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 11.4 g/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 11.3 g/L butyric acid was achieved. However, the butyric acid productivity increased to 0.251 g/L∙hr with a butyric acid yield of 0.29 g/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.

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, 120 min, and 1:8 (w:v), the ratio between corn stover solids and extracting liquid. Under the optimal condition, 33.92 g of lignin yield per 100 g 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.

Production of Ethanol From Newly Developed and Improved Winter Barley Cultivars.

Winter barley has attracted strong interest as a potential feedstock for fuel ethanol production in regions with mild winter climate such as the mid-Atlantic and northeastern USA. Ten recently developed and improved winter barley cultivars and breeding lines including five hulled and five hull-less lines were experimentally evaluated for potential ethanol production. The five hulled barley lines included three released cultivars (Thoroughbred, Atlantic, and Secretariat) and two breeding lines (VA09B-34 and VA11B-4). The five hull-less lines also included three released cultivars (Eve, Dan, and Amaze 10) and two breeding lines (VA08H-65 and VA13H-34). On the average, the hull-less barley cultivars produced more ethanol per unit mass because of their higher starch and ß-glucan contents. However, since the hulled barley cultivars had higher agronomic yield, the potential ethanol production per acre of land for the two types were approximately equal. Among the ten cultivars tested, the hull-less cultivar Amaze 10 was the best one for ethanol production. The ethanol yield values obtained for this cultivar were 2.61 gal per bushel and 292 gal per acre.

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 3 months. 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 9.5 wt% 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.

Pretreatment of Dried Distiller Grains with Solubles by Soaking in Aqueous Ammonia and Subsequent Enzymatic/Dilute Acid Hydrolysis to Produce Fermentable Sugars.

Dried distillers grains with solubles (DDGS), a co-product of corn ethanol production in the dry-grind process, was pretreated by soaking in aqueous ammonia (SAA) using a 15 % w/w NH4OH solution at a solid/liquid ratio of 1:10. The effect of pretreatment on subsequent enzymatic hydrolysis was studied at two temperatures (40 and 60 °C) and four reaction times (6, 12, 24, and 48 h). Highest glucose yield of 91 % theoretical was obtained for the DDGS pretreated at 60 °C and 24 h. The solubilized hemicellulose in the liquid fraction was further hydrolyzed with dilute H2SO4 to generate fermentable monomeric sugars. The conditions of acid hydrolysis included 1 and 4 wt% acid, 60 and 120 °C, and 0.5 and 1 h. Highest yields of xylose and arabinose were obtained at 4 wt% acid, 120 °C, and 1 h. The fermentability of the hydrolysate obtained by enzymatic hydrolysis of the SAA-pretreated DDGS was demonstrated in ethanol fermentation by Saccharomyces cerevisiae. The fermentability of the hydrolysate obtained by consecutive enzymatic and dilute acid hydrolysis was demonstrated using a succinic acid-producing microorganism, strain Escherichia coli AFP184. Under the fermentation conditions, complete utilization of glucose and arabinose was observed, whereas only 47 % of xylose was used. The succinic acid yield was 0.60 g/g total sugar consumed.

Capture of carbon dioxide from ethanol fermentation by liquid absorption for use in biological production of succinic acid.

Previously, it was shown that the gas produced in an ethanol fermentor using either corn or barley as feedstock could be sparged directly into an adjacent fermentor as a feedstock for succinic acid fermentation using Escherichia coli AFP184. In the present investigation, it was demonstrated that the CO2 produced in a corn ethanol fermentor could be absorbed in a base solution and the resultant carbonate solution used both for pH control and supply of the CO2 requirement in succinic acid fermentation. Thus, the CO2 produced in a 5-L corn mash containing 30 wt% total solids was absorbed in a packed column containing 2 L of either 5 M NaOH, 5 M KOH, or 15 wt% NH4OH, and the resultant carbonate solutions were used for pH control in a succinic acid fermentor. The results obtained indicated no significant differences between succinic acid production in these experiments and when 2.5 M solutions of Na2CO3, K2CO3, and (NH4)2CO3 from commercial sources were used. In a commercial setting, the demonstrated capture of CO2 in liquid form will allow transportation of the carbonate solutions to locations not in the immediate vicinity of the ethanol plant, and excess carbonate salts can also be recovered as value-added products.

