Progress 10/01/14 to 09/30/19

Outputs
Progress Report Objectives (from AD-416): 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 (from AD-416): 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. 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 ÿ- 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 01 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.

Impacts
(N/A)

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.
• 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.
• 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.
• 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.
• Thomas, S.M., Franqui-Villanueva, 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.
• 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.
• 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.

Progress 10/01/17 to 09/30/18

Outputs
Progress Report Objectives (from AD-416): 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 (from AD-416): 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. Sub-objective 1A. Vitamin C (ascorbate) is made in plants in a series of reactions from galacturonic acid, which occurs in large quantities in citrus and beet processing waste. ARS scientists in Albany, California, identified a galacturonate oxidoreductase enzyme (PcOD) that catalyzes the first step of the reaction from galacturonate by producing keto- sugars which are then processed by additional enzymes to ascorbate. The kinetic and biophysical properties of PcOD were characterized for use in industrial processes or for in-vivo metabolic engineering of yeast for fermentative production of ascorbate from food waste. Its low thermal stability indicates PcOD is an excellent candidate for the thermal stability enzyme engineering. Sub-objective 1B. Poly-3-hydroxybutyrate (PHB) is a carbon storage molecule in prokaryotes and can serve as a bio-based plastic replacement; however, the handling and material properties of virgin PHB are not as robust as polypropylene, a traditional petroleum-based plastic. A second difficulty with PHB is that recycling the material can be difficult since the temperatures of melting and degradation are almost identical, which results in partial breakdown of the polymer. ARS scientists in Albany, California, developed a strategy that addresses both issues. Degraded PHB fragments derived from the recycling process were used as bio-based additives to virgin PHB. The incorporation of the degraded PHB resulted in plasticizing and toughening of virgin PHB, thus expanding the practical range of virgin PHB and a strategy for using recycled PHB. Sub-objective 1C: Efficient bioconversion of hemicellulose from agricultural residues and forest harvest waste to value-added chemicals is instrumental to the success of a biorefinery. ARS scientists in Albany, California, have successfully deployed an enzyme complex, known as the rosettazyme (a nano-assembly enzyme construct), to efficiently convert hemicellulose to xylonic acid (a desirable substrate). A key enzyme in the complex is xylose dehydrogenase from Caulobacter crescentus, a bacterium living in fresh-water lakes and streams. Although xylose dehydrogenase is commonly used by many researchers to successfully degrade hemicellulose, little is known about how xylose dehydrogenase functions. Detailed kinetic studies were conducted on the enzyme to clarify its functionality. This data will prove useful to improve the activity of this key enzyme. Sub-objective 2A: Polypropylene, polyethylene terephthalate, and polyethylene are commodity plastics commonly used in a wide array of applications. Widespread use of plastics has resulted in active recycling programs to re-use plastics. Unfortunately, most recycled plastics are degraded with a significant loss in mechanical properties. To improve or broaden the range of properties of recycled plastics, certain additives are melt blended into recycled plastics via extrusion. Additives can displace the cost of adding virgin polymer and include minerals, glass fibers, and clays to improve stiffness, impact strength, and heat stability. ARS scientists have successfully added torrefied almond shells into recycled plastics. Almond shells were subjected to a pretreatment known as torrefaction where the shells undergo thermal conversion, under limited oxygen, at 200-300 degrees Celsius. The thermal process improves the adhesion between the torrefied shells and recycled plastics, thereby reducing the use of petroleum-based industrial additives. Adding torrefied shells to recycled plastic provided additional advantageous mechanical properties beyond adding color to the resulting plastic composite. When 2 to 20 percent torrefied shells were added to recycled polypropylene, the heat deflection temperature and rigidity of the resulting polymer were improved. The improvements were significantly higher than those of the usual fillers combined with recycled plastic. Currently, scientists in Albany, California, are collaborating with two industrial partners who show an interest in incorporating the torrefied almond shells or other biomass into their current technologies. One of the partners specializes in the production of pallets and bins using recycled plastics. The main objective is to improve the mechanical properties of the pallets by adding torrefied biomass at a certain percentage. The other partner specializes in the production of protective packaging called slip sheets using recycled and virgin high-density polyethylene. The main objective of this partner is to displace the cost of adding a lamination to the slip sheet. The lamination prevents containers from sliding during transport. The goal is that the addition of the torrefied biomass will create the necessary �roughness� on the surface of the film, which translates to higher coefficient of friction. Sub-objective 2B: ARS scientists in Albany, California, have been using molecular engineering to improve enzyme thermal stability for conversion of food processing waste streams to cleaning product and fabric building blocks. Food waste from citrus and sugar beet processing contains copious amounts of a pectin that is enzymatically converted to sugar diacids. Sugar diacids are included in the Department of Energy�s Top 10 renewable resource chemical feedstocks, can be used as metal chelators in detergents or as polymer building blocks. Molecular breeding was used to significantly improve thermal stability of this enzyme, Better thermostability allows more cost-effective and efficient industrial processes to be developed. Development of the highly thermal stable enzyme facilitates additional engineering for other desirable properties that are process-specific at the optimal pH. This technology has now been transferred to Department of Energy collaborators at Lawrence Berkeley National Laboratories Advanced Light Source for enzyme structure work. Sub-objective 2C: Combinatorial chemistry has been a focus of intense activity in modern drug discovery. The central idea is to synthesize a vast population of diverse molecular structures and screen for the few variants that exhibit the desired target property. ARS scientists in Albany, California, have developed the concept of combinatorial chemistry applied to enzymes for the bioconversion of plant fibers. Combinatorial enzyme digestion of pectic materials produced libraries of oligosaccharides. Rapid fractionation and screening resulted in the isolation of an active species with antimicrobial activity. The active species may be useful as alternatives for antimicrobial growth promoters or a new source of high-value preservatives. Direct cloning of metagenomes has proven to be a powerful tool for the exploration of the diverse sequence space of a microbial community leading to many recent gene discovery and biocatalyst development. The key to the success of direct cloning is the development and use of rapid, sensitive, and reliable high-throughput screening. ARS scientists in Albany, California, developed a novel method for rapid and sensitive metagenomic activity screening. The approach represents a radical departure from conventional methods, and significantly increases the success rate in gene discovery. Accomplishments 01 A novel method for rapid and sensitive metagenomic activity screening. Cloning large regions of DNA has proven to be a powerful tool for exploring the diversity of a microbial community and can lead biorefinery development via high-throughput screening methods. ARS scientists in Albany, California, developed a novel method for rapid and sensitive screening of many clones. The approach is a radical departure from conventional methods. The successful gene discovery rate will increase significantly with use of this new process. 02 Molecular engineering to improve enzyme thermal stability. Waste streams from citrus and sugar beet processing contain copious amounts of pectin. ARS researchers in Albany, California, converted pectin, a renewable resource, to sugar diacids via pectinase. They used molecular breeding to significantly increase the thermal stability of pectinase to develop more cost-effective and efficient industrial processes. The thermally-stable pectinase will facilitate additional engineering for process-specific properties such as operation under acidic or alkaline conditions. 03 Adding torrefied almond shells to improve recycled plastics. Recycled plastics often exhibit a significant loss in mechanical properties. Additives displace the cost of adding virgin polymer and traditionally include minerals, glass fibers, and clays to improve stiffness, impact strength, and heat stability. ARS scientists in Albany, California, processed almond shells, a harvest residue, by heating at 200-300 degrees Celsius under limited oxygen. The thermal process (torrefaction) improved the adhesion between the torrefied shells and recycled plastics, thereby reducing the use of petroleum-based industrial additives. Adding torrefied shells to recycled plastic increased heat stability and added stiffness relative to traditional fillers.

