mass per year and per area. Therefore, pretreatment, saccharification, and fermentation processes for ethanol production from Napier grass have been extensively studied. As pretreatment method, acid, alkali, PBHW (pressurized batch hot water), and LMAA (low-moisture anhydrous ammonia) pretreatments were reviewed. As saccharification and fermentation process, saccharification followed by co-fermentation of hexose and pentose, simultaneous saccharification and fermentation (SSF) followed by pentose fermentation, simultaneous saccharification and co-fermentation (SSCF) process were proposed. The SSCF was most advantageous process since the SSCF can prevent contamination risks of other microorganism and can construct simple processing procedure. An example of ethanol production from Napier grass was a combination process of LMAA-pretreatment with SSCF which was performed for of LMAA-treated Napier grass at 36℃ for 96 h using cellulase, xylanase, Saccharomyces cerevisiae, and Escherichia coli KO11. The ethanol yield reached 74.1%. Thus, Napier grass was thought to be a promising biomass for ethanol production.

Effect of low-moisture anhydrous ammonia (LMAA) pretreatment on biomass quality and enzymatic hydrolysis for long-term storage.

Biomass storage is essential for effective utilization of lignocellulosic biomass. Although long-term storage of biomass is as important as the conversion process, no effective method has been developed. This study investigated the effects of low-moisture anhydrous ammonia (LMAA) pretreatment on biomass quality for long-term storage with moisture content from 30 to 70 %. Carbohydrate loss due to fungal growth was observed in the moisturized corn stover without ammonia loading, after 12 weeks of storage. This also resulted in reduced enzymatic digestibility. Both ammoniation and LMAA pretreatment reduced the decomposition of carbohydrates by fungal growth, during storage in the presence of moisture. LMAA pretreatment rendered 83 % of glucan and 67 % of xylan digestibility after 24 weeks of storage. Ammoniated corn stover also resulted in 63 % of glucan and 48 % of xylan digestibility at 0 week of storage, and hydrolysis yields increased to 78 % for glucan and 64 % for xylan, without pretreatment, after 12 weeks of storage.