Research Project: Improved Conversion of Sugar Crops into Food, Biofuels, Biochemicals, and Bioproducts

Location: Commodity Utilization Research

2024 Annual Report

Objectives
Objective 1 Quantify the impact of new sugar processing aids (chemical oxidizers) in combination with existing ones (e.g., enzymes) on raw cane sugar manufacturing. The research in Objective 1 will address the following sub-objectives: 1.A Evaluation of oxidizing agents and amylase enzyme for the degradation of soluble and insoluble starch in sugarcane juice. 1.B Evaluation of oxidizing agents and dextranase enzyme to control microbial growth and dextran levels in sugarcane juice. 1.C Evaluation of oxidizing agents to reduce sessile microbial growth and film formation on equipment. Objective 2 Develop sustainable, commercially viable, biobased products from sugar cane and sugar beets byproducts (e.g., sugarcane bagasse, sugar beet pulp, clarifier mud/cake). The research in Objective 2 will address the following sub-objectives: 2.A Development of commercially sustainable biobased products from processing byproducts to improve soil health, reduce waste disposal costs for the U.S. sugar manufacturers, and address the food-water-energy nexus. 2.B Development of commercially sustainable biobased products from processing byproducts (bagasse and beet pulp) for high end horticulture products and soil health applications. 2.C Production of biobased polymers from sugarcane molasses, bagasse, and clarifier mud/cake. Objective 3 Enable commercially viable, renewable, biofuels and chemicals from sugar cane and sugar beet processing byproducts (e.g., molasses, clarifier mud/cake). The research in Objective 3 will address the following sub-objectives: 3.A Production of solvents and jet fuel pre-cursors from sugarcane molasses and clarifier mud/cake. 3.B Production of acetoin and 2,3-butanediol from molasses and sugar beet extract. 3.C Recovery of aconitic acid from sugarcane molasses and testing its potential for nematode bioactivity.

Approach
Modern sugar production for human consumption, and as a starting point for fermentations, has existed for centuries. While the sugar manufacturing technology is well known, there is a need by the sugar industry to improve processing and develop new coproducts to increase the profitability for farmers and processors. This can be accomplished by * reducing chemicals used to control starch and dextran in raw sugar manufacturing, * improving the quality of the raw sugar to minimize refining costs, * reducing sugar losses by microbial action by improving sanitation, and * more effectively utilizing the byproducts to make coproducts. Therefore, to address these goals our research will focus on * identify impact and optimize the use of processing aids to improve the sugar quality while reducing the cost, * develop biobased products from sugar manufacturing byproducts, and * develop renewable biofuels and biochemicals from sugar manufacturing byproducts. The byproducts targeted to produce the bioproducts and biochemicals are * bagasse, clarifier mud/cake, and molasses from sugarcane processing, and * pulp, and beet extract from sugarbeet processing. In any research effort, it is important that performance metrics are established. Thus, each sub-objective has its own performance metrics. In all cases, an economic analysis will be performed to determine cost of implementation. In addition, the impact of byproduct use or process changes will be evaluated in collaboration with local (or impacted) industry using a limited life cycle assessment around the system altered. The outcome of this research will result in the following anticipated products: * a lower cost, cleaner process, and improved raw sugar from sugarcane factories, * advanced fertilizers and high-end soil amendments, * composite polymers, and * biochemicals for solvents and fuels, as precursors to other chemicals, and for pest control.

