Progress 10/29/14 to 10/28/19

Outputs
Progress Report Objectives (from AD-416): 1: Develop technologies that enable the integrated processing of sorghum grains and sweet sorghum juice at existing biofuels production facilities and that enable the commercial production of new co-products at sorghum- based biorefineries. 1A: Develop technologies that enable the integrated processing of sorghum grains at existing biofuels production facilities. 1B: Develop technologies that enable the integrated processing of sweet sorghum juice at existing biofuels production facilities. 1C: Develop technologies that enable the commercial production of new co- products at sorghum-based biorefineries. 2: Develop technologies that enable the commercial production of marketable C5-rich and C6-rich sugar streams from sorghum lignocellulosic components. 2A: Develop technologies that enable the commercial production of marketable C5-rich sugar streams from sorghum lignocellulosic components. 2B: Develop technologies that enable the commercial production of marketable C6-rich sugar streams from sorghum lignocellulosic components. 3: Develop technologies that enable the commercial conversion of sorghum lignocellulosic components into fuels and industrial chemicals. 3A: Develop technologies that enable the commercial production of industrial chemicals from the C5-rich sugar stream obtained from the enzymatic hydrolysis of pretreated sorghum cellulosic components. 3B: Develop technologies that enable the commercial production of additional ethanol and industrial chemicals from the C6-rich sugar stream obtained from the enzymatic hydrolysis of the cellulose-enriched residue. 3C: Develop technologies that enable the use of byproducts and wastes generated in ethanol and other fermentation processes in the sorghum biorefinery for production of energy and chemicals. Approach (from AD-416): In conjunction with collaborators, develop technologies that enable commercially-preferred bio/chemical processes for converting all components of sorghum plants, including grains, juice, and bagasse, into fuels, industrial chemicals and consumer products. Develop commercially viable processes for incorporation of sorghum grains into existing commercial corn-based ethanol plants and evaluate the effects of this process modification on overall water balances in the existing plants. Develop commercially viable technologies for using sweet sorghum juice and sorghum biomass, including both carbohydrates and lignin, for the production of important platform chemicals, i.e. chemicals that can be used as precursors for production of a wide range of industrial chemicals and consumer products. Develop technologies for capturing the carbon dioxide gas generated in ethanol fermentation for use in other fermentation processes that requires CO2 as a secondary feedstock in addition to fermentable sugars. Develop technologies for conversion of the wastes generated in cellulosic ethanol and industrial fermentation processes into methane for internal use as an energy source. Develop an integrated process combining the aforementioned process components for a sorghum-based biorefinery. Considerable progress was made on all objectives, all of which fall under National Program 306 ⿿ Product Quality and Uses, Component 3 - Biorefining. Objective 1a: We have adapted our existing corn to ethanol model developed at the Eastern Regional Research Center (ERRC) to utilize grain sorghum. This model has been further adapted to include mixtures of corn with grain sorghum. These models will be utilized for economic and production comparison with the base case corn-to-ethanol model. The comparison will help determine the impact on ethanol and coproduct yields as well as the potential benefits of grain sorghum utilization. Objective 1b: Fermentations utilizing sweet sorghum juice mixed with corn were conducted. Sufficient quantities of modified distillers dried grains with solubles (DDGS) were produced during these fermentations for detailed compositional analysis. Important animal nutrient profiles of the DDGS were measured and evaluated. Results indicated that the modified DDGS composition changed little from the base case (i.e. corn-to-ethanol). However, the overall DDGS yields were reduced that could potentially decrease coproduct revenue. Objective 1c: A simple process using boiling ethanol under reflux conditions to extract wax from sorghum grains was developed. The extracted wax was recovered as a value-added co-product. Wax removal was found to improve enzymatic starch hydrolysis and subsequent ethanol yield. The de-waxed grains then were treated with dilute sulfuric acid (1 and 2 wt %) and subjected to mashing for ethanol fermentation. Commercial cellulases were added to the mash to hydrolyze the cellulose fraction of the hulls to produce glucose for additional ethanol production. A net improvement of 36.8 % in ethanol production over the raw grains was obtained. This work was completed and previously reported in the 2017 annual report. Objective 2c: Lignin has been isolated from sorghum bagasse insoluble residue obtained after enzymatic hydrolysis. Initial characterization by analytical pyrolysis GC/MS has indicated it was enriched in G and H-type lignin monomers such as phenol, o-cresol, guaiacol, and vanillin. Currently, sorghum bagasse lignin is being extracted and recovered in larger quantities to perform pyrolysis at larger scale to generate a representative sample of bio-oil. This substance will be characterized for chemical composition and overall water content. Additionally, a lignin sample will be characterized by an outside collaborator to determine if this lignin type is a good material candidate to be used as a replacement for bisphenol-A in epoxy resins. Objective 3b: Pretreated sweet sorghum bagasse by the low moisture anhydrous ammonia (LMAA) process has been hydrolyzed to generate a sugar hydrolysate enriched in both glucose and xylose. This hydrolysate will form the primary substrate in which the microorganisms Clostridium acetobutylicum ATCC 824 and C. beijerinckii ATCC 55025 will be cultivated to determine butanol production titers. C. tyrobutyricum ATCC 25755 will be another strain investigated, but this strain produces butyric acid instead of butanol. Different fermentation processing schemes will be investigated to determine if the butyric acid produced from C. tyrobutyricum can be fermented downstream by the other two organisms to butanol. Objective 3c: Cellulosic ethanol stillage has been collected after ethanol fermentation for utilization as a substrate for anaerobic digestion. Local farms are being contacted to provide a sample of cow manure to be used as a starter inoculum for anerobic digestion. The cellulosic ethanol stillage will be inoculated with bacteria from the starter inoculum to determine production rates of biogas (e.g. methane, carbon dioxide, and other gases).

