Location: Bioproducts Research
2019 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 agriculturally-derived 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 project 2030-41000-054-00D, which is currently undergoing NP 306 review.
In partnership with major stakeholders to optimize the value of agriculturally-derived coproduct streams, research in Objective 1 was directed toward developing commercially-viable technologies for converting agriculturally-derived biomass, crop residues, biogas, and underutilized waste streams into marketable chemicals. Research on converting all products, including biogas conversion, involved significant collaboration with (and support from) multiple partners.
Under Sub-objective 1A, ARS researchers successfully provided process models for integrated biorefineries that utilized available solid waste from California agriculture. Target outputs included ethanol, biogas and commercially-viable coproducts. This collaborative team built a pilot-plant biorefinery at the Salinas Crazy Horse Landfill that converts municipal solid waste and/or food waste into bioenergy and other coproducts, including recycled paper and compostable ground cover. This research site continues as a working model of a landfill-located biorefinery. Touted as an “energy park” that handles both rural and urban solid waste to produce ethanol, biogas, compost, and/or value-added recyclables, the Salinas Valley Solid Waste Authority has committed to scale the system up to 300 tons a day at the Johnson Canyon Landfill. The project has been featured in the popular press, on TV and in newspapers. ARS researchers hosted visits or made numerous public presentations on this. The city of San Francisco (SF), represented by the waste handler, Recology, has also developed preliminary plans to install the team’s autoclave systems for biomass pretreatment based, in part, on our data and recommendations.
Sub-objective 1B involved converting biogas and digestor-derived acids into the sustainable, commercial plastics, specifically polyhydroxyalkanoate plastics. In this research USDA researchers worked under two cooperative research and development agreement (CRADA) partnerships with commercial partners for methane conversion and for acid conversion. The commercial partner used novel methanotrophic bacteria, co-developed with ARS researchers, to make the biopolymer poly-3-hydroxybutyrate (PHB) from methane, a gas that can be produced renewably from anaerobic digestion of agricultural waste. Since the crystallinity of PHB makes it harder for this material to compete with traditional plastics in many commercial applications, the ARS team worked with the commerical partner to improve the quality of their PHB. The ARS researchers developed a bio-based plasticizer that could be blended with PHB to improve the polymer’s properties, such as decreasing viscosity, stiffness, and melting temperature, while also increasing its toughness. A strategy was developed for co-feeding alternate carbon substrates to methanotrophs to produce different copolymers that had improved performance characteristics compared to those of the PHB homopolymer. ARS researchers also improved production of PHB from organic acids with a commercial partner in their on-site scale-up efforts from lab-scale to pilot-scale. Both partners are building demonstration plants in the SF Bay Area to produce industrial quantities of bioplastics, all based off or our joint research.
In support of Sub-objective 1C, ARS researchers compared two ß-xylosidase enzymes from differing sources that are very similar to each other and are both critical in breaking down straw residues to sugars for biofuel production. Both shared very similar amino acid structure. One enzyme (RS223-BX) was from a rice straw metagenomic library, and the other, (BoXA) was derived from the bacteria, Bacteroides ovatus. They shared similar amino acid sequences with 19 of 20 identical active-site residues. These two were compared by using site-directed mutagenesis of aspartic acid (Asp) and histidine (His) residues implicated in metal binding for their enzymatic activity. The logic was that the RS223-BX is strongly activated by divalent-metal cations and the previously published X-ray structure of this enzyme shows that a Ca2+ cation is chelated by an active-site. The hypothesis that mutation at that site change the enzyme activity for RS223 proved correct. Mutation (from His to Ala at the active metal-binding site) causes 20% loss of activity for the His mutant and 40% gain of activity for the Asp mutant, indicating the lack of importance for activity of the native residues. Yet, for the other enzyme (BoXA) there was a lack of metal-dependency. The results strengthen our conclusion that these two very similar proteins differ in one being metal ion dependent and one not based on the activity of a metal-binding site.
