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ARS Home » Midwest Area » Peoria, Illinois » National Center for Agricultural Utilization Research » Bioenergy Research » Research » Research Project #438816

Research Project: New Bioproducts for Advanced Biorefineries

Location: Bioenergy Research

2023 Annual Report

Objective 1: Generate enzymes required to hydrolyze recalcitrant xylan structures to increase sugar availability for biorefinery processes. Objective 2: Develop an improved biorefinery process for production of itaconic acid from lignocellulose. Objective 3: Develop a biorefinery process for production of butyric acid from lignocellulose. Objective 4: Develop technologies that enable non-Saccharomyces yeast-based processes for bioconversion of lignocellulose to advanced biofuels and value-added bioproducts. Objective 5: Enable production of biocontrol co-products to add value to biorefinery process streams.

The last few decades have seen a dramatic growth in biofuels and bioproducts. Bioethanol accounts for over one-third of United States corn consumption and bio-based products (e.g., apart from ethanol) add $369 billion and 1.5 million jobs to the national economy (USDA, 2015). Yet the transition to lignocellulose feedstocks, with an estimated availability of 1 billion tons per year, has been slow and halting. One reason for their slow adoption has been a lack of bioproducts. The objectives of this plan share the common goal of developing microbial bioproducts for advanced bioenergy plants. Sugar conversion efficiency will be increased by using sophisticated analytical techniques to identify recalcitrant xylan structures and to use this knowledge for enzyme discovery. Structural analysis of carbohydrates is technically challenging and will rely on novel methods developed by ARS researchers. It is proposed to convert the generated sugars using either bacterial, yeast, or fungal cultures to butyrate, itaconate, and lipids. Butyrate is a widely used commodity chemical, itaconate can be used to manufacture bio-plastics, and lipids can be used either as a bioproduct or as a feedstock to manufacture biodiesel or green diesel. Finally, a unique set of Pseudomonas of proven efficacy as biocontrol agents for effectively combating fungal potato dry rot (and other plant diseases) will be evaluated for valorizing agriculture and lignocellulose associated process streams. The biocontrol agent is a substitute for azole-based chemicals and, so, this goal is medically beneficial in combating overuse of anti-fungal azole-based chemicals, which are blamed for raising fungal resistance in clinical settings. Taken together, the success of this project will advance the use of lignocellulose to the benefit of the U.S., especially the rural economy, and advance national environmental goals.