Impacts
(N/A)

Publications

• Norvell, K.L., Nghiem, N.P. 2018. Soaking in aqueous ammonia (SAA) pretreatment of whole corn kernels for cellulosic ethanol production from the fiber fractions. Fermentation. 4(87):1-10.
• Stoklosa, R.J., Johnston, D., Nghiem, N.P. 2019. Phaffia rhodozyma cultivation on structural and non-structural sugars from sweet sorghum for astaxanthin generation. Process Biochemistry. 83:9-17.

Progress 10/01/17 to 09/30/18

Outputs
Progress Report Objectives (from AD-416): 1: Develop technologies that enable the integrated processing of sorghum grains and sweet sorghum juice at existing biofuels production facilities and that enable the commercial production of new co-products at sorghum- based biorefineries. 1A: Develop technologies that enable the integrated processing of sorghum grains at existing biofuels production facilities. 1B: Develop technologies that enable the integrated processing of sweet sorghum juice at existing biofuels production facilities. 1C: Develop technologies that enable the commercial production of new co- products at sorghum-based biorefineries. 2: Develop technologies that enable the commercial production of marketable C5-rich and C6-rich sugar streams from sorghum lignocellulosic components. 2A: Develop technologies that enable the commercial production of marketable C5-rich sugar streams from sorghum lignocellulosic components. 2B: Develop technologies that enable the commercial production of marketable C6-rich sugar streams from sorghum lignocellulosic components. 3: Develop technologies that enable the commercial conversion of sorghum lignocellulosic components into fuels and industrial chemicals. 3A: Develop technologies that enable the commercial production of industrial chemicals from the C5-rich sugar stream obtained from the enzymatic hydrolysis of pretreated sorghum cellulosic components. 3B: Develop technologies that enable the commercial production of additional ethanol and industrial chemicals from the C6-rich sugar stream obtained from the enzymatic hydrolysis of the cellulose-enriched residue. 3C: Develop technologies that enable the use of byproducts and wastes generated in ethanol and other fermentation processes in the sorghum biorefinery for production of energy and chemicals. Approach (from AD-416): In conjunction with collaborators, develop technologies that enable commercially-preferred bio/chemical processes for converting all components of sorghum plants, including grains, juice, and bagasse, into fuels, industrial chemicals and consumer products. Develop commercially viable processes for incorporation of sorghum grains into existing commercial corn-based ethanol plants and evaluate the effects of this process modification on overall water balances in the existing plants. Develop commercially viable technologies for using sweet sorghum juice and sorghum biomass, including both carbohydrates and lignin, for the production of important platform chemicals, i.e. chemicals that can be used as precursors for production of a wide range of industrial chemicals and consumer products. Develop technologies for capturing the carbon dioxide gas generated in ethanol fermentation for use in other fermentation processes that requires CO2 as a secondary feedstock in addition to fermentable sugars. Develop technologies for conversion of the wastes generated in cellulosic ethanol and industrial fermentation processes into methane for internal use as an energy source. Develop an integrated process combining the aforementioned process components for a sorghum-based biorefinery. Progress was made on all objectives, all of which fall under National Program 306 - Product Quality and New Uses, Component 3. Biorefining. Addressing Problem Statement 3.B. Technologies that reduce risks and increase profitability in existing industrial biorefineries. Objective 1a: Utilizing our base process model for corn ethanol production (developed at the Eastern Regional Research Center (ERRC) and distributed to many people who have requested it), an updated model for utilizing grain sorghum was developed. This model is still undergoing validation to determine the accuracy of the model relative to laboratory data. Initial evaluations show that the yields of distillers grains and ethanol are representative. Energy usage and yields for oil as a co- product are still being evaluated. Objective 1b: Utilizing the developed process models we are beginning to address the economics of incorporating sweet sorghum juice into existing corn ethanol facilities. The data generated will be used to understand how to improve the integration and to identify areas that may substantially impact the production costs. We have already identified that alterations in the water balance of the existing plants will be an area that has a substantial negative impact on the operation. Objective 1c: A fermentation system for the production of lysine using sweet sorghum juice was developed. The juice was found to contain sufficient sugars (carbon source). However, there were inadequate nutrients for growth of the production organism. The juice required the addition of these nutrients (mainly amino acids provided by adding yeast extract) before the organism could be grown. Once the nutrients were added in sufficient quantities to the juice, the organism was able to grow and produce significant amounts of lysine. However, additional sugar would still need to be added to obtain commercially viable levels. Objectives 2a and 2b: Xylitol production was investigated using the yeast Candida mogii. Conditions that favored xylitol production were investigated in defined media using shake-flasks. The results of these studies indicated that high initial xylose concentrations around 35-40 g/ L gave the highest yields. The pH of the medium also had significant effect on xylitol production. An initial pH of 5 was found to be the optimum. Hydrolysates then were prepared from sweet sorghum bagasse (SSB). Thus SSB was first