A comparison of two milling strategies to reduce the mycotoxin deoxynivalenol in barley.

Winter barley (Hordeum vulgare L.), a potential feedstock for fuel ethanol production, may be contaminated with the trichothecene mycotoxin deoxynivalenol (DON). DON is a threat to feed and food safety in the United States and may become concentrated during the production of distillers dried grains with solubles (DDGS). DDGS is a coproduct of fuel ethanol production and is increasingly being used as feed for domestic animals. Therefore, new strategies to reduce the threat of DON in DDGS need to be developed and implemented for grain destined for fuel ethanol production. It is known that large concentrations of DON accumulate in the hulls of wheat and barley. Consequently, improved methods are needed to carefully remove the hull from the grain and preserve the starchy endosperm. Whole kernels from five Virginia winter barley genotypes were used to evaluate the abilities of two different milling strategies (roller milling and precision milling (FitzMill)) for their ability to remove the hull-enriched tissue from the kernel while maintaining starch levels and reducing DON levels in the endosperm-enriched tissue. After whole kernels were milled, DON and starch levels were quantified in the hull-enriched fractions and endosperm-enriched fractions. Initial milling experiments demonstrated that the precision mill system (6 min run time) is able to reduce more DON than the roller mill but with higher starch losses. The average percent DON removed from the kernel with the roller mill was 36.7% ± 5.5 and the average percent DON removed from the dehulled kernel with the precision mill was 85.1% ± 9.0. Endosperm-enriched fractions collected from the roller mill and precision mill contained starch levels ranging from 49.0% ± 12.1 to 59.1% ± 0.5 and 58.5% ± 1.6 to 65.3% ± 3.9, respectively. On average, the precision mill removed a mass of 23.1% ± 6.8 and resulted in starch losses of 9.6% ± 6.3, but produced an endosperm-enriched fraction with relatively very little average DON (5.5 ± 2.7 µg g(-1)). In contrast, on average, the roller mill removed a mass of 12.2% ± 1.6 and resulted in starch losses of 2.1% ± 0.5, but produced an endosperm-enriched fraction with high average DON (20.7 ± 13.5 µg g(-1)). In a time course precision milling experiment, we tested barley genotypes Nomini, Atlantic, and VA96-44-304 and attempted to reduce the starch loss seen in the first experiment while maintaining low DON concentrations. Decreasing the run time of the precision mill from 5 to 2 min, reduced starch loss at the expense of higher DON concentrations. Aspirated fractions revealed that the precision milled hull-enriched fraction contained endosperm-enriched components that were highly contaminated with DON. This work has important implications for the reduction of mycotoxins such as DON in barley fuel ethanol coproducts and barley enriched animal feeds and human foods.

Enzymatic fractionation of SAA-pretreated barley straw for production of fuel ethanol and astaxanthin as a value-added co-product.

Barley straw was used to demonstrate an integrated process for production of fuel ethanol and astaxanthin as a value-added co-product. Barley straw was pretreated by soaking in aqueous ammonia using the previously determined optimum conditions, which included 77.6 °C treatment temperature, 12.1 h treatment time, 15 wt% ammonia concentration, and 1:8 solid-to-liquid ratio. In the newly developed process, the pretreated barley straw was first hydrolyzed with ACCELLERASE® XY (a commercial hemicellulase product) to generate a xylose-rich solution, which contained 3.8 g/l glucose, 22.9 g/l xylose, and 2.4 g/l arabinose, with 96 % of the original glucan being left intact. The xylose-rich solution was used for production of astaxanthin by the yeast Phaffia rhodozyma without further treatment. The resulting cellulose-enriched solid residue was used for ethanol production in a fed-batch simultaneous saccharification and fermentation using ACCELLERASE® 1500 (a commercial cellulase product) and the industrial yeast Saccharomyces cerevisiae. At the end of the fermentation, 70 g/l ethanol was obtained, which was equivalent to 63 % theoretical yield based on the glucan content of the solid substrate.

Maximum production of fermentable sugars from barley straw using optimized soaking in aqueous ammonia (SAA) pretreatment.