Progress Report
Objective 1: Producing biofuels and chemicals from cellulosic biomass is a long running national priority. A critical step in processing biomass is using enzymes to break down the fibers into sugars, which are used for fermentation. One barrier is sourcing enzyme formulations that are sufficiently comprehensive to release all the sugars. The issue is that the fibers are very complex at a molecular level and vary among different plants. Therefore, the enzyme mixtures used for this step need to be complex as well. Though commercial enzyme producers have made huge strides, they are still missing enzymes needed for complete hydrolysis. Absence of these minor enzyme components results in lower sugar yields and slow processing times. Earlier, ARS researchers developed the necessary analytical tools to identify the type of enzymes missing by analyzing biomass samples treated with commercial enzymes for what remained intact. It was discovered that commercial enzymes were deficient in enzymes for two different activities. This year we targeted enzymes with the missing activities. For the first target, enzymes from crude mixtures that showed the desired activity were enriched, via multi-step separations, and analyzed using proteomics; however, the protein database indicated over 50 possible candidates. Therefore, the enzyme mixture is being further purified to reduce the number of candidates to a manageable number. To target the second enzymatic activity, genes for four candidate enzymes were generated and recombinant microbes are in development to produce the four enzymes in a purified form for further testing for improvement of commercial enzymes. Objective 2: Itaconic acid is a versatile renewable industrial chemical used in the production of various resins, coatings, polymers, and clear plastics. However, its widespread use is limited by high production costs relative to petrochemical alternatives, such as acrylic acid. Waste agricultural residues can be used as a low-cost source of sugars for its production. ARS researchers in Peoria, Illinois, have identified an excellent fungal strain for conversion of cellulosic sugars to itaconic acid at high concentrations and yields, have identified and developed a plan to overcome obstacles to producing itaconic acid, and have been able to produce itaconic acid in a larger bioreactor using refined glucose. The research unit has secured the research records and biological materials and the research will resume pending a new hire. Objective 3: Butyric acid is an organic acid that can be upgraded for use in jet fuel, which is an 18 billion gallon market. Currently over 80 thousand metric tons are used each year. The goal of the project is to produce butyric acid using cellulosic feedstocks. Earlier, ARS researchers in Peoria, Illinois, isolated a bacterium from nature that was able to selectively produce butyric acid and was robust for fermentation of hydrolysate sugars. However, the fermentation took too long. This year the shortcoming was solved by implementing a cell recycle reactor. The culture is continuously filtered to remove the butyric acid while retaining the bacterium cells. The result is to reduce the concentration of butyric acid, concentrate the number of bacterium cells in the bioreactor, and greatly accelerate the rate of fermentation. In this case, production of butyric acid was increased over six-fold. Furthermore, the bioreactor ran for two weeks without incident; the culture remained productive, and the filter did not plug up. The strain was discovered not to ferment xylose. Fermentation of xylose is critical because it can account for 30-40%w/w of the sugars refined from fibrous biomass. This problem was solved by obtaining another bacterium strain of the same species. Fortuitously, this strain ferments glucose and xylose at the same time, which is a rare trait in microbes. Objective 4: ARS researchers in Peoria, Illinois, are working on yeast fermentation processes that convert sugars into single cell oils that can be potentially used to produce biodiesel and renewable jet fuels from cellulosic biomass. ARS researchers have identified exceptional yeast candidates for microbial conversion of cellulosic sugars to lipids from a large yeast screen, have begun to intensify the production of lipids to achieve higher concentrations and yields, and broaden the scope of research to include oil production from formate. Rhodotorula includes multiple species of yeast, many of which have been reported to be very good producers of lipids. Utilizing the ARS culture collection, fifty Rhodotorula yeast and related lipid-producing yeast were screened for lipid production on unrefined biomass hydrolysate sugars. This screen combined growth robustness on a challenging feedstock with lipid production capacity. Notably, of the top ten lipids producers, nine were Rhodotorula toruloides strains (R. toruloides). The R. toruloides yeast were further studied for lipid production using two different sources of pretreated biomass sugars. The best overall strains for lipid production were Y-6984, 6987, 27102, and 27013. One of these yeast strains was used by a collaborator to process industrially produced cellulosic sugars for production of lipids. ARS researchers in Peoria, Illinois, are seeking to further improve the properties of the R. toruloides yeast by combining them to form diploids which contain two copies instead of one copy of their genomic DNA. In preparation, this year, ARS researchers developed a DNA-based assay to determine the mating type (e.g., A1 or A2) and a separate assay to determine their ploidy (e.g., copies of genomic DNA). These assays were used to confirm that our most robust yeast (Y-6987) is a diploid. Our yeast cultures typically yield only 10 (grams/Liter) of lipids, which is too low for commercial interest. This problem has been solved by implementing a two-stage culture process. In the first stage, the yeast is grown to a very high cell density and in the second stage the yeast are cultured under conditions that favor lipid production. Lipid production was further increased by periodically feeding in sugars during the second stage. Through optimization of this process, the lipid concentration has been increased to over 35 g/l without lengthening the fermentation. ARS researchers have also begun to increase production volume to generate sufficient lipid samples to test for use in making biodiesel. Microbial fermentation of formate to value added products is of interest because formate can be manufactured directly from carbon dioxide. Therefore, fermentation of formate to value-added products is a possible strategy to sequester carbon dioxide to combat climate change. While R. toruloides yeast cannot grow directly on formate, quantifying the amount of formate it can tolerate will determine if it is an acceptable candidate for genetic engineering. We identified two strains that are promising to engineer for formate utilization. Objective 5: Fusarium dry rot causes greater potato losses than any other postharvest disease, costing approximately $500 million in the U.S. and approximately $10 billion globally. Dry rot decay incited by Fusarium sambucinum impacts potato quality and trim losses, and affected tubers are more susceptible to secondary disease infections. Treatment methods are limited. Because the dry rot pathogen infects tubers through wounds, treating tubers before storage with desiccation tolerant Pseudomonas triculture biocontrol agent (BCA), which coats and colonizes wounds, has shown great promise for disease control. This year small pilot and lab scale storage trials tested tricultures produced on either an expensive refined medium or one that uses unrefined sugars extracted from switchgrass. Switchgrass is being developed by ARS plant researchers as a dedicated bioenergy crop. The production cost was also lowered by successfully drying BCAS using fructose on the carrier. Trials showed significant impacts of disease pressure, potato cultivars, and storage temperature on disease development. However, low volume BCA application rates favored by the industry, led to concentrating insoluble components in sprays, which clogged nozzles in our large-scaled tests. Overall, results from ARS in Peoria, Illinois, and collaborators in Kimberly, Idaho, suggested superior performance by the synthetic versus switchgrass based medium. Although BCAs were not as effective as the leading chemical fungicide, further testing of mixtures of the chemical and bio-control products is warranted and this approach has shown promise in preliminary studies. Mixtures will broaden mode of action and may achieve more complete disease control and reduce risk of pathogen resistance. Jar testing BCA mixtures with three relevant chemical fungicides showed that suspensions could be maintained with continuous mixing, which did not appear harmful to cell viability during 24 hour exposure. Future research will seek to resolve observed spraying issues. Results from these studies advanced BCA development for the potato industry by revealing key factors contributing to product variability at large scale.

1. Identified yeast for production of oil suitable for conversion to fuels and chemicals. The aviation industry is seeking to lower their environmental footprint by using sustainable aviation fuel produced from vegetable oil. However, there is not enough vegetable oil available to meet their demand (18 billion gallons per year). ARS researchers in Peoria, Illinois, had earlier developed processes for producing oil similar to vegetable oil using a yeast that grew well on cellulosic sugars. ARS researchers in Peoria, Illinois, have now identified a set of four yeast from the ARS microbial culture collection that produce more lipids from cellulosic sugars than the original yeast. These new yeasts are expected to lower the cost for making biofuel using this process by producing more yeast oil per ton of cellulosic biomass.

