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

Location: Commodity Utilization Research

2023 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 Sub-objective 1.A, ARS researchers in New Orleans, Louisiana, conducted experiments to test for interactions between processing aides. Processing aides include enzymes to break down polysaccharides, and biocides to control microbial growth. Enzyme tests were conducted in sugarcane juice. Results show that oxidizing biocides may inhibit enzymes. Non-oxidizing biocides did not affect enzymes. These biocides may also be effective in lower amounts for control of certain microbes. In support of Sub-objective 1.B, ARS researchers in New Orleans, Louisiana, collected juice and biofilm samples from sugarcane and sugar beet processors. They isolated and identified microbes from these samples. Several of these microbes are being tested for susceptibility to conventional and novel biocides. For experiments to measure control of microbial growth in juices, the researchers used an alternative approach to get around some challenges with using seasonal freshly collected juice at harvest time. For example, juices collected from factories tend to vary in the amount and types of microbes. To overcome challenges and use a more reproducible approach, they isolated dextran-producing bacteria from various sugarcane factory juices and biofilms. The researchers tested these isolates for susceptibility to 5 ppm and 10 ppm of bleach. These low levels of oxidizer had no impact on growth of these isolates. They published these results in a manuscript. Additional testing of microbes for sensitivity to oxidizers and biocides is ongoing to determine concentrations that may be more effective at controlling microbial growth. In support of Sub-objectives 1.B and 1.C, ARS researchers in New Orleans, Louisiana, established an incoming agreement (number 72589) with the American Sugar Cane League. This agreement is titled “Molecular and Microbiological Characterization of Dextran/Fructan-Forming Organisms from Sugar Cane.” Additional funding was provided by the Beet Sugar Development Foundation for a related project. These agreements allow for detailed characterization of the microorganisms in sugar factory streams. Progress on other agreements (number 63451, 64764, 64768) with the American Sugar Cane League and related to Objective 1 included the hire of a chemical engineering student to help complete the experiments under the agreements. Supplies are being purchased and experiments ongoing. In support of Sub-objective 2.A, ARS researchers in New Orleans, Louisiana, developed fertilizer quality assessment indicators using chemistry and genetic approaches. They assessed plant hormone-like structures in organic fertilizer using fluorescence and near infrared spectrometry. Wavelength ratios for the chemically stable and unstable structures enabled the comparison of different fertilizer feedstocks. Then, the researchers used quinones (chemicals, often with a ring structure) and other electron-transfer catalysts to simulate the aging of characterized fertilizers. Based on those chemistry evaluations, oxidative and photochemical parameters were incorporated in the fertilizer quality estimation tools. To compare the biochemical performance of different fertilizers, the researchers used metagenomic sequencing to estimate potential functions. They compared several fresh and aged clarifier mud samples with composts to evaluate the commercial viability of mud-based fertilizers. Metagenome-assembled genome of composted clarifier mud revealed previously unknown types of bacteria potentially capable of diverse metabolic functions observed in complex soil/sediment environments. In support of Sub-objective 2.A, under agreement 6054-41000-114-003S with Cornell University, ARS researchers in New Orleans, Louisiana, extracted DNA and RNA from clarifier mud-amended root soils for metagenome and meta-transcriptome sequencing. Time course of soybean pod count was used to understand when clarifier mud enhances the plant growth. The efficacy of the mud was evaluated against different biochemical pathways, the origin of the mud, and various soil treatments. Under agreement 63890 with Danimer Scientific, the collaborator completed the Phase II expansion of its Kentucky facility to franchise resins for disposable food and beverage utensils. Product cycle models are being revised to add sugar feedstocks to the resin inventory currently relying on canola and soybean oils. Localized supply of sugar feedstocks in marginal lands is being investigated to compensate for the higher feedstock to product ratio of sugars than lipids. In support of Sub-objective 2.B, ARS researchers in New Orleans, Louisiana, characterized several biochars produced from sugar beet pulp and sugarcane bagasse at different pyrolysis temperatures. Analyses included surface area, ash content, fixed carbon, volatile matter, pH, particle size distribution, and elemental composition. Also, in support of Sub-objective 2.B, in collaboration with ARS scientists from Houma, the researchers carried out a study to determine the impact of application rates of bagasse-based biochar on physical, chemical, and biological properties of the soil. Five different soils used to grow sugarcane in Louisiana were tested and amendments included biochar alone, with starter fertilizer, and field residue. Adding biochar with mineral nitrogen decreased evolved CO2, indicating a negative priming effect. Overall, results indicate that lower levels of bagasse biochar minimally impacted soil properties and crop yield; however, the biochar was stable in the soil and represents a carbon-rich amendment. They submitted a manuscript with the research results. Also, in support of this objective, ARS researchers in New Orleans, Louisiana, began sample analysis on soil samples (before and after biochar application) from 16 acres of sugarcane producing land owned by a local raw sugar factory. Half the acreage received 2 ton/acre of biochar. The other half were controls without biochar. Sugarcane yield data will be collected in the fall 2023 upon harvesting. The researchers completed two other studies involving biochar application onto cotton and corn fields and submitted results for publication. Also, related to Objective 2.B, under research agreement number 68450, with West Virginia State University, ARS researchers in New Orleans, Louisiana, produced a series of biochars from woody and herbaceous bioenergy crops, specifically miscanthus, switchgrass and willow. Biochars are developed with the objective to maximize their efficacy as soil amendment, specifically to enhance marginal soils nutrient and water holding capacity and improve efficiency of runoff mitigation practices in retaining and removal of contaminants. In support of Sub-objective 3.A, ARS researchers in New Orleans, Louisiana, mixed clarifier mud/cake with sugarcane molasses and fermented it into acetone, butanol, and ethanol. The result indicated that, 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. This was demonstrated on two different sources of molasses. Typical levels of acetone, butanol, and ethanol was obtained without mud, when compared to control experiments with pure sugar as raw material. In support of Sub-objective 3.B, ARS researchers in New Orleans, Louisiana, conducted large scale fermentations (2 L) to produce acetoin from Bacillus bacteria. We carried out small scale extractions (10-15 mL) to separate acetoin from the fermentation broth. These extractions used mixtures of acetone and butanol. Larger extractions (500 mL) are planned with optimized solvent conditions. In support of Sub-objective 3.C, ARS researchers in New Orleans, Louisiana, optimized a process to extract aconitic acid with mixtures of acetone, butanol, and ethanol. These mixtures were anticipated products from research carried out in Sub-objective 3.A. After extraction, the researchers recovered the aconitic acid as a concentrated solution by a second extraction (called back-extraction) with sodium carbonate. They performed an economic analysis for the proposed industrial process, and they tested the extract as a nematicide with mixed results. The results indicated that, contrary to literature, the aconitic acid appeared to have little impact on the tested nematodes. While the extract was effective in reducing mobility and increasing mortality of nematodes, the results could not conclusively be linked to aconitic acid. The results were included in a published manuscript. Also, related to Objective 3, ARS researchers in New Orleans, Louisiana, made progress under agreement number 71883 with Cotton Incorporated. The researchers investigated the possibility of using sugar solutions obtained from solubilized discarded textiles to produce a food-quality liquor. The results were shared with Cotton Incorporated in quarterly reports.