Soaking in aqueous ammonia (SAA) pretreatment was investigated to improve enzymatic digestibility and consequently to increase total fermentable sugar production from barley straw. Various effects of pretreatment process parameters, such as reaction temperature, reaction time, solid:liquid ratio, and ammonia concentration, were evaluated, and the optimum conditions for two of the most important factors, reaction temperature and time were determined using response surface methodology. Optimized reaction conditions were 77.6 °C treatment temperature, 12.1 h. treatment time, 15 wt.% ammonia concentration, and 1:8 solid-to-liquid ratio, which gave a sugar recovery yield of 71.5 % (percent of theoretical sugar recovered from the untreated barley straw) with enzyme loading of 15 FPU/g-glucan. In the optimization of the SAA pretreatment process, ammonia concentration, reaction temperature, and reaction time were determined to be the most significant factors correlated to subsequent enzyme digestibility. Based on tested conditions exhibiting high sugar recovery yields of >60 %, it appeared that reaction temperature affected total fermentable sugar production more significantly than reaction time.

Conversion of deoxynivalenol to 3-acetyldeoxynivalenol in barley-derived fuel ethanol co-products with yeast expressing trichothecene 3-O-acetyltransferases.

BACKGROUND: The trichothecene mycotoxin deoxynivalenol (DON) may be concentrated in distillers dried grains with solubles (DDGS; a co-product of fuel ethanol fermentation) when grain containing DON is used to produce fuel ethanol. Even low levels of DON (≤ 5 ppm) in DDGS sold as feed pose a significant threat to the health of monogastric animals. New and improved strategies to reduce DON in DDGS need to be developed and implemented to address this problem. Enzymes known as trichothecene 3-O-acetyltransferases convert DON to 3-acetyldeoxynivalenol (3ADON), and may reduce its toxicity in plants and animals. RESULTS: Two Fusarium trichothecene 3-O-acetyltransferases (FgTRI101 and FfTRI201) were cloned and expressed in yeast (Saccharomyces cerevisiae) during a series of small-scale ethanol fermentations using barley (Hordeum vulgare). DON was concentrated 1.6 to 8.2 times in DDGS compared with the starting ground grain. During the fermentation process, FgTRI101 converted 9.2% to 55.3% of the DON to 3ADON, resulting in DDGS with reductions in DON and increases in 3ADON in the Virginia winter barley cultivars Eve, Thoroughbred and Price, and the experimental line VA06H-25. Analysis of barley mashes prepared from the barley line VA04B-125 showed that yeast expressing FfTRI201 were more effective at acetylating DON than those expressing FgTRI101; DON conversion for FfTRI201 ranged from 26.1% to 28.3%, whereas DON conversion for FgTRI101 ranged from 18.3% to 21.8% in VA04B-125 mashes. Ethanol yields were highest with the industrial yeast strain Ethanol Red®, which also consumed galactose when present in the mash. CONCLUSIONS: This study demonstrates the potential of using yeast expressing a trichothecene 3-O-acetyltransferase to modify DON during commercial fuel ethanol fermentation.

Pretreatment of corn stover using low-moisture anhydrous ammonia (LMAA) process.

A simple pretreatment method using anhydrous ammonia was developed to minimize water and ammonia inputs for cellulosic ethanol production, termed the low moisture anhydrous ammonia (LMAA) pretreatment. In this method, corn stover with 30-70% moisture was contacted with anhydrous ammonia in a reactor under nearly ambient conditions. After the ammoniation step, biomass was subjected to a simple pretreatment step at moderate temperatures (40-120°C) for 48-144 h. Pretreated biomass was saccharified and fermented without an additional washing step. With 3% glucan loading of LMAA-treated corn stover under best treatment conditions (0.1g-ammonia+1.0 g-water per g biomass, 80°C, and 84 h), simultaneous saccharification and cofermentation test resulted in 24.9 g/l (89% of theoretical ethanol yield based on glucan+xylan in corn stover).

Scale-up of ethanol production from winter barley by the EDGE (enhanced dry grind enzymatic) process in fermentors up to 300 l.