Progress Report
This is the third annual report. The project focuses on issues related to the processing of sugar crops for food use. Also investigated is the use of byproduct streams for food- and nonfood-based products. Progress was made on the project objectives, all of which fall under National Program 306, Component 1 – Foods, Problem Statement 1A: Define, Measure, and Preserve/Enhance/Reduce Factors that Impact Quality and Marketability; Component 2 – Nonfood (fibers including hides), Problem Statement 2B: Enable Technologies to Produce New and Expand Marketable Nonfood, Nonfuel Biobased Products Derived from Agricultural Feedstocks; and Component 3 – Biorefining, Problem Statement 3A: Viable Technologies for Producing Advanced Biofuels (including Biodiesel), or other Marketable Biobased Products. In support of Objective 1, ARS researchers in New Orleans, Louisiana, completed isolation and identification of numerous microorganisms (isolates) from sugarcane and sugar beet factories. In particular, one study on microbes present in North American sugar beet factory juices and biofilms resulted in 612 isolates for investigation. Selected microbial isolates were studied further for production of dextran (a large and sticky polysaccharide chemical) and impact on liquid viscosity. This is an ongoing challenge during raw sugar manufactoring. Understanding how various microbial polysaccharides contribute to operational challenges during sugar crop processing is central to solving these problems. Additional work was also done in collaboration with University of Georgia, under agreement 6054-41000-114-008S, to determine the structure of these sticky polysaccharides. This work was documented in a manuscript and will continue in future years. Also in support of Objective 1, ARS researchers in New Orleans, Louisiana, studied several isolates from sugarcane factories and measured sensitivity to industrial biocides in order to determine optimal level required to prevent microbial growth and minimize sucrose losses. These ARS researchers also used factory data to determine “breakeven” prices of biocides as neutral thresholds above which it would be profitable to apply a particular biocide. The results were written up in a manuscript submitted for publication. In support of Objective 1, ARS researchers in New Orleans, Louisiana, treated sugarcane billets with two common processing aids (the biocide carbamate and the oxidizer bleach). Each study included controls with no treatment (water control) and dry controls (no treatment dipping). Billets were infected with a common bacteria called Leuconostoc mesenteroides, which had been found at a raw sugar factory and grown in the laboratory. The impact of the processing aids were monitored over 24 hours. In summary, both the biocide and the bleach were effective in reducing the microbial population for the first 6 hours after treatment (from 57% to 85%) but after 24 hours, differences were no longer observed between treatment and controls. By that time, the treatment was no longer effective for the overwhelming population of bacteria. Follow up experiments are planned once harvesting season starts again. In support of Objective 1, ARS researchers in New Orleans, Louisiana, collected raw sugar manufacturing data from processors in Louisiana. In total, several sugar mills have shared complete daily logs of relevant data, which includes quantitative information on the use of processing aids (enzymes and biocides). Computational analyses are ongoing to highlight how the use of processing aids impacts raw sugar production and quality. Follow-up conversations are planned with sugar beet processing facilities to collect analogous data for similar analysis. In support of Objective 2, ARS researchers in New Orleans, Louisiana, produced additional biochars from Florida sugarcane factory bagasse and field residues. Initial testing revealed that these feedstocks have different properties as compared to those from Louisiana. Biochars produced were analyzed by various methods. These biochars, together with biochars previously produced from Louisiana sugarcane factories will be utilized in greenhouse studies. Also, related to Objective 2, under agreement 58-6054-1-007 (Log 68450) with West Virginia State University, ARS researchers in New Orleans, Louisiana, fully characterized the series of biochars that had been produced, from woody and herbaceous bioenergy crops (miscanthus, switchgrass and willow). Biochars are developed with the objective to maximize their efficacy as soil amendment, specifically to enhance marginal soil nutrient and water holding capacity and improve efficiency of runoff mitigation practices in retaining and removal of contaminants. The West Virginia State University collaborator will be testing the biochars in greenhouse experiments. In support of Objective 2, different types of transformation pathways were determined for sugarcane mill mud and bagasse, which are byproducts of sugarcane factories. ARS researchers in New Orleans, Louisiana, determined how parts of the chemicals in the sugar processing byproducts were affected by different natural degradation pathways, such as by light or by microorganisms. It was found that under normal factory storage conditions in piles, the amount of reactive chemicals decreased, but when some of the byproducts were illuminated by light, the amount of reactive chemicals increased. This increase in reactive chemicals should be beneficial when the byproducts are applied to fields and may stimulate plant growth for months. Also in support of Objective 2, and under agreement 6054-41000-114-003S with Cornell University, greenhouse experiments were designed to test the effects of pretreatments (such a maturation) on the performance of sugarcane factory mud as a plant growth stimulator. A detailed study of the microorganisms and their internal biological machinery suggested that this type of investigation could be used as a tool to monitor maturation stages and predict how the mature material may benefit soil health. By extension, the developed tool could also be used to evaluate the general health of soil. In support of Objective 2, under agreement 58-6054-3-015 with American Sugarcane League, multiple indicators of sugarcane’s freeze tolerance were discovered in the plant parents used in the ongoing cultivar development program. The indicators (which included amino acids, plant fats, and other plant components) explained why some sugarcane varieties were more tolerant against winter freeze than others. Now, selection protocols are being developed to incorporate these indicators in the commercial sugarcane breeding program. In support of Objective 3, ARS researchers in New Orleans, Louisiana, conducted large scale fermentations (2 liters) to produce acetoin from Bacillus bacteria. Small scale (10 milliliters) and large scale (500 milliliter) extractions were carried out to separate acetoin from the fermentation broth. Experiments show that using acetone and potassium salt for extraction, 90 to 95% of acetoin can be recovered consistently. These results were then used to estimate the cost of bio-based acetoin production and recovery and a draft manuscript was written. In support of Objective 3, under agreement 58-6054-3-005 (Log 71883) with Cotton Incorporated, high-quality alcohol was produced by fermentation with yeast. The work will continue in the next fiscal year as non-funded collaboration.

