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ARS Home Midwest Area Peoria, Illinois National Center for Agricultural Utilization Research Bioenergy Research Research





- Dien, Bruce
- Bowman, Michael
- Qureshi, Nasib
- Slininger, Patricia - Pat
- 2023
- 2022
- 2021
- Publications
- Product Quality and New Uses (306)

Research Project: New Bioproducts for Advanced Biorefineries **Location:
                            Bioenergy Research** 2022 Annual Report **Objectives**
                                

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. **Approach**
                                

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: Production of sugars for fermentation to biofuels requires enzymes to release them from biomass fibers. These formulations contain multiple enzymes needed to act on the complex fibers within the plant cell where the sugars reside. Earlier research showed that commercial enzyme formulations are incomplete for release of these sugars. However, ARS researchers in Peoria, Illinois, were able to discover which enzymes were missing by screening various enzymes in combination with commercial mixtures and using novel and sophisticated analytical techniques to characterize residual fiber material that was only partially digested. As expected, adding in crude enzyme mixtures containing the putative missing enzymes resulted in more complete breakdown of fibers. This year, we sought to isolate and determine the protein sequence of specific enzymes from these crude mixtures. This involved running a complex scheme of isolation steps, starting with large batches of crude enzymes and evaluating after each step where the targeted enzymes ended up. Once enriched, the proteins present in these fractions were sequenced and individual proteins identified. The next step will be to generate genes to express these enzymes in either Escherichia coli or yeast to generate pure enzyme for detailed kinetic testing and validating benefits of the individual enzymes when used to supplement commercial enzyme formulations. The eventual outcome will be enzyme formulations that give higher sugar yields and require a lower loading of expensive enzymes. After feedstocks, enzymes are the costliest input for producing cellulosic biofuels.
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, but certain metal ions and non-organic materials present in the biomass inhibit itaconic acid production by a fungus. To address this, we developed techniques that allow for removal of metal ions throughout the process, thereby, producing metal-free sugars. The heart of the process was treating wheat straw with hot water, which is advantageous compared to other similar treatments because it avoids adding chemicals, such as acid or base. Adding chemicals incurs greater input costs and complicates waste treatment of process water. Sugars for fermentation were subsequently generated by treating the hot water treated wheat straw using standard commercial enzymes marketed for this purpose, but that had also been specially treated with techniques developed by ARS researchers to remove metals; this is the first time we are aware of that this task has been accomplished. The process was optimized and demonstrated that wheat straw can be used to produce itaconic acid. 
Objective 3: Butyric acid is a small weight organic acid. Currently over 80 thousand metric tons are used each year and it can also be upgraded for use in jet fuel, which is an 18 billion gallon annual market. However, butyric acid production by fermentation needs further refinement to make it commercially attractive by allowing for high productivities and titers. Last year, growth conditions were optimized to increase fermentation rates by 300%. However, these experiments used an expensive refined medium. This year, several commercial nitrogen sources were considered, including those prepared from corn steep liquor (CSL) and soy, either alone or in combination. An optimized medium based solely on CSL gave yields similar to the refined medium and better than either adding soy or the mixtures. CSL is an inexpensive byproduct of corn milling that is commonly used for industrial fermentations. Use of CSL lowers medium costs by an order of three-fold.