pretreated by the low moisture anhydrous ammonia method. The pretreated SSB then was hydrolyzed with a commercial hemicellulase to produce a C5-rich sugar solution. The cellulose-enriched solid residue then was hydrolyzed with a commercial cellulase to produce a C6-rich sugar solution. The C5-rich solution was used for xylitol production and the C6-rich sugar solution was used for production of succinic acid and glutamic acid. All these experiments currently are in progress. In another process option, the pretreated SSB was hydrolyzed with both hemicellulase and cellulase in sweet sorghum juice instead of in water as in the aforementioned case. The entire mixture then was used for ethanol fermentation using a commercial Saccharomyces cerevisiae yeast strain. At the end of the fermentation, ethanol was removed by gentle boiling to simulate ethanol recovery by distillation in a typical commercial ethanol plant. The obtained solution, which contained xylose and glycerol, then was used for xylitol production. These experiments also are in progress. Objectives 3a and 3b: Based on our previous successful cultivation of Phaffia rhodozyma in sweet sorghum juice, enzymatic hydrolysate obtained from pretreated sweet sorghum bagasse was utilized as a medium to also grow P. rhodozyma and produce astaxanthin (a pink carotenoid pigment which must be added to the feed of farm-raised salmon to ensure that the meat is pink). Defined media experiments were conducted to assess P. rhodozyma growth on C5-sugars, i.e. xylose and arabinose. Although the organism could metabolize the C5-sugars, the consumption rate of these sugars is much slower than glucose. Furthermore, combining even a low concentration of glucose with xylose and arabinose could increase C5- sugar consumption rate and biomass growth. P. rhodozyma was next cultivated in shake flasks with sweet sorghum bagasse hydrolysate. After multiple trials with undiluted and diluted hydrolysate, and higher inoculation loadings it was found that P. rhodozyma could not grow in the nutrient supplemented hydrolysate. To determine the cause of this, the hydrolysate was detoxified using activated carbon. By treating the hydrolysate with 10% (w/w) activated carbon for 2 hours at 50 degree C, P. rhodozyma was successfully grown in the bagasse hydrolysate. Experiments are currently ongoing to assess P. rhodozyma growth restriction in the bagasse hydrolysate. Dissolved aromatic compounds originating from lignin are known to be inhibitory during fermentation with certain organisms. Raw hydrolysate and detoxified hydrolysate will be analyzed by LC-MS to see which aromatic compounds exist in the hydrolysate that could be detrimental to P. rhodozyma. Additionally, enzymatic detoxification with laccase or peroxidase enzymes will be investigated for the removal or detoxification of aromatic compounds or other inhibitors. Objective 3c: Lignin is a major byproduct of biomass fermentation to produce biofuels and bioproducts and currently it has a very low value because most of it is burned in boilers. To increase the net profitability of biomass fermentation to biofuels and bioproducts, it is important to identify higher value applications for lignin. Pretreated sweet sorghum bagasse utilizing the low moisture anhydrous ammonia (LMAA) process was enzymatically hydrolyzed to produce an aqueous solution enriched in monomeric sugars and an insoluble residue comprised mostly of lignin. The insoluble residue after hydrolysis was washed, dried, and extracted with NaOH at elevated temperature (80 degree C) for 1 hour to remove as much lignin as possible. After extraction, the pH of the alkaline solution was lowered to a pH around 2 through the addition of acid to precipitate the dissolved lignin. This lignin sample was washed and lyophilized. Around 90% of the lignin in the bagasse residue after enzymatic hydrolysis could be extracted with NaOH. Elemental analysis on the recovered lignin indicated a composition of 54.2% carbon, 5.6% hydrogen, 3.5% nitrogen, and 36.7% oxygen. The oxygen content is more than likely an inflated value since it was indirectly quantified based on the difference of the summed values for carbon, hydrogen, and nitrogen, and accounts for residual inorganic (i.e. ash) content as well. It is noteworthy that the lignin contains 3.5% nitrogen, most of which probably originates from the ammonia utilized for bagasse pretreatment. Analytical pyrolysis followed by GC-MS of the recovered lignin showed the usual aromatic monomers associated with H and G-lignin types. The lignin produced high quantities of phenol, 4-ethylphenol, and guaiacol monomers. The pyrolysis of lignin also produced large quantities of furan based compounds such as furfural and 2-furanmethanol. This is an indication that the lignin is not �pure� and contains some quantity of residual sugars from the bagasse. Currently, larger quantities of lignin are being recovered from pretreated sweet sorghum bagasse to perform larger scale lignin pyrolysis to obtain a true bio-oil sample for analysis and characterization. Objective 3c: Because about one third of the carbon from biomass is released as CO2 during fermentation, new processes are needed to capture and convert this wasted CO2 (which is also an undesirable greenhouse gas) and convert it into energy and/or valuable chemicals. The growth conditions of the oil-producing microalgae Scenedesmus obliquus were investigated at various pH values and with sodium carbonate supplementation (which is easier to control than using CO2 supplementation) at 0.2, 2 and 20 g/L. Similar cell yields were observed at all three pH levels tested (pH 6, 8 and 10). Slightly higher cell yields were observed with sodium carbonate addition compared to the control (without sodium carbonate and opened to air). Additional experiments will be performed using CO2-rich off-gas from an ethanol fermentor and also sodium carbonate solutions prepared by absorption of CO2 from an ethanol fermentor in NaOH solution.

