Research Project: Technologies for Improving Industrial Biorefineries that Produce Marketable Biobased Products

Location: Bioproducts Research

2020 Annual Report

Objectives
This project provides technological solutions to the biofuels industry to help the U.S. meet its Congressionally mandated goal of doubling advanced biofuels production within the next decade. The overall goal is to develop optimal strategies for converting agricultural biomass to biofuels and to create value-added products (bioproducts) that improve the economics of biorefining processes. Specific emphasis is to develop strategies for biorefineries located in the Western United States by using regionally-specific feedstocks and crops, including sorghum, almond byproducts, citrus juicing wastes, pomace, municipal solid wastes (MSW), and food processing wastes. These feedstocks will be converted into biofuels, bioenergy and fine chemicals. Objective 1: Develop commercially-viable technologies for converting agriculturallyderived biomass, crop residues, biogas, and underutilized waste streams into marketable chemicals. Research on converting biogas will involve significant collaboration with one or more industrial partners. Sub-objective 1A: Provide data and process models for integrated biorefineries that utilize sorghum and available solid waste to produce ethanol, biogas and commercially-viable coproducts. Sub-objective 1B. Convert biogas from biorefining processes into polyhydroxyalkanoate plastics. Sub-objective 1C: Apply the latest tools in immobilized enzymes, nano-assemblies, to convert biomass to fermentable sugars, formaldehyde, and other fine chemicals. Objective 2: Develop commercially-viable fractionation, separation, de-construction, recovery and conversion technologies that enable the production of marketable products and co-products from the byproducts of large-scale food production and processing. Sub-objective 2A: Add value to almond byproducts. Sub-objective 2B: Apply bioenegineering of bacteria and yeast to produce diacids, ascorbic acid and other value-added products from pectin-rich citrus peel waste. Sub-objective 2C: Convert biomass into commercially-viable designer oligosaccharides using combinatorial enzyme technology.

Approach
Objective 1, referred to by some as Gen 1.5 Biorefineries, involves development of processes that will generate advanced biofuels using the “cheapest source of carbons” within a given region. Sub-objective 1A provides data about the properties of grain, forage, and sweet sorghum grown in California.Compositional analysis of cellulose, lignin and hemicellulose for grain, forage, and sweet sorghum varieties grown in California provides growers information to decide whether sorghum will become a viable biofuels feedstock in integrated biorefineries that also include anaerobic digestion. Sub-objective 1B is goal-driven research toward improving methanotrophic bacteria for commercial production of commodity and fine chemicals. High throughput mutagenesis is employed to enrich production of polyhydroxyalkanoate, PHA, from mixed populations. Sub-objective 1C tests the hypothesis that bioconversion of biomass substrates into value-added products will be achieved more efficiently with enzymes anchored to nano-assemblies, compared with using the same enzymes free in solution. The basic nano-assembly building block, termed the Rosettasome, will spontaneously assemble into an 18-subunit, double-ring structure that holds up to 18 different enzymes. Proposed research involves developing optimized Rosettazymes for hydrolyzing various biomass substrates into value-added bioproducts using multiple tethered enzymes. Objective 2 will provide data and technology that will add value to food processing byproducts. Sub-objective 2A consists of a goal-driven series of engineering developments to recover value-added free sugars, hemicellulose, and gums from almond byproducts. Release and utilization of free sugar and sugar alcohol can be improved by optimizing extraction parameters (time, temperature, particle size of the hulls,etc.) during hot water isolation. This process releases fermentable sugars, hemicellulose molasses and gums from almond shells and hulls. Equations and their corresponding parameters will be developed into process models for recovery of water soluble sugars in almond hulls. The goal is to add increased value to all components of the almond processing industry. Research in sub-objective 2B is driven by the hypothesis that whole cells can be engineered to convert pectin and other specific oligosaccharides into value-added products more efficiently than using multi-step chemical or enzymatic reactions.This will be achieved by applying bioenegineering of bacteria and yeast to produce diacids, ascorbic acid, and other value-added products from pectin-rich citrus peel waste. The general hypothesis driving sub-objective 2C is that bioconversion research is that specific well-defined enzymes can be applied to "surgically" remove selective branching groups from individual polysaccharide substrates via controlled enzymatic debranching and cleavage of main chain polymers.