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 sustainable aviation fuel from cellulosic biomass. A technical barrier is finding a yeast that will grow and produce lipids when fed unrefined biomass sugars and achieves high oil titers. To address this, researchers developed a method that allows for convenient screening of lipid-producing yeast for the best strains based on growth rate, lipid production, fatty acid composition, final yeast biomass, and sugar consumption. This method was used to screen 72 yeast strains and selected strains were further screened using multiple sources of biomass sugars. While analysis of results is continuing, researchers have already identified one yeast that produced the same amount of lipids in half the time as the next best yeast. This yeast strain is being actively researched to elicit the source of its enhanced efficiency. ARS researchers in Peoria, Illinois, also made important progress in development of a new pretreatment that combines simultaneous pressurized ammonia and ball milling to extract sugars that are readily fermented by the lipid producing yeast. The method was also used to convert residual fibers from pressed sugarcane to fermentable sugars. In contrast to results with more conventional pretreatment methods, leftover sugar present in the unwashed fibers made it through the process intact. The pretreated fiber was subsequently digested with commercial enzymes and the extracted sugars successfully converted to yeast oil. Other successes, achieved with collaborators include: identifying a new fermentation medium that increased lipid yields and lowered ingredient costs, further reducing costs by cutting the amount of buffer added by ten-fold without effecting product yield, and demonstrating that the ARS preferred oil producing yeast performed as good or better on cellulosic sugars than a comparable genetically modified organism (GMO) yeast.
Objective 5: Fusarium dry rot causes greater potato losses than any other postharvest disease. Over 80% of pathogenic strains are resistant to marketed chemicals. Biological control agents use microbes to combat plant diseases and are an accepted and environmentally friendly alternative to use of chemicals.  Three Pseudomonas species have been discovered to work together as a biological control agent (BCA) to protect potatoes against sprouting and several especially problematic fungal diseases for the potato industry. Evolution yielded desiccation tolerant (DT) strain variants (U.S. patent issued April 2021), a trait allowing longer shelf-life for better economy and convenience. In second-year studies, cocultures, produced in biomass hydrolysate media, performed well in small pilot trials at University of Idaho.  The microbes were grown on unrefined cellulosic sugars, which are expected to be less expensive than refined corn sugars and to provide a valuable co-product for the cellulosic based industry. Funded by the ARS-State Partnership Potato Research Program, several production/application practices were also tested in order to enhance BCA performance, improve economics and handling convenience, and promote adoption by the potato industry.  Practices tested included:  BCA growth media compatible with lignocellulose biorefineries; surfactants to improve spray coverage at minimal volumes for effective BCA dosage; and rehydration techniques for reactivating dry stored BCA products.
Overall, results showed a clear benefit of BCA in lowering potential for dry rot decay of Clearwater Russets in storage. Circa 30% reduction in the incidence of tubers with 5% decay was observed, and up to 78% reduction of disease severity in best treatments. This is a significant finding, which demonstrates that the BCA can be effectively applied to  guard against a disease that is difficult to control in potatoes. Production of a broad-spectrum antifungal agent on low-cost renewable substrates and improved dry storage formulations of the biocontrol product are expected to lower costs and expedite application by growers. This new technology benefits agriculture by providing an antifungal microbial alternative to chemicals no longer effective to prevent dry rot and as a potential co-product for generation of cellulosic biofuels. **Accomplishments**
                                