Accomplishments
1. Individual microbes isolated and identified from sugarcane factories. ARS researchers in New Orleans, Louisiana, performed genome sequencing of microbes from sugarcane factory juices and biofilms. DNA from 17 of these microorganisms was extracted and sent to a service lab to sequence the genomes. These genomes were used to determine the identity of the microbes down to the species level. Analysis was also performed to locate genes involved in formation of problematic polysaccharides during sugarcane processing in factories that may also play a role in biofilms that form on equipment and factory surfaces. Understanding how biofilms are formed through the production of polysaccharides is an important step in overcoming challenges during sugar crop processing caused by polysaccharides such as dextrans.

2. Understanding the fauna of microbes in sugar factories. The “microbiome” present in sugarcane factory juices and biofilms were identified by ARS researchers in New Orleans, Louisiana. Microbes in sugarcane factory juice and biofilm degrade sucrose and make problematic sugar polymers. Together these cause economic loss. Our work used a more advanced technique that had not been previously applied to sugarcane processing. This novel application provided a more comprehensive description of microbes. This work fills a knowledge gap about the overall composition of microbes present in sugarcane factories. Previous knowledge was limited to a small sample number of studies with a smaller number of microbes identified. The results showed a wider possibility of microbes that are likely involved in sucrose losses during sugarcane processing in the factory than previously thought. This accomplishment will have impact on future research aimed at reducing sucrose losses and improving profitability. For each 0.1% increase in sugar yield, an additional $1,000/day of additional revenue is realized by a factory.

3. Survival of microorganisms throughout the product cycle is key to the success of bio-fertilizer for food crops. ARS scientists at New Orleans, Louisiana, in collaboration with Cornell University, developed a bio-stimulation method to directly increase the edible part (pods) yields of soybeans. This new technique uses high-temperature pasteurization of organic byproducts (clarifier mud) during raw sugar production. Clarifier mud fosters plant growth-promoting microbes, while potential pathogens are sterilized. The new consortium-based yield enhancement technique replaces older inoculation methods and industrial composts. This advancement will help domestic facilities manufacture the renewable fertilizer products cost-effectively.

4. Removal of color compounds from sugar refinery streams using bagasse biochars. Refineries often struggle with color removal and can either use powdered activated carbons or ion exchange resins. Both products are expensive and either fossil fuel derived or generate waste streams. Increased interest exists in sustainable products, particularly those that can be made from sugar crops by-products. ARS researchers in New Orleans, Louisiana, made several biochars from fresh and aged bagasse. These were further processed to impart the necessary functionality for removal of color compounds. The researchers characterized all the biochars and tested them in the laboratory for their ability to remove color (either native to the sugarcane or processing color due heat). Researchers identified the best performing color removing materials as the acid activated biochars produced from aged bagasse as well as the physical activated biochars also from aged bagasse. Their performance was slightly lower to that one of a commercial activated carbon, however due to their lower cost and sustainability, it can be potentially competitive.