Work in Objective 2 was based around the fact that processing losses for nuts, especially almonds, can exceed 40%. Under Sub-objective 2A, research was directed toward the almond industry, spearheaded by the Almond Board of California, which has committed to zero waste by 2025. ARS researchers worked with the industry to create viable end-uses for their coproducts, everything from orchard to table. Sugars were extracted from almond hulls showing that they contain up to 40% “free sugar”. It was then shown that hulls have a higher sugar content than sugar beets; they have a broader harvesting window than sugar beets; they can be stored longer than sugar beets; and almond hulls can be processed in the same equipment as sugar beets using essentially identical protocols. Research continues toward scale-up to show that hulls and sugar beets could potentially complement each other very well in a multi-sugar fermentation process. Although human consumption of almond hulls is limited by their high tannin content, making them too bitter, ARS-based research showed that these tannin levels can be reduced effectively by simple food-grade processing technologies.
In support of Sub-objective 2B, research continued on converting food waste to Vitamin C (ascorbate). Plants make ascorbate in a series of enzyme-mediated steps that starts with a pectin derivative that is present in large amounts in citrus and sugar beet byproducts. ARS researchers identified an enzyme that catalyzes the first step of the pathway that converts pectin into an important keto-sugar intermediate, which is then processed by additional enzymes to ascorbate. This first enzyme was characterized for both development of in vitro industrial processing and for cloning into a modified yeast for fermentative production of ascorbate from food waste. It was shown that the enzyme exhibits the most significant specificity when the starting material is pure pectin, derived either from citrus and beet waste. The team then measured the amount of inhibition of the enzyme under model process conditions and determined that the enzyme is an excellent candidate for engineering thermal stability for commercial efforts. Our enzyme team continues to be leading pioneering research on multi-functional enzyme arrays for biomass de-construction and conversion, resulting in applications of Rosettasomes. This includes continued collaborations with partners to create bi- and tri-functional enzymes that exhibit synergies and developing cloning techniques with improved screening to apply multi-functional enzymes via combinatorial enzyme reactions to create bioproducts.
Food waste from citrus and sugar beet processing, which contains copious amounts of a pectin derivative, was converted enzymatically to sugar diacids, specifically glucaric acid and other aldaric acid derivatives. ARS researchers converted pectin-rich feedstocks into aldaric acids. These acids are of interest to commercial partners, and they were listed among the Department of Energy’s (DOE) Top 10 chemical feedstocks from renewable resources. This is because they can be used as metal chelators in, for example, laundry detergents, or as polymer building blocks. ARS researchers used molecular breeding to significantly improve the thermal stability of this enzyme by 18 degrees Celsius (C), allowing more cost-effective and efficient industrial processes at higher temperatures. Development of this highly thermal stabile enzyme facilitates additional engineering for other desirable properties that are process-specific, for example, unit operation pH optima. This technology has now been transferred to DOE collaborators for enzyme structure work and one specific aldaric acid was tested by a commercial producer of soaps and detergents to test its efficacy.
In support of Sub-objective 2C, fine chemicals were created from agricultural coproducts. The ARS researchers, with a commercial partner, created “Reversible” Antibiotics. Specifically, along with the commercial partner, the ARS team invented a class of broad-spectrum, fast acting antimicrobials that are “reversible”, which allows them to revert into “benign” (non-antibiotic) chemicals after use. This reduces their persistence in the environment. The pervasive use of antibiotics has triggered major human and environmental issues including antibiotic resistance of microbes. The ARS team created natural antimicrobial agents that “fall apart after use”. When active, they are as effective as the commercial standard used in shampoos, for example, isothiazolinone, but because they are reversible, they overcome the toxic effects of isothiazolinone, which has triggered sensitization epidemics in Europe and North America. This should reduce the risk of long-term danger of subinhibitory exposure, which leads to antibiotic-resistant bacteria. This technology has won multiple awards, including the GC3 Challenge, an Industry-wide award for safer antibiotics. The research was also featured in C&E News.