Accomplishments
1. Production and recovery of actetoin from sugar crop byproducts. Acetoin is a chemical that serves as a starting material for many other chemicals in chemical processing. ARS scientists in New Orleans, Louisiana, evaluated several byproduct streams from sweet sorghum, sugar beet, and sugar cane processing and showed that acetoin can be produced from sugar-rich streams by a bacterium called Bacillus subtilis in fermentations. The scientists then showed, with minor assistance from Tulane University students, that the acetoin can be extracted by acetone, which is a solvent that can also be produced from sugar crop byproducts. Finally, the ARS scientists designed a fermentation and recovery process using SuperPro Designer software and estimated the cost of production and environmental impact for different scales of production. The work was documented in a draft manuscript that will be submitted to a peer-reviewed journal. The work will benefit stakeholders in the sugar crop industry who are interested in the use of sugar-rich byproducts from their processes.

2. Modeling molecular properties of biofuel compounds. Conversion of renewable resources into biofuels can produce new compounds that are difficult to characterize. Using machine learning techniques, ARS researchers in New Orleans, Louisiana, developed a flexible modeling approach that can estimate properties of complex, bio-based molecules. These properties are related to solubility, heating, and combustion for biofuels. This advancement will make it easier to understand the performance of new biofuels. The work was published in a peer reviewed journal. Overall, this will improve the processing of agricultural byproducts and other renewable resources into carbon neutral, bio-based products.

3. Identification of polysaccharide-producing bacteria in sugar mills. Microbes present during raw sugar manufacturing can result in lost revenue and significant operational challenges. ARS researchers in New Orleans, Louisiana, isolated and identified several individual microbes from sugar crop factories. Next, they purified the sticky polysaccharide material that the bacteria produce. Although the industry has historically focused on only one type of polysaccharide/biofilm material, known as dextran, ARS research supported by the Beet Sugar Development Foundation and the American Sugar Cane League identified many different types of microbes (about 700 isolates) that produce sticky products. One prominent and overlooked type is levan fructan. Levan fructan from sugarcane factory bacteria was produced and thoroughly characterized in the laboratory and potential mitigation strategies were identified. Successfully controlling microbes and their sticky polysaccharides has potential to result in additional revenue of roughly $3 million per year for Louisiana raw sugar manufacturing, which makes up nearly 25% of the United States production market.

4. Electro-catalytic method to measure the environmental fate of soil amendments. Electron transfer (redox) reactions control the performance of renewable fertilizers. Traditional analyses lack sensitivity to detect the chemical structures causing the desirable functions of soil amendments. ARS researchers in New Orleans, Louisiana, utilized redox catalysts to directly measure the reactivity of agricultural byproducts. The developed method is useful to grade the quality of biomass feedstocks for soil application and to predict soil health effects. The new approach offers a high-throughput decision tree to divert an estimated 350 million tons of biomass left unused in the U.S. annually.