5. Extraction and recovery of aconitic acid from sweet sorghum syrup. Aconitic acid naturally occurs in sweet sorghum and sugar cane. If recovered, it can serve as raw material for many chemical and biochemical products. However, the approach has not been used for over 70 years due to alternative fossil fuel petroleum-based technologies. With increased interest in non-petroleum-based approaches, ARS researchers at New Orleans, Louisiana, developed a process by which aconitic acid was extracted from concentrated sweet sorghum syrups with bio-chemicals. Once extracted, the researchers recovered the aconitic acid using a water solution with carbonate. While demonstrated for sweet sorghum, the technique should also work for sugar cane syrups and is a clear alternative to other proposed methods of extraction that use petroleum-based chemicals. The accomplishment demonstrates to the sugar cane industry the technical feasibility of recovering aconitic acid from by-products (molasses) that are often of low value to the industry. The cost of production was estimated to be about $16 per kilogram.

Review Publications
Uchimiya, S.M. 2022. Diversification in the nexus of sugar production, environmental stewardships, and sustainable agriculture. International Sugar Journal. 124(1486):608-613.
Pires, A.P., Olarte, M., Terrell, E.C., Garcia-Perez, M., Han, Y. 2023. Co-hydrotreatment of yellow greases and the water insoluble fraction of pyrolysis oil part I: Experimental design to increase kerosene yield and reduce coke formation. Energy and Fuels. 37(3):2100-2114. https://doi.org/10.1021/acs.energyfuels.2c03250.
Uchimiya, S.M., Vilanova, B., Derito, C., Hay, A.G. 2023. Bioinformatics for sugar industry: metabolic potentials of microorganisms in sugarcane mill mud. International Sugar Journal. 125 (1490):104-111.
Klasson, K.T., Qi, Y., Bruni, G.O., Watson, T.T., Pancio, B.T., Terrell, E. 2023. Recovery of aconitic acid from sweet sorghum plant extract using a solvent mixture, and its potential use as a nematicide. Life. 13(3). Article 724. https://doi.org/10.3390/life13030724
Denson, M.D., Terrell, E., Kostetskyy, P., Olarte, M., Broadbelt, L., Garcia-Perez, M. 2023. Elucidation of structure and physical properties of pyrolytic sugar oligomers derived from cellulose depolymerization/dehydration reactions: A density functional theory study. Energy and Fuels. 37(11):7834-7847. https://doi.org/10.1021/acs.energyfuels.3c00641.
Pires, A.P.P., Garcia-Perez, M., Olarte, M.O., Kew, W., Schmidt, A., Zemaitis, K., Denson, M., Terrell, E., McDonald, A., Han, Y. 2023. Comparison of the chemical composition of liquids from the pyrolysis and hydrothermal liquefaction of lignocellulosic materials. Energy and Fuels. 37(10):7221-7236. https://doi.org/10.1021/acs.energyfuels.2c03239.
Bruni, G.O., Qi, Y., Klasson, K.T., Lima, I.M., Terrell, E. 2022. Isolation and analysis of microbial contaminants from Louisiana raw sugarcane factories. International Sugar Journal. 124(1485):530-538.
Uchimiya, M., Hay, A.G., LeBlanc, J. 2022. Chemical and microbial characterization of sugarcane mill mud for soil applications. PLoS ONE. 17(8). Article e027013. https://doi.org/10.1371/journal.pone.0272013.
Jian, X., Uchimiya, M., Orlov, A. 2019. Particle size- and crystallinity-controlled phosphorus release from biochars. Energy and Fuels. 33(6):5343-5351. https://doi.org/10.1021/acs.energyfuels.9b00680.
Wang, Y.-M., Tang, D.-D., Yuan, X.-Y., Uchimiya, M., Li, J.-Z., Li, Z.-Y., Luo, Z.-C., Xu, Z.-W., Sun, S.-G. 2020. Effect of amendments on soil Cd sorption and trophic transfer of Cd and mineral nutrition along the food chain. Ecotoxicology and Environmental Safety. 189. Article 110045. https://doi.org/10.1016/j.ecoenv.2019.110045.
Wang, Y.-M., Liu, Q., Li, M., Yuan, X.-Y., Uchimiya, M., Wang, S.-W., Zhang, Z.-Y., Ji, T., Wang, Y., Zhao, Y.-Y. 2020. Rhizospheric pore-water content predicts the biochar-attenuated accumulation, translocation, and toxicity of cadmium to lettuce. Ecotoxicology and Environmental Safety. 208. Article 111675. https://doi.org/10.1016/j.ecoenv.2020.111675.
Li, M., Long, T., Tian, K., Wei, C., Liu, M., Wu, M., Li, Z., Uchimiya, M. 2022. Temperature and moisture mediated changes in chemical and microbial properties of biochars in an Anthrosol. Science of The Total Environment. 845. Article 157219. https://doi.org/10.1016/j.scitotenv.2022.157219.