Location: Sustainable Biofuels and Co-products Research
2018 Annual Report
Objectives
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
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 Report
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.
Accomplishments
Review 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.