Impacts
(N/A)

Publications

• Guo, M., Jin, Z.T., Nghiem, N.P., Fan, X., Qi, P.X., Jang, C., Shao, L., Wu, C. 2018. Assessment of antioxidant and antimicrobial properties of lignin from corn stover residue pretreated with low-moisture anhydrous ammonia and enzymatic hydrolysis process. Applied Biochemistry and Biotechnology. 184:350-365.
• Phongpreecha, T., Hool, N.C., Stoklosa, R.J., Klett, A.S., Foster, C.E., Bhalla, A., Holmes, D., Thies, M.C., Hodge, D.B. 2017. Predicting lignin depolymerization yields from quantifiable properties using fractionated biorefinery lignins. Green Chemistry. 19(21):5131-5143.
• Stoklosa, R.J., Johnston, D., Nghiem, N.P. 2018. Utilization of sweet sorghum juice for the production of astaxanthin as a biorefinery co- product by phaffia rhodozyma. ACS Sustainable Chemistry & Engineering. 3(6) :3124-3134.
• Nghiem, N.P., O'Connor, J., Hums, M.E. 2018. Integrated process for extraction of wax as a value-added co-product and improved ethanol production by converting both starch and cellulosic components in sorghum grains. Fermentation. 4:1-12.
• Pham, H.T., Nghiem, N.P., Kim, T.H. 2018. Near theoretical saccharification of sweet sorghum bagasse using simulated green liquor pretreatment and enzymatic hydrolysis. Energy. 157:894-903.