2. Developed low-cost formulation of desiccation stable biological control agents for potatoes. Potato losses from fungal spoilage during storage are approximately $500 million in the U.S. and approximately $10 billion globally. The majority of losses are caused by the fungus Fusarium sambucinum. Chemical solutions are limited, and their efficacy threatened by pathogen resistance. ARS researchers in Peoria, Illinois, have developed an effective environmentally friendly treatment to stem these losses based on bacteria that are naturally antagonistic to this fungus. The major barrier to applying this “bio-control” is formulating it as a long-lasting dried product that is easy to apply. This problem was solved by developing strains to be more robust to drying and creating a special drying formulation in which simple inexpensive fructose sugar is blended with the bacteria to protect them during drying. Furthermore, the fructose can be used by the bacteria when they are revived before being sprayed onto the potatoes. The result is a product that can be stored for longer than 7 months and reduces potato storage disease by 80%. This research was supported by U.S. potato producers through an ARS State Partnership Potato Research Program grant.

Review Publications
Rozina, Ahmad, M., Qureshi, N., Zafar, M., Ullah, S.A., Ul Abidin, S.Z. 2022. Renewable energy production from novel and non-edible seed oil of Cordia dichotoma using nickel oxide nano catalyst. Fuel. 332(1). Article 126123.
Okonkwo, C.C., Duduyemi, A., Ujor, V.C., Atiyeh, H.K., Iloba, I., Qureshi, N., Ezeji, T.C. 2022. From agricultural wastes to fermentation nutrients: A case study of 2,3-butanediol production. Fermentation. 9(1). Article 36.
Deshavath, N., Dien, B.S., Slininger, P.J., Jin, Y., Singh, V. 2022. A chemical-free pretreatment for biosynthesis of bioethanol and lipids from lignocellulosic biomass: An industrially relevant 2G biorefinery approach. Fermentation. 9(1). Article 5.
Liu, S., Lu, S.Y., Qureshi, N., El Enshasy, H.A., Skory, C.D. 2022. Antibacterial property and metagenomic analysis of milk kefir. Probiotics and Antimicrobial Proteins. 14:1170-1183.
Saha, B.C., Kennedy, G.J., Bowman, M.J., Qureshi, N., Nichols, N.N. 2022. Itaconic acid production by Aspergillus terreus from glucose up to pilot scale and from corn stover and wheat straw hydrolysates using new manganese tolerant medium. Biocatalysis and Agricultural Biotechnology. 43. Article 102418.
Ashby, R.D., Qureshi, N., Strahan, G.D., Johnston, D., Msanne, J.N., Lin, X. 2022. Corn stover hydrolysate and levulinic acid: mixed substrates for short-chain polyhydroxyalkanoate production. Biocatalysis and Agricultural Biotechnology. 43:102391.
Qureshi, N., Liu, S., Saha, B.C. 2022. Butyric acid production by fermentation: Employing potential of the novel Clostridium tyrobutyricum strain NRRL 67062. Fermentation. 8(10). Article 491.
Palmer, N.A., Sarath, G., Bowman, M.J., Saathoff, A.J., Edme, S.J., Mitchell, R., Tobias, C.M., Madhavan, S., Scully, E.D., Sattler, S.E. 2023. Divergent metabolic changes in rhizomes of lowland and upland switchgrass (panicum virgatum) from early season through dormancy onset. Plants. 12(8). Article 1732.
Cardona, J.B., Grover, S., Bowman, M.J., Busta, L., Sarath, G., Sattler, S.E., Louis, J. 2023. Sugars and cuticular waxes impact sugarcane aphid (melanaphis sacchari) colonization on two different developmental stages of sorghum. Plant Science. 330.Article 111646.
Jia, Y., Kumar, D., Winkler-Moser, J.K., Dien, B.S., Rausch, K., Tumbleson, M.E., Singh, V. 2022. Coprocessing corn germ meal for oil recovery and ethanol production: a process model for lipid-producing energy crops. Processes. 10(4). Article 661.
Singh, R., Dien, B.S., Singh, V. 2022. Solvent-free enzymatic esterification of free fatty acids with glycerol for biodiesel application: Optimized using the Taguchi experimental method. Journal of the American Oil Chemists' Society. 99(9):781-790.
Cheng, M.H., Maitra, S., Carr Clennon, A.N., Appell, M., Dien, B.S., Singh, V. 2022. The effects of sequential hydrothermal-mechanical refining pretreatment on cellulose structure changes and sugar recoveries. Biomass Conversion and Biorefinery.
Slininger, P.J., Schoepke, A.R., Dien, B.S. 2022. Production of biological pest control agents on hydrolysates of switchgrass. Bioresource Technology Reports. 21. Article 101312.
Cheng, M.H., Singh, S., Carr-Clennon, A.N., Dien, B.S., Singh, V. 2023. Production of designer xylose-acetic acid enriched hydrolysate from bioenergy Sorghum, oilcane, and energycane bagasses. Bioresource Technology. 380. Article 129104.