Progress Report
This is the final report for bridge project 2030-41000-065-00D, "Technologies for Improving Industrial Biorefineries that Produce Marketable Biobased Products," which replaced project 2030-41000-054-00D which expired on 9/30/2019. This project is being replaced by new project 2030-41000-068-00D, “Zero Waste Agricultural Processing”, which was certified on August 24, 2020. Accomplishments of Objective 1 from the expired project 2030-41000-054-00D involved developing commercially-viable technologies for converting agriculturally derived biomass, crop residues, biogas, and underutilized waste streams into marketable chemicals. In continuation of research in support of Objective 1, ARS scientists developed a new strategy to produce a copolymer containing 4-hydroxybutyrate monomers in genetically-engineered Gram-positive bacteria. This work represents a significant advance in this field for several reasons. Most commercial biopolymer production utilizes Gram-negative bacteria which have endotoxic lipopolysaccharides that must be removed with additional processing before the polymer can be utilized in applications involving human contact. Furthermore, the newly engineered bacteria were designed to utilize low-cost, simple sugar substrates instead of standard expensive precursors, such as butanediol. Finally, the genes in the new bacterial strains were designed to be inducible thus allowing the scientists to adjust the levels of 4-hydroxybutyrate monomers to produce customized copolymers with the desired physical properties. Research in support of Objective 2 from the expired project 2030-41000-054-00D involved developing commercially-viable fractionation, separation, de-construction, recovery and conversion technologies that enable the production of marketable products and co-products from the byproducts of large-scale food production and processing. In continuation of research in support of Objective 2, ARS scientists used enzymes to develop combinatorial libraries of oligosaccharides from agricultural waste fibers. These libraries can be screened to discover variants that exhibit a desired target property, such as antimicrobials. ARS scientists discovered and characterized a unique enzyme (alpha-L-arabinofuranosidase) which has improved activity against disubstituted xylosyl residues found in arabinoxylan polysaccharides, a common food-processing waste stream. Utilization of this enzyme will result in more varied oligosaccharide libraries thus increasing the probability of discovering active variants. In further support of Objective 2, ARS scientists conducted research to identify optimal torrefaction conditions of almond shells which represent 1.5 billion pounds of yearly waste from the almond industry. ARS scientists conducted six field trials with various companies to incorporate torrefied biomass into plastic and rubber composites. Using this material, they successfully produced shipping pallets from recycled plastic and rubber pads for almond farm tree shakers.

Accomplishments
1. Research creates new applications for almond shell waste. California is the world’s largest producer of almonds and the market continues to increase. California’s industry generates 1.5 billion pounds of almond shell waste. ARS researchers at Albany, California, have demonstrated that almond shell waste can be processed into a filler to strengthen plastic and rubber composites. The processed almond shells can displace the traditional, non-renewable fillers that are currently used to modify plastics and rubber. ARS researchers have collaborated with commercial partners to create prototypes of commercial products.