Progress 10/01/16 to 09/30/17

Outputs
Progress Report Objectives (from AD-416): 1: Develop technologies that enable the integrated processing of sorghum grains and sweet sorghum juice at existing biofuels production facilities and that enable the commercial production of new co-products at sorghum- based biorefineries. 1A: Develop technologies that enable the integrated processing of sorghum grains at existing biofuels production facilities. 1B: Develop technologies that enable the integrated processing of sweet sorghum juice at existing biofuels production facilities. 1C: Develop technologies that enable the commercial production of new co- products at sorghum-based biorefineries. 2: Develop technologies that enable the commercial production of marketable C5-rich and C6-rich sugar streams from sorghum lignocellulosic components. 2A: Develop technologies that enable the commercial production of marketable C5-rich sugar streams from sorghum lignocellulosic components. 2B: Develop technologies that enable the commercial production of marketable C6-rich sugar streams from sorghum lignocellulosic components. 3: Develop technologies that enable the commercial conversion of sorghum lignocellulosic components into fuels and industrial chemicals. 3A: Develop technologies that enable the commercial production of industrial chemicals from the C5-rich sugar stream obtained from the enzymatic hydrolysis of pretreated sorghum cellulosic components. 3B: Develop technologies that enable the commercial production of additional ethanol and industrial chemicals from the C6-rich sugar stream obtained from the enzymatic hydrolysis of the cellulose-enriched residue. 3C: Develop technologies that enable the use of byproducts and wastes generated in ethanol and other fermentation processes in the sorghum biorefinery for production of energy and chemicals. Approach (from AD-416): In conjunction with collaborators, develop technologies that enable commercially-preferred bio/chemical processes for converting all components of sorghum plants, including grains, juice, and bagasse, into fuels, industrial chemicals and consumer products. Develop commercially viable processes for incorporation of sorghum grains into existing commercial corn-based ethanol plants and evaluate the effects of this process modification on overall water balances in the existing plants. Develop commercially viable technologies for using sweet sorghum juice and sorghum biomass, including both carbohydrates and lignin, for the production of important platform chemicals, i.e. chemicals that can be used as precursors for production of a wide range of industrial chemicals and consumer products. Develop technologies for capturing the carbon dioxide gas generated in ethanol fermentation for use in other fermentation processes that requires CO2 as a secondary feedstock in addition to fermentable sugars. Develop technologies for conversion of the wastes generated in cellulosic ethanol and industrial fermentation processes into methane for internal use as an energy source. Develop an integrated process combining the aforementioned process components for a sorghum-based biorefinery. Progress was made on all objectives, all of which fall under NP 213 (Biorefining), Problem Statement 1.1 � �Technologies for producing advanced biofuels or other marketable biobased products�; Problem Statement 1.2 - �Technologies that reduce risks and increase profitability in existing industrial biorefineries�; and Problem Statement 1.3 - �Accurately estimate the economic value of biochemical technologies�. Objective 1a: The Eastern Regional Research Center (ERRC) corn dry grind process model has been updated with current economic and processing data. The process model now includes distillers corn oil recovery, which is currently being used in the majority of corn ethanol facilities. Objective 1b: Utilization of sweet sorghum juice for the production of ethanol and for value-added products is ongoing. Incorporation of the sugar rich juice from sweet sorghum into existing corn ethanol facilities has been tested on a small scale in the lab. Results clearly show that the juice can be incorporated into the ethanol process; however, alterations to the co-product yield and water balances are significant. A process model is being developed to investigate alternative approaches to solving these significant problems in an economically viable manner. Objective 1c: Shake-flasks fermentations have been used to thoroughly study production of astaxanthin by the yeast Phaffia rhodozyma strain ATCC 74219. Synthetic media were prepared to mimic the expected sugar concentrations in sweet sorghum juice to assess yeast growth and astaxanthin production with different sources and levels of nitrogen supplementation. Preliminary fermentations in synthetic media showed best growth and astaxanthin production with yeast extract and urea supplementation. Similar results were obtained when sweet sorghum juice was fermented with P. rhodozyma. Supplementation with yeast extract and urea produced cell productivities of about 1500 and 800 mg of astaxanthin per kg of dry cells, respectively. It is expected that a greater amount of astaxanthin will be produced in larger scale fermentations whereby variables such as pH or nitrogen supplementation can be controlled during the entire fermentation process. Currently, experiments are being developed to test astaxanthin production in fermenters with P. rhodozyma and sweet sorghum juice. The obtained results will identify fermentation process parameters that give the best yields of astaxanthin. Objective 2b: Sweet sorghum bagasse was washed to extract residual sugars. The washed bagasse then was subjected to low moisture anhydrous ammonia (LMAA) pretreatment and subsequently hydrolyzed with commercial xylanases to produce a 5-carbon sugar-rich solution from the hemicellulose fraction with little hydrolysis of the cellulose fraction. The remaining cellulose-rich solids then were hydrolyzed with commercial cellulases to produce a 6-carbon sugar-rich solution. The feasibility of this process has been demonstrated. Work is being continued to produce larger quantities of 6-carbon sugar-rich solution for use in fermentation experiments to demonstrate fermentability of this sugar solution. Objective 2c: The remaining solids obtained after hydrolysis of the sweet sorghum bagasse with cellulases as described in Objective 2b were rich in lignin. The lignin was extracted with sodium hydroxide solution and purified by precipitation with ethanol. The conditions for lignin extraction and recovery had previously been determined with corn stover and will be used in lignin extraction and recovery from sweet sorghum bagasse. The recovered lignin will be characterized using standard procedures. Objective 3a: Itaconic acid production using the microorganism Aspergillus terreus has been demonstrated with solutions containing glucose (a 6-carbon sugar), xylose (a 5-carbon sugar) and mixture of the two sugars in shake-flasks. The experiments have been successfully scaled up in 2-liter fermentors using glucose media and the effects of pH and dissolved oxygen (DO) levels will be studied under controlled conditions. Experiments are now being performed with 5-carbon sugar-rich solutions obtained from sweet sorghum bagasse as described under Objective 2b. Objective 3b: Washed and unwashed sweet sorghum bagasse were pretreated by the low moisture anhydrous ammonia (LMAA) method. The pretreated materials then were combined with sweet sorghum juice for ethanol fermentation. Commercial cellulases were added to generate glucose for additional ethanol production. To attempt to enhance ethanol production from sorghum grains, the grains were de-waxed with boiling ethanol under reflux conditions to recover wax as a value-added co-product and improve enzymatic starch hydrolysis. The de-waxed grains then were treated with dilute sulfuric acid (1 and 2 wt %) and subjected to mashing for ethanol fermentation. Commercial cellulases were added to the mash to hydrolyze the cellulose fraction of the hulls to produce glucose for additional ethanol production. A net improvement of 36.8 % in ethanol production over the raw grains was obtained. Accomplishments 01 Production of wax and ethanol from sorghum grains. Some sorghum contains wax that negatively affects enzymatic hydrolysis of starch during ethanol fermentation. Removal of wax by ethanol extraction, which resulted in more efficient starch hydrolysis to glucose and subsequently better ethanol yield. Following wax extraction the grains were treated with dilute sulfuric acid under mild conditions and commercial cellulase was added during fermentation to produce more ethanol from the additional sugar coming from the hulls. An integrated process for production of wax as a value-added co-product and ethanol at higher yields using sorghum grains as feedstock.