Accomplishments
1. Developed a novel application for spent almond hulls. Almond hulls can be a viable source of industrial sugars, considering that they contain more “free”, extractable sugar than sugar beets; however viable end-uses must be found for the biomass remaining after sugar extraction, the so-called “spent hulls”. ARS researchers in Albany, California, have recently developed a novel application for spent hulls, which is to use them as ground cover for commercial production of mushrooms. Propagation of vegetative mycelium from mushrooms generally requires application of a specific peat moss mix (called casing) with physical and chemical properties including high water-holding capacity, even pore distribution for gas exchange, and balanced minerals. It has been shown that spent almond hulls possess these important traits, with a water-holding capacity of greater than 500 percent, numerous pores in the size range optimal for gas exchange, and high mineral content that is suitable for mushrooms. ARS researchers and their industrial collaborators are now exploring the use of spent almond hulls as a ground cover (casing) in commercial mushroom production at an industrial scale.
Review Publications
McCaffrey, Z., Torres, L.F., Flynn, S.M., Cao, T.K., Chiou, B., Klamczynski, A.P., Glenn, G.M., Orts, W.J. 2019. Recycled polyproplene-polyethylene torrefied almond shell biocomposites. Industrial Crops and Products. 125:425-432. https://doi.org/10.1016/j.indcrop.2018.09.012.
Arantes, A., Silva, L., Wood, D.F., Almeida, C., Tonoli, G., Oliveira, J., Silva, J., Williams, T.G., Orts, W.J., Bianchia, M. 2018. Bio-based thin films of cellulose nanofibrils and magnetite for application in green electronics. Carbohydrate Polymers. 207(1):100-107. https://doi.org/10.1016/j.carbpol.2018.11.081.
Lee, C.C., Jordan, D.B., Stoller, R.J., Kibblewhite, R.E., Wagschal, K.C. 2018. Biochemical characterization of caulobacter crescentus xylose dehydrogenase. International Journal of Biological Macromolecules. 118:1362-1367. https://doi.org/10.1016/j.ijbiomac.2018.06.124.
Minelli, M., Hart-Cooper, W.M., Sinnwell, J.G., Blumberg, D.T., Guzei, I.A., Spencer, L.C., Saucedo-Vasquez, J., Solano-Peralta, A., Sosa-Torres, M. 2018. Synthesis, structure, and characterization of molybdenum(VI) imido complexes with N-salicylidene-2-aminothiophenol. Polyhedron. 146:26-34. https://doi.org/10.1016/j.poly.2018.02.017.
Thomas, S.M., Franquivillanueva, D.M., Hart-Cooper, W.M., Waggoner, M., Glenn, G.M. 2019. Lactic acid production from almond hulls. Journal of Food & Industrial Microbiology. 5(1):128.
Wong, D., Feng, D., Batt, S.B., Orts, W.J. 2018. Combinatorial enzyme approach to produce Oligosaccharides of diverse structures for functional screen. Advances in Enzyme Research. 6(2):11-20. https://doi.org/10.4236/aer.2018.62002.
Cal, A.J., Grubbs, B., Torres, L.F., Riiff, T.J., Orts, W.J., Lee, C.C. 2019. Nucleation and plasticization with recycled low-molecular-weight poly-3-hydroxybutyrate toughens virgin poly-3-hydroxybutyrate. Journal of Applied Polymer Science. 136(17):47432. https://doi.org/10.1002/app.47432.
Shah, T.A., Tabassum, R., Orts, W.J., Lee, C.C. 2019. Isolation of ligninolytic Bacillus sp. strains for depolymerization of alkali lignin. Journal of Environmental Progress and Sustainable Energy. 38(3):e13036. https://doi.org/10.1002/ep.13036.