1. Developed new resource for biofuel production. It has long been known that the sugar laden juice collected from sweet sorghum can be used to make butanol by fermentation. However, this leaves behind the bagasse or pressed plant stalks. ARS researchers in Peoria, Illinois, developed a new process that also converts the bagasse to butanol. Notable aspects of the process are combining steps for converting the treated stalks to sugars, fermentation, and recovery of the butanol into a single tank. Using a single tank saves money, and continuous butanol recovery greatly accelerates butanol production. This work supports  expanded U.S. production of sweet sorghum, a crop which requires low agricultural inputs and is highly drought tolerant. This will benefit the biofuels industry by increasing the yield that can be realized for butanol when growing sorghum on arid lands. 2. Developed novel method for co-production of two valuable chemicals from corn stover. 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, but certain metal ions and organic inhibitors generated during biomass pretreatment suppress itaconic acid production by the fungus. ARS researchers in Peoria, Illinois, have solved this problem by developing a novel method to fractionate corn stover into an ash free glucose syrup and largely non-glucose containing sugar syrup.
The glucose was conveniently fermented to itaconic acid and the other sugars microbially converted to xylitol, which is a sugar substitute that is prized as a food ingredient. The results demonstrate that processed corn stover sugars can be separated to produce two value-added products. Annually, about 75 million tons of corn stover residue is produced in the United States. This research seeks to increase farmers’ profits by developing a market for this underutilized and widespread crop residue. 3. Developed a new biological control agents for postharvest control of Fusarium dry rot of potatoes. Fusarium dry rot causes about $500 million in annual losses for stored potatoes. It is estimated to be the costliest postharvest disease for U.S. producers.  However, pathogens have developed resistance to available azole-based chemical treatments, a concern for control of both agriculturally and medically important fungal diseases. A biological control agent (BCA) developed by ARS scientists in Peoria, Illinois, is produced as a dry product using a combination of three desiccation tolerant Pseudomonas species. Originally found in suppressive potato fields, the BCA colonizes potato wounds and prevents Fusarium dry rot decay. Additionally, it helps ward off other potato diseases and prevent sprouting during storage. It does so via multiple mechanisms that minimizes risk of pathogen resistance. Funded by the ARS-State Partnership Potato Research Program, a two-year collaborative study was carried out with University of Idaho scientists in Kimberly, Idaho, using Clearwater Russet potatoes (a cultivar having average dry rot susceptibility) under simulated commercial storage conditions. The study tested if the BCA bacteria could be grown using inexpensive cellulosic sugars as well as several improvements in applying the BCA to further reduce costs and improve handling convenience. Test results are expected to promote adoption of this technology by the potato industry.  Results demonstrated a clear benefit for using BCA on stored potatoes. Circa 30% reduction in the incidence of tubers with 5% decay was observed, and up to 78% reduction of disease severity in best treatments. This is a significant finding, which identifies how to produce and apply the BCA to effectively guard against a disease that is difficult to control in potatoes. This new technology benefits agriculture by providing both a biological alternative for dry rot disease control that will be resilient to pathogen resistance and a novel use for cellulosic sugars. **Review Publications**
                            *Qureshi, N., Saha, B.C., Liu, S., Ezeji, T.C., Nichols, N.N. 2021. Cellulosic butanol biorefinery: production of biobutanol from high solid loadings of sweet sorghum bagasse - simultaneous saccharification, fermentation, and product recovery. Fermentation. 7(4): Article 310. https://doi.org/10.3390/fermentation7040310.*
                                *Hay, W.T., Anderson, J.A., McCormick, S.P., Hojilla-Evangelista, M.P., Selling, G.W., Utt, K.D., Bowman, M.J., Doll, K.M., Ascherl, K.L., Berhow, M.A., Vaughan, M.M. 2022. Fusarium head blight resistance exacerbates nutritional loss of wheat grain at elevated CO2. Scientific Reports. 12. Article 15. https://doi.org/10.1038/s41598-021-03890-9.*
                                *Ispirli, H., Bowman, M.J., Skory, C.D., Dertli, E. 2021. Synthesis and characterization of cellobiose-derived oligosaccharides with bifidogenic activity by glucansucrase E81. Food Bioscience. 44(Part A). Article 101388. https://doi.org/10.1016/j.fbio.2021.101388.*
                                *Zhao, S., Dien, B.S., Lindemann, S.R., Chen, M. 2021. Controlling autohydrolysis conditions to produce xylan-derived fibers that modulate gut microbiota responses and metabolic outputs. Carbohydrate Polymers. 271. Article 118418. https://doi.org/10.1016/j.carbpol.2021.118418.*
                                *Singh, R., Dien, B.S., Singh, V. 2021. Response surface methodology guided adsorption and recovery of free fatty acids from oil using resin. Biofuels, Bioproducts, & Biorefining (Biofpr). 15(5): 1485-1495. https://doi.org/10.1002/bbb.2255.*
                                *Maitra, S., Dien, B., Long, S.P., Singh, V. 2021. Development and validation of time-domain 1H-NMR relaxometry correlation for high-throughput phenotyping method for lipid contents of lignocellulosic feedstocks. Global Change Biology Bioenergy. 13(7): 1179-1190. https://doi.org/10.1111/gcbb.12841.*
                                *Walker, C., Dien, B.S., Giannone, R., Slininger, P.J., Thompson, S.R., Trinh, C. 2021. Exploring proteomes of robust Yarrowia lipolytica isolates cultivated in biomass hydrolysate reveals key processes impacting mixed sugar utilization, lipid accumulation, and degradation. mSystems. 6(4). Article e00443-21. https://doi.org/10.1128/mSystems.00443-21.*
                                *Liu, Z., Dien, B.S. 2022. Cellulosic ethanol production using a dual function novel yeast. International Journal of Microbiology. 2022: Article 7853935. https://doi.org/10.1155/2022/7853935.*


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