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

Location: Commodity Utilization Research

2022 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 second annual report. The project focuses on issues related to the processing of sugar crops for food use and the use of byproduct streams for food-based products and nonfood biobased fuels, chemicals and products. Progress was made 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 Sub-objective 1.A, ARS researchers at New Orleans, Louisiana,contacted a commercial vendor and asked for samples of chemicals and enzymes used at raw sugar factories. These samples include the enzyme amylase which is part of the future studies. Experimental setup for future milestones on this work was prepared. Procurement of necessary supplies is ongoing. In support of Sub-objectives 1.B and 1.C, several samples were collected from raw sugar factories. We used the microorganism in the samples make chemical compounds called dextrans and fructans, which make up biofilms. Special biofilm reactors were purchased and will be used in future studies. Also, in support of Sub-objective 1.B, an incoming agreement (number 70626) was formed with the American Sugar Cane League, entitled “Molecular and Microbiological Characterization of Dextran/Fructan-Forming Organisms from Sugar Cane”. Here, we are characterizing microbes from sugar cane factories. These individual microbes are then tested for sugar use and formation of byproducts that are problematic for sugar processing. Progress on other agreements (number 61080, 63451, 64764) with the American Sugar Cane League and related to Objective 1 have been on hold due to Covid restrictions. The research will be re-started in Fall/Winter 2022. In support of Sub-objective 2.A and to understand what causes environmental decay of sugarcane byproducts applied to soil, we conducted several chemistry and microbial tests. The microbiology approach used gene sequencing (16S), and chemistry approach focused on the effects of sunlight and free radicals. A total of 15 byproduct samples were collected from 5 different cane sugar factories in Louisiana and Florida. Microbial community that survived factory operation was unstable, and quickly diversified. This change in byproduct microbiology could be predicted from different chemistry, particularly nutrients (phosphorus and organic carbon) and acidity. Those simple chemistry metrices can be used as the indicator for how stable can sugar byproducts are in soils and other outdoor environments, e.g., storage piles and waste ponds. Because sunlight and free radicals are the key factors in the byproducts decay, different light intensities and ranges were tested. Changes in chemical structures were highly time-dependent. Identified light and redox parameters are used to add values to byproducts to develop potting mix and higher-end gardening products, in collaboration with compost producers. Related to Sub-objective 2.A, research under agreement number 6054-41000-114-003S with Cornell University, we did genetic sequencing of microbes in raw sugar factory mud and bagasse, and on soils samples where the mud and bagasse had been added. We used a method called random forest analysis to understand what the microbes from the different raw sugar factory byproducts may do when they are added to soil. In support of Sub-objective 2.B, several biochars were produced from sugar beet pulp and sugarcane bagasse at different pyrolysis temperatures to produce a range of biochars of varying surface functional properties and chemical composition. Characterization of the resulting biochars has been initiated but not completed. Also, in support of this objective, a large field application of biochar was carried out, covering 16 acres of sugarcane producing land owned by a local raw sugar factory. Half the acreage received 2 ton/acre of biochar applied directly on top of row, the remaining were controls without biochar. The field study is being done in collaboration with various stakeholders. Soil samples were collected before and after biochar application for future analysis. Also under this sub-objective, we did an extensive literature review on characterization of the byproducts of sugar crop production. We also did an in-depth review of conversion technologies and reuse. The reviews were published. In support of Objective 2 to and under agreement 63890 with Danimer Scientific, the collaborator is currently undergoing a billion-dollar facility expansion for biobased and bio-degradable plastic (bio-plastics) production in Georgia. As diversification is a long-term interest of biobased industry, a life cycle inventory for potential sugar feedstocks are being constructed. Different boundary conditions are considered for sweet sorghum and other resistant sugar crops against lipid-based feedstocks. In support of Sub-objective 3.A, we mixed clarifier mud/cake was with sugarcane molasses and fermented it into acetone/butanol/ethanol. The result indicated without the addition of mud, butanol was the preferred product. With mud (5-20%), acetone was the preferred product. More mud tended to favor more acetone production but not significantly. In support of Sub-objective 3.B, we added (one-by-one) yeast extract, corn steep liquor, and urea in a solution used to feed an acetoin-producing bacteria. The studies showed that, when grown on molasses, yeast extract could be eliminated without significant impact on acetoin production. This is a major step, as yeast extract is an expensive source of nitrogen that does not need to be added in the future. Also, under this objective, we did an extensive literature review on the production of valuable chemicals (such as acetoin) from agricultural byproducts. The review was published and was featured on the cover of the journal. In support of Sub-objective 3.C, we analyzed for aconitic acid in raw sugar factory streams samples collected October to early December to see if there was a seasonal variability of aconitic acid in the streams. We evaluated a large amount of data and showed that there was no strong trend in the levels of aconitic acid across the season. This would suggest that aconitic acid could be recovered at any time during the season from the sugarcane stream investigated. We shared the results with stakeholders at a workshop in June 2021. We published the data together with an extensive literature review about aconitic acid, its uses, and production methods. We also made progress on optimizing aconitic acid extraction and recovery to make a concentrated extract. Also, in support of this objective, we made progress toward testing the extract as nematicide in a nematode model. The results will be included in an upcoming manuscript. Also related to Objective 3, we completed research under agreement number 66450 with Cotton Incorporated in December 2021 and a final report was submitted. Here, we investigated the need for contaminant removal from sugar solutions obtained from solubilized discarded textiles and fermentation of the sugar solutions to biochemicals. The work was successful and, in some cases, contaminant removal was needed. The research was presented with posters at several scientific meetings. The results were shared with Cotton Incorporated in quarterly reports.