A fermentation process, which was designated the enhanced dry grind enzymatic (EDGE) process, has recently been developed for barley ethanol production. In the EDGE process, in addition to the enzymes normally required for starch hydrolysis, commercial ß-glucanases were used to hydrolyze (1,3)(1,4)-ß-D: -glucans to smaller molecules, thus reducing the viscosity of the mash to levels sufficiently low to allow transport and mixing in commercial equipment. Another enzyme, a developmental ß-glucosidase, then was used to hydrolyze the resulting oligomers to glucose, which subsequently was fermented to produce additional ethanol. The EDGE process was developed with Thoroughbred, a winter hulled barley, using a shake flask model. To move toward commercialization, it is necessary to prove that the developed process would be applicable to other barley varieties and also to demonstrate its scalability. Experiments were performed in 7.5, 70, and 300-l fermentors using Thoroughbred and Eve, a winter hull-less barley. It was shown that the process was scalable for both barley varieties. Low levels of glucose throughout the course of the fermentations demonstrated the high efficiency of the simultaneous saccharification and fermentation process. Final ethanol concentrations of 14% (v/v) were achieved for initial total solids of 28.5-30% (w/w), which gave an ethanol yield of 83-87% of the theoretical values. The distillers dried grains with solubles co-products contained very low levels of ß-glucans and thus were suitable for use in feed formulations for all animal species.

Economic analysis of fuel ethanol production from winter hulled barley by the EDGE (Enhanced Dry Grind Enzymatic) process.

A process and cost model was developed for fuel ethanol production from winter barley based on the EDGE (Enhanced Dry Grind Enzymatic) process. In this process, in addition to ß-glucanases, which are added to reduce the viscosity of the mash, ß-glucosidase is also added to completely hydrolyze the oligomers obtained during the hydrolysis of ß-glucans to glucose. The model allows determination of capital costs, operating costs, and ethanol production cost for a plant producing 40 million gallons of denatured fuel ethanol annually. A sensitivity study was also performed to examine the effects of ß-glucosidase and barley costs on the final ethanol production cost. The results of this study clearly demonstrate the economic benefit of adding ß-glucosidase. Lower ethanol production cost was obtained compared to that obtained without ß-glucosidase addition in all cases except one where highest ß-glucosidase cost allowance and lowest barley cost were used.

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 ß-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.

Production of astaxanthin from cellulosic biomass sugars by mutants of the yeast Phaffia rhodozyma.

Astaxanthin is a potential high-value coproduct in an ethanol biorefinery. Three mutant strains of the astaxanthin-producing yeast Phaffia rhodozyma, which were derived from the parent strain ATCC 24202 (UCD 67-210) and designated JTM166, JTM185, and SSM19, were tested for their capability of utilizing the major sugars that can be generated from cellulosic biomass, including glucose, xylose, and arabinose, for astaxanthin production. While all three strains were capable of metabolizing these sugars, individually and in mixtures, JTM185 demonstrated the greatest sugar utilization and astaxanthin production. Astaxanthin yield by this strain (milligrams astaxanthin per gram of sugar consumed) was highest for xylose, followed by arabinose and then glucose. The kinetics of sugar utilization by strain JTM185 was studied in fermenters using mixtures of glucose, xylose, and arabinose at varied concentrations. It was found that glucose was utilized preferentially, followed by xylose, and lastly, arabinose. Astaxanthin yield was significantly affected by sugar concentrations. Highest yields were observed with sugar mixtures containing the highest concentrations of xylose and arabinose. Hydrolysates produced from sugarcane bagasse and barley straw pretreated by the soaking in aqueous ammonia method and hydrolyzed with the commercial cellulase preparation, Accellerase™ 1000, were used for astaxanthin production by the mutant strain JTM185. The organism was capable of metabolizing all of the sugars present in the hydrolysates from both biomass sources and produced similar amounts of astaxanthin from both hydrolysates, although these amounts were lower when compared to yields obtained with reagent grade sugars.

Consolidated conversion of hulled barley into fermentable sugars using chemical, thermal, and enzymatic (CTE) treatment.

A novel process using chemical, thermal, and enzymatic treatment for conversion of hulled barley into fermentable sugars was developed. The purpose of this process is to convert both lignocellulosic polysaccharides and starch in hulled barley grains into fermentable sugars simultaneously without a need for grinding and hull separation. In this study, hulled barley grains were treated with 0.1 and 1.0 wt.-% sulfuric acid at various temperatures ranging from 110 to 170 °C in a 63-ml flow-through packed-bed stainless steel reactor. After sulfuric acid pretreatment, simultaneous conversion of lignocellulose and starch in the barley grains into fermentable sugars was performed using an enzyme cocktail, which included α-amylase, glucoamylase, cellulase, and ß-glucosidase. Both starch and non-starch polysaccharides in the pre-treated barley grains were readily converted to fermentable sugars. The treated hulled barley grains, including their hull, were completely hydrolyzed to fermentable sugars with recovery of almost 100% of the available glucose and xylose. The pretreatment conditions of this chemical, thermal, and enzymatic (CTE) process for achieving maximum yield of fermentable sugars were 1.0 wt.% sulfuric acid and 110 °C. In addition to starch, the acid pretreatment also retained most of the available proteins in solid form, which is essential for subsequent production of fuel ethanol and high protein distiller's dried grains with solubles co-product.