Impacts
(N/A)

Publications

• Nghiem, N.P., Ellis, C.W., Montanti, J.M. 2016. The effects of ethanol on hydrolysis of cellulose and pretreated barley straw by some commercial cellulolytic enzyme products. AIMS Bioengineering. 3(4):441-453.
• Challi, R.K., Zhang, Y.B., Johnston, D., Singh, V., Engeseth, N.J., Tumbleson, M., Rausch, K.D. 2017. Evaporator fouling tendencies of thin stillage and concentrates from the dry grind process. Heat Transfer Engineering. 38(7-8):743-752.
• Johnston, D., Moreau, R.A. 2017. A comparison between corn and grain sorghum fermentation rates, distillers dried grains with solubles composition, and lipid profiles. Bioresource Technology. 226:118-124.
• Nghiem, N.P., Montanti, J., Tae, H. 2016. Pretreatment of dried distillers grains with solubles by soaking in aqueous ammonia and subsequent enzymatic/dilute acid hydrolysis to produce fermentable sugars. Applied Biochemistry and Biotechnology. 179(2):237-250.
• Nghiem, N.P., Senske, G.E., Kim, T.H. 2016. Pretreatment of corn stover by low moisture anhydrous ammonia (LMMA) in a pilot-scale reactor and bioconversion to fuel ethanol and industrial chemicals. Applied Biochemistry and Biotechnology. 179(1):111-125.
• Nghiem, N.P., Brooks, W.S., Griffey, C.A., Toht, M.J. 2017. Production of ethanol from newly developed and improved winter barley cultivars. Applied Biochemistry and Biotechnology. 182:400-410.
• Yoo, C., Nghiem, N.P., Kim, T. 2016. Production of fermentable sugars from corn fiber using soaking in aqueous ammonia (saa) pretreatment and fermentation to succinic acid by Escherichia coli afp184. Korean Journal of Chemical Engineering. 33(10):2863-2868.
• Nghiem, N.P., Kleff, S., Schegmann, S. 2017. Succinic acid: technology development and commercialization. Fermentation. 3(26):1-14.