Accomplishments
1. Causes of biomass aging explained by bioinformatics. ARS researchers in New Orleans, Louisiana, and collaborators utilized gene sequencing techniques to understand the long-term effects (the aging) of soil amendments derived from sugar processing byproducts. In its pure form, heat-resistant bacteria (Firmicutes) dominated the biosolid generated from high-temperature conversion of sugarcane to raw sugar. Upon environmental exposure when mixed with soil, the native microbes in the soils quickly replaced Firmicutes. Different chemistry of byproducts predicted the turnover of microbial community. A decision tree framework was developed that can be used by producers to predict the nutrient availability and other microbial community-level functions of soil amendments after application.

2. New irradiation method to convert agricultural wastes to biostimulants. Biostimulants have grown to a multi-billion dollar global market; however, the feedstock is largely non-agricultural. Claimed plant hormone-like effects of biostimulant are attributable to the bioactive chemical components. ARS researchers in New Orleans, Louisiana, developed a low-cost and energy-efficient photochemistry method to add reactive chemical structures that would improve the effectiveness of biostimulants. Controlled wavelength and intensity of light irradiation reproducibly added biostimulant functions to agricultural wastes via electron transfer and additional chemistry pathways. The conversion method developed by this work opens an avenue for farmers to become the raw material supplier for biostimulant production.

3. Reviews of agricultural byproducts utilization. The production of sugar from sugar cane and sugar beet generates significant amounts of byproducts. Often, these are treated as waste, or some may be sold as low-cost animal feed. It is desirable to add value to these products so that they can be used to improve the economics of sugar production. ARS researchers in New Orleans, Louisiana, wrote and published three reviews on the possibility of producing a variety of products from sugar production byproducts. At least one of the reviews was the first of its kind. Another was featured on the front cover of a scientific journal. Providing information about the need for byproduct use and potential alternatives for their use will encourage scientist around the world to come up with innovative solution that will help the sugar-producing industry.

Review Publications
Velazquez-Martinez, V., Quintero-Quiroz, J., Rodriguez-Uribe, L., Valles-Rosales, D.V., Reyes-Jaquez, D., Klasson, T., Delgado, E. 2022. Effect of glandless cottonseed meal protein and maltodextrin as microencapsulating agents on spray-drying of sugar cane bagasse phenolic compounds. Journal of Food Science. 87(2):750-763. https://doi.org/10.1111/1750-3841.16032.
Terrell, E. 2022. Estimation of Hansen solubility parameters with regularized regression for biomass conversion products: An application of adaptable group contribution. Chemical Engineering Science. 248(B). Article 117184. https://doi.org/10.1016/j.ces.2021.117184.
Klasson, K.T., Sturm, M.P., Cole, M.R. 2022. Acid hydrolysis of sucrose in sweet sorghum syrup followed by succinic acid production using a genetically engineered Escherichia coli. Biocatalysis and Agricultural Biotechnology. 39:102231. https://doi.org/10.1016/j.bcab.2021.102231.
Klasson, K.T., Cole, M.R., Pancio, B.T., Heckemeyer, M. 2022. Development of an enzyme cocktail to bioconvert untapped starch in sweet sorghum processing by-products: Part II. Application and economic potential. Industrial Crops and Products. 176:114370. https://doi.org/10.1016/j.indcrop.2021.114370.
Tews, I.J., Terrell, E., Mood, S.H., Garcia-Perez, M. 2022. Wet oxidation of thermochemical aqueous effluent utilizing char catalysts in microreactors. Journal of Cleaner Production. 351. Article 131222. https://doi.org/10.1016/j.jclepro.2022.131222.
Wright, M.S., Lima, I.M. 2021. Identification of microbial populations in blends of worm castings or sugarcane filter mud compost with biochar. Agronomy. 11(8):1671. https://doi.org/10.3390/agronomy11081671.
Uchimiya, M. 2022. Aromaticity of secondary products as the marker for sweet sorghum [Sorghum bicolor (L.) Moench] genotype and environment effects. Journal of Agriculture and Food Research 9. Article 100338. https://doi.org/10.1016/j.jafr.2022.100338.