Integration of succinic acid and ethanol production with potential application in a corn or barley biorefinery.

Production of succinic acid from glucose by Escherichia coli strain AFP184 was studied in a batch fermentor. The bases used for pH control included NaOH, KOH, NH(4)OH, and Na(2)CO(3). The yield of succinic acid without and with carbon dioxide supplied by an adjacent ethanol fermentor using either corn or barley as feedstock was examined. The carbon dioxide gas from the ethanol fermentor was sparged directly into the liquid media in the succinic acid fermentor without any pretreatment. Without the CO(2) supplement, the highest succinic acid yield was observed with Na(2)CO(3), followed by NH(4)OH, and lowest with the other two bases. When the CO(2) produced in the ethanol fermentation was sparged into the media in the succinic acid fermentor, no improvement of succinic acid yield was observed with Na(2)CO(3). However, several-fold increases in succinic acid yield were observed with the other bases, with NH(4)OH giving the highest yield increase. The yield of succinic acid with CO(2) supplement from the ethanol fermentor when NH(4)OH was used for pH control was equal to that obtained when Na(2)CO(3) was used, with or without CO(2) supplementation. The benefit of sparging CO(2) from ethanol fermentation on the yield of succinic acid demonstrated the feasibility of integration of succinic acid fermentation with ethanol fermentation in a biorefinery for production of fuels and industrial chemicals.

Bioethanol production from corn stover using aqueous ammonia pretreatment and two-phase simultaneous saccharification and fermentation (TPSSF).

An integrated bioconversion process was developed to convert corn stover derived pentose and hexose to ethanol effectively. In this study, corn stover was pretreated by soaking in aqueous ammonia (SAA), which retained glucan ( approximately 100%) and xylan (>80%) in the solids. The pretreated carbohydrates-rich corn stover was converted to ethanol via two-phase simultaneous saccharification and fermentation (TPSSF). This single-reactor process employed sequential simultaneous saccharification and fermentation (SSF), i.e. pentose conversion using recombinant Escherichia coli KO11 in the first phase, followed by hexose conversion with Saccharomyces cerevisiae D5A in the second phase. In the first phase, 88% of xylan digestibility was achieved through the synergistic action of xylanase and endo-glucanase with minimal glucan hydrolysis (10.5%). Overall, the TPSSF using 12-h SAA-treated corn stover resulted in the highest ethanol concentration (22.3g/L), which was equivalent to 84% of the theoretical ethanol yield based on the total carbohydrates (glucan+xylan) in the untreated corn stover.

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 degrees 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.

Production of astaxanthin from corn fiber as a value-added co-product of fuel ethanol fermentation.

Five strains of the yeast Phaffia rhodozyma, NRRL Y-17268, NRRL Y-17270, ATCC 96594 (CBS 6938), ATCC 24202 (UCD 67-210), and ATCC 74219 (UBV-AX2) were tested for astaxanthin production using the major sugars derived from corn fiber. The sugars tested included glucose, xylose, and arabinose. All five strains were able to utilize the three sugars for astaxanthin production. Among them, ATCC 74219 was the best astaxanthin producer. Kinetics of sugar utilization of this strain was studied, both with the individual sugars and with their mixtures. Arabinose was found to give the highest astaxanthin yield. It also was observed that glucose at high concentrations suppressed utilization of the other two sugars. Corn fiber hydrolysate obtained by dilute sulfuric acid pretreatment and subsequent enzyme hydrolysis was tested for astaxanthin production by strain ATCC 74219. Dilution of the hydrolysate was necessary to allow growth and astaxanthin production. All the sugars in the hydrolysate diluted with two volumes of water were completely consumed. Astaxanthin yield of 0.82 mg/g total sugars consumed was observed.