Review Publications
Wong, D., Chan, V.J., Liao, H. 2019. Metagenomic discovery of feruloyl esterases from rumen microflora. Applied Microbiology and Biotechnology. 103:8449-8457. https://doi.org/10.1007/s00253-019-10102-y.
Castro, J., Nobre, J.C., Napoli, A., Trigilho, P., Tonoli, G.D., Wood, D.F., Bianchi, M. 2020. Pretreatment affects activated carbon from piassava. Polymers. 12(7):1483. https://doi.org/10.3390/polym12071483.
Bilbao-Sainz, C., Sinrod, A., Powell-Palm, M., Dao, L.T., Takeoka, G.R., Williams, T.G., Wood, D.F., Ukpai, G., Aruda, J., Bridges, D.F., Wu, V.C., Rubinsky, B., McHugh, T.H. 2018. Preservation of sweet cherry by isochoric (constant volume) freezing. Innovative Food Science and Emerging Technologies. 52:108-115. https://doi.org/10.1016/j.ifset.2018.10.016.
Quispe-Fuentes, I., Vega-Galvez, A., Aranda, M., Poblete, J., Pasten, A., Bilbao-Sainz, C., Wood, D.F., McHugh, T.H., Delporte, C. 2020. Effects of drying processes on composition, microstructure and health aspects from maqui berries. Journal of Food Science and Technology. 57:2241-2250. https://doi.org/10.1007/s13197-020-04260-5.
Castro, J.P., Nobre, J.C., Napoli, A., Bianchi, M., Moulin, J.C., Chiou, B., Williams, T.G., Wood, D.F., Avena Bustillos, R.D., Orts, W.J., Tonoli, G.D. 2019. Massaranduba sawdust: a potential source of charcoal and activated carbon. Polymers. 11(8). Article 1276. https://doi.org/10.3390/polym11081276.
Wagschal, K.C., Jordan, D.B., Hart-Cooper, W.M., Chan, V.J. 2019. Penicillium camemberti galacturonate reductase: C-1 xidation/reduction of uronic acids and substrate inhibition mitigation by aldonic acids. International Journal of Biological Macromolecules. 153:1090-1098. https://doi.org/10.1016/j.ijbiomac.2019.10.239.
Castro, J.P., Nobre, J.C., Bianchi, M., Trugilho, P., Napoli, A., Chiou, B., Williams, T.G., Wood, D.F., Avena Bustillos, R.D., Orts, W.J., Tonoli, G.D. 2018. Activated carbons prepared by physical activation from different pretreatments of amazon piassava fibers. Journal of Natural Fibers. 16(7):961-976. https://doi.org/10.1080/15440478.2018.1442280.
Chiou, B., Cao, T.K., Bilbao-Sainz, C., Vega-Galvez, A., Glenn, G.M., Orts, W.J. 2020. Properties of gluten foams containing different additives. Industrial Crops and Products. 152. Article 112511. https://doi.org/10.1016/j.indcrop.2020.112511.
Flynn, A., Torres, L.F., Hart-Cooper, W.M., McCaffrey, Z., Glenn, G.M., Wood, D.F., Orts, W.J. 2020. Evaluation of biodegradation of polylactic acid mineral composites in composting conditions. Journal of Applied Polymer Science. 137. Article 48939. https://doi.org/10.1002/app.48939.
McCaffrey, Z., Thy, P., Long, M., Oliveira, M., Wang, L., Torres, L.F., Aktas, T., Chiou, B., Orts, W.J., Jenkins, B. 2019. Air and steam gasification of almond residues. Fuel Processing Technology. 7(21):84. https://doi.org/10.3389/fenrg.2019.00084.
Wagschal, K.C., Chan, V.J., Pereira, J.H., Zwart, P.H., Sankaran, B. 2020. Chromohalobacter salixigens Uronate Dehydrogenase: directed evolution for improved thermal stability and mutant. Process Biochemistry. https://doi.org/10.1016/j.procbio.2020.02.013.
Placido, D.F., Dierig, D.A., Cruz, Von, M.V., Ponciano, G.P., Dong, C., Dong, N., Huynh, T.T., Williams, T.G., Cahoon, R.E., Wall, G.W., Wood, D.F., Mcmahan, C.M. 2020. Downregulation of an allene oxide synthase gene improves photosynthetic rate and alters phytohormone homeostasis in field-grown guayule. Industrial Crops and Products. 153:112341. https://doi.org/10.1016/j.indcrop.2020.112341.
Bilbao-Sainz, C., Sinrod, A., Dao, L.T., Takeoka, G.R., Williams, T.G., Wood, D.F., Bridges, D.F., Powell-Palm, M., Ukpai, G., Chiou, B., Wu, V.C., Rubinsky, B., McHugh, T.H. 2019. Preservation of spinach by isochoric (constant volume) freezing. International Journal of Food Science and Technology. 55(5):2141–2151. https://doi.org/10.1111/ijfs.14463.