5. Treatment of billeted cane in cane yards is achievable and reduces microbial contamination. The ability to treat billets ahead of processing at the raw sugar factory can translate into significant decreases of microbial contamination and sugar loss the raw sugar factory. ARS researchers in New Orleans, Louisiana, demonstrated that both the chemicals carbamate and bleach were effective at reducing microbial degradation for up to 6 hours. This accomplishment demonstrates that microbial loads can be better managed if the treatments start in the cane yard, ahead of processing of the sugarcane at the factory.

6. Application of sugarcane biochar onto cotton and corn fields resulted in increased yields. ARS researchers in New Orleans, Louisiana, showed that cotton fields had up to 22 % increase in lint yield and up to 13% increase in cotton seed yield after three years of biochar application to soil. After the third year of successive biochar applications, corn yield increased 16 % when compared to control. These studies demonstrated that successive biochar applications can benefit crop production.

Review Publications
Uchimiya, S.M. 2023. Electrocatalytic method to quantify the redox reactivity of agricultural byproducts. Biomass and Bioenergy. 181. Article 107006. https://doi.org/10.1016/j.biombioe.2023.107006.
Klasson, K.T. 2024. A discussion and evaluation of statistical procedures used by JIMB authors when comparing means. Journal of Industrial Microbiology and Biotechnology. 51. Article kuae001. https://doi.org/10.1093/jimb/kuae001.
Uchimiya, M., Elliott, L.E., Derito, C.M., Hay, A.G. 2023. Metagenome-assembled genomes from sugarcane mill mud. Microbiology Resource Announcements. 12(11). Article e00568-23. https://doi.org/10.1128/MRA.00568-23.
Bruni, G.O., Qi, Y., Terrell, E., Dupre, R.A., Mattison, C.P. 2024. Characterization of levan fructan produced by a Gluconobacter japonicus strain isolated from a sugarcane processing facility. Microorganisms. 12(1). Article 107. https://doi.org/10.3390/microorganisms12010107.
Uchimiya, S.M., Reis, A., Lago, B., Kimbeng, C. 2023. Early detection cold markers to expedite the sugarcane (Saccharum spp. hybrids) breeding. Journal of Agricultural Science and Technology. 3(12): 1117-1124. https://doi.org/10.1021/acsagscitech.3c00209.
Terrell, E. 2024. Estimation of fuel properties for the heavy fraction of biomass pyrolysis oil consisting of proposed structures for pyrolytic lignin and humins. Energies. 17(9). Article 2011. https://doi.org/10.3390/en17092011.
Qi, Y., Bruni, G.O., Klasson, K.T. 2023. Microbiome analysis of sugarcane juices and biofilms from Louisiana raw sugar factories. Microbiology Spectrum. 11(3). Article e04345-22. https://doi.org/10.1128/spectrum.04345-22.
Qi, Y., Bruni, G.O. 2023. Draft genomes of 17 bacterial isolates from Louisiana raw sugarcane factory juices and biofilms. Microbiology Resource Announcements. 12(9). Article e00416-23. https://doi.org/10.1128/MRA.00416-23.
Terrell, E., Qi, Y., Bruni, G.O., Heck, E. 2023. Influence of oxidizing and non-oxidizing biocides on enzymatic and microbial activity in sugarcane processing. Processes. 11(9). Article 2693. https://doi.org/10.3390/pr11092693.
Denson, M.D., Terrell, E., Kostetskyy, P., Broadbelt, L., Olarte, M., Garcia-Perez, M. 2023. Theoretical insights on the fragmentation of cellulosic oligomers to form hydroxyacetone and hydroxyacetaldehyde. Energy and Fuels. 37(18):13997-14005. https://doi.org/10.1021/acs.energyfuels.3c01924.