Progress 10/01/15 to 09/30/16

Outputs
Progress Report Objectives (from AD-416): 1: Develop technologies that enable the integrated processing of sorghum grains and sweet sorghum juice at existing biofuels production facilities and that enable the commercial production of new co-products at sorghum- based biorefineries. 1A: Develop technologies that enable the integrated processing of sorghum grains at existing biofuels production facilities. 1B: Develop technologies that enable the integrated processing of sweet sorghum juice at existing biofuels production facilities. 1C: Develop technologies that enable the commercial production of new co- products at sorghum-based biorefineries. 2: Develop technologies that enable the commercial production of marketable C5-rich and C6-rich sugar streams from sorghum lignocellulosic components. 2A: Develop technologies that enable the commercial production of marketable C5-rich sugar streams from sorghum lignocellulosic components. 2B: Develop technologies that enable the commercial production of marketable C6-rich sugar streams from sorghum lignocellulosic components. 3: Develop technologies that enable the commercial conversion of sorghum lignocellulosic components into fuels and industrial chemicals. 3A: Develop technologies that enable the commercial production of industrial chemicals from the C5-rich sugar stream obtained from the enzymatic hydrolysis of pretreated sorghum cellulosic components. 3B: Develop technologies that enable the commercial production of additional ethanol and industrial chemicals from the C6-rich sugar stream obtained from the enzymatic hydrolysis of the cellulose-enriched residue. 3C: Develop technologies that enable the use of byproducts and wastes generated in ethanol and other fermentation processes in the sorghum biorefinery for production of energy and chemicals. Approach (from AD-416): In conjunction with collaborators, develop technologies that enable commercially-preferred bio/chemical processes for converting all components of sorghum plants, including grains, juice, and bagasse, into fuels, industrial chemicals and consumer products. Develop commercially viable processes for incorporation of sorghum grains into existing commercial corn-based ethanol plants and evaluate the effects of this process modification on overall water balances in the existing plants. Develop commercially viable technologies for using sweet sorghum juice and sorghum biomass, including both carbohydrates and lignin, for the production of important platform chemicals, i.e. chemicals that can be used as precursors for production of a wide range of industrial chemicals and consumer products. Develop technologies for capturing the carbon dioxide gas generated in ethanol fermentation for use in other fermentation processes that requires CO2 as a secondary feedstock in addition to fermentable sugars. Develop technologies for conversion of the wastes generated in cellulosic ethanol and industrial fermentation processes into methane for internal use as an energy source. Develop an integrated process combining the aforementioned process components for a sorghum-based biorefinery. Objective 1: Fermentation studies utilizing grain sorghum and sweet sorghum juice have been ongoing. Evaluation of enzyme addition during fermentation has demonstrated several important differences relative to corn that will be beneficial in determining the best practices for sorghum integration at existing ethanol facilities. Sweet sorghum juice studies have shown that the juice alone is deficient in nutrients needed by the yeast for rapid fermentation and requires nutrient supplementation. When the juice is mixed with corn, the nutrient levels routinely added for corn are sufficient for fermentation. Microbial contamination in the incoming sweet sorghum juice is a significant concern. Contamination control must be adequately addressed to prevent widespread contamination if the juice is to be effectively used at existing ethanol facilities. Fractionation studies utilizing grain sorghum are also ongoing. Recovery of a protein rich fraction that is low in fiber has been accomplished. Further analysis and yield determinations are currently being conducted. Alternative fractionation methods are also being evaluated. The protein rich fraction has potential uses in animal diets where high fiber content is undesirable. Objective 2: Sweet sorghum bagasse was washed with water to recover the residual sugars (sucrose, glucose, fructose) that remained in the solids after juice extraction. The optimum conditions such as solid/water ratio, washing time and washing temperature were determined for maximizing sugar recovery. The washed solids were subjected to low moisture anhydrous ammonia (LMAA) pretreatment to increase the efficiency of the subsequent enzymatic hydrolysis. The pretreated solids were hydrolyzed with a commercial hemicellulase enzyme product to produce a xylose-rich solution, which could be used in fermentation processes for production of various chemicals using suitable xylose-metabolizing microorganisms. Another objective was to produce a solution of multiple fermentable sugars, which included both six-carbon sugars such as glucose and sucrose and five-carbon sugars such as xylose. To achieve this objective, both washed and unwashed sweet sorghum bagasse were pretreated with the LMAA process and subsequently subjected to enzymatic hydrolysis. The results indicated that LMAA pretreatment did not degrade the residual sugars in the unwashed bagasse to a significant extent and hence washing was not necessary for total sugar production by enzymatic hydrolysis. The optimal conditions of the LMAA pretreatment process such as reaction time and reaction temperature were determined for maximizing total sugar production from the pretreated bagasse in the subsequent enzymatic hydrolysis step. Objective 3: Ethanol fermentation was performed and the carbon dioxide co-product was absorbed in a sodium hydroxide solution in a glass absorption column to form a sodium carbonate solution. Six stainless- steel reactors were constructed for study of the use of the sodium carbonate solution for pretreatment of sweet sorghum bagasse to enhance the subsequent enzymatic hydrolysis for fermentable sugar production. Preliminary pretreatment experiments were performed. Statistics software was used to design the experiments to be performed on the optimization of the pretreatment process using sodium carbonate as the pretreatment chemical. These experiments are in progress and will be completed in the next fiscal year.