Impacts
(N/A)

Publications

• Jordan, D.B., Stoller, J.R., Lee, C.C., Chan, V.J., Wagschal, K.C. 2016. Biochemical characterization of a GH43 �-xylosidase from Bacteroides ovatus. Applied Biochemistry and Biotechnology. 182:250-260. doi: 10.1007/ s12010-016-2324-0.
• Shang, M., Chan, V.J., Wong, D., Hans, L. 2018. A novel method for rapid and sensitive metagenomic activity screening. MethodsX.
• Fonseca, A.O., Raabe, J., Dias, L.S., Baliza, A.T., Costa, T., Silva, L., Vasconcelos, R.P., Marconcini, J., Savastano, H.J., Mendes, L., Yu, A., Orts, W.J., Tonoli, G. 2018. Main characteristics of underexploited Amazonian palm fibers for using as potential reinforcing materials. Waste and Biomass Valorization. 2018:1-18.
• Riff, T.J., Webb, M.A., Orts, W.J., Aramthanapon, K. 2017. Small-angle neutron scattering studies on an idealized diesel biofuel platform. Energy and Fuels. 31(4):3995-4002.
• Lee, C.C., Jordan, D.B., Stoller, J.R., Kibblewhite, R.E., Wagschal, K.C. 2018. Biochemical characterization of caulobacter crescentus xylose dehydrogenase. International Journal of Biological Macromolecules.

Progress 10/01/16 to 09/30/17

Outputs
Progress Report Objectives (from AD-416): 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 (from AD-416): 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. 3a. Pectin rich biomass is an underutilized waste stream from the sugar and juice industry that can be converted to value added products. Pectin from citrus peels, for example, is mainly a polymer termed homogalacturonan that consists of esterified galacturonic acid. With some simple enzymatic conversions, steps have been taken to convert galacturonic acid into ascorbic acid (Vitamin C), and steps are now being taken to make adipic acid, which can be used as a feedstock to make �green� nylon. One of the main enzymes responsible for the depolymerization of pectin is exo-polygalacturonase, which removes one galacturonic acid residue at a time from the chain. A report was published on the biophysical and kinetic characterization of a hyper- thermostable polygalacturonase (termed RmGH28) that exhibits the highest rates of depolymerize activity ever reported for breaking down pectin. A great advantage of this enzyme is that it is hyper-thermostable, thus able to withstand temperatures of 93.9 degrees Celsius for 1 hour and lose only half of the initial activity, indicating the enzyme would be stable for extended periods of time at elevated reactor temperatures. This would have significant industrial application because higher temperatures increase the rate of reaction, lower the viscosity to save mixing costs, and reduce the risk of contamination; all of which potentially increase the economic viability of the process. 3b. Plant cell wall polysaccharides, which consist of long-chain backbones with various types of sugar-based polymer substituents, can potentially provide a wide array of valuable chemicals, if only they can be de-polymerized cleanly and specifically. ARS researchers from Albany, California, have become pioneers in a type of specific depolymerization process they term �combinatorial enzyme conversion� in which an array of enzymes are used to transform polysaccharides in a manner similar to combinatorial chemistry. Specifically, an array of enzymes are tested in a combinatorial approach to target a specific property or product. Via this combinatorial approach, an active oligo-polysaccharide species has been isolated from citrus-pectin that is a potent antimicrobial agent. In microbial tests of antibiotic behavior, a short-chain derivative of pectin suppressed the growth of the test microorganism, Escherichia coli ATCC 8739, a known pathogen at levels similar to those used as preservatives. It was found that the inhibitory effect increased