Impacts
(N/A)

Publications

• Nghiem, N.P., Montanti, J.M., Johnston, D. 2016. Sorghum as a renewable feedstock for production of fuels and industrial chemicals. Current Biochemical Engineering. 3(1):75-91.

Progress 10/01/14 to 09/30/15

Outputs
Progress Report Objectives (from AD-416): 1: Develop technologies that enable the integrated processing of sorghum grains and sweet sorghum juice at existing biofuels production facilities and that enable the commercial production of new co-products at sorghum- based biorefineries. 1A: Develop technologies that enable the integrated processing of sorghum grains at existing biofuels production facilities. 1B: Develop technologies that enable the integrated processing of sweet sorghum juice at existing biofuels production facilities. 1C: Develop technologies that enable the commercial production of new co- products at sorghum-based biorefineries. 2: Develop technologies that enable the commercial production of marketable C5-rich and C6-rich sugar streams from sorghum lignocellulosic components. 2A: Develop technologies that enable the commercial production of marketable C5-rich sugar streams from sorghum lignocellulosic components. 2B: Develop technologies that enable the commercial production of marketable C6-rich sugar streams from sorghum lignocellulosic components. 3: Develop technologies that enable the commercial conversion of sorghum lignocellulosic components into fuels and industrial chemicals. 3A: Develop technologies that enable the commercial production of industrial chemicals from the C5-rich sugar stream obtained from the enzymatic hydrolysis of pretreated sorghum cellulosic components. 3B: Develop technologies that enable the commercial production of additional ethanol and industrial chemicals from the C6-rich sugar stream obtained from the enzymatic hydrolysis of the cellulose-enriched residue. 3C: Develop technologies that enable the use of byproducts and wastes generated in ethanol and other fermentation processes in the sorghum biorefinery for production of energy and chemicals. Approach (from AD-416): In conjunction with collaborators, develop technologies that enable commercially-preferred bio/chemical processes for converting all components of sorghum plants, including grains, juice, and bagasse, into fuels, industrial chemicals and consumer products. Develop commercially viable processes for incorporation of sorghum grains into existing commercial corn-based ethanol plants and evaluate the effects of this process modification on overall water balances in the existing plants. Develop commercially viable technologies for using sweet sorghum juice and sorghum biomass, including both carbohydrates and lignin, for the production of important platform chemicals, i.e. chemicals that can be used as precursors for production of a wide range of industrial chemicals and consumer products. Develop technologies for capturing the carbon dioxide gas generated in ethanol fermentation for use in other fermentation processes that requires CO2 as a secondary feedstock in addition to fermentable sugars. Develop technologies for conversion of the wastes generated in cellulosic ethanol and industrial fermentation processes into methane for internal use as an energy source. Develop an integrated process combining the aforementioned process components for a sorghum-based biorefinery. Grain Sorghum was utilized for fermentations utilizing a process that was analogous to the corn dry grind ethanol process. Fermentation of grain sorghum using the identical process for corn resulted in small but statistically significant reductions in both rate and yield of ethanol relative to corn. This would have a serious negative economic impact for fuel ethanol facilities. Further testing showed that modification to grinding and enzyme supplementation could improve the conversion. Additional work is continuing to address the necessary modifications for successful implementation of grain sorghum into existing ethanol facilities. Sweet sorghum juice was used in ethanol fermentation using an industrial yeast strain of Saccharomyces cerevisiae. Final ethanol concentrations obtained with and without addition of commercial starch hydrolysis enzymes were the same, which indicated that the sweet sorghum juice used in these experiments did not contain significant levels of starch. The fermentation rates, however, were significantly faster when nutrients were added, which indicated the sweet sorghum juice required nutrients for efficient ethanol fermentation.

Impacts
(N/A)

Publications

• Zhang, X., Nghiem, N.P. 2014. Pretreatment and fractionation of wheat straw for production of fuel ethanol and value-added co-products in a biorefinery. Mathematical Biosciences and Engineering (MBE) Journal. 1(1) :40-52.
• Nghiem, N.P., Senske, G.E. 2014. Capture of carbon dioxide from ethanol fermentation by liquid absorption for use in biological production of succinic acid. Applied Biochemistry and Biotechnology. 175:2104-2113.
• Challa, R., Johnston, D., Singh, V., Tumbleson, M., Rausch, K. 2014. Fouling characteristics of model carbohydrate mixtures and their interaction effects. Journal of Food and Bioproducts Processing. 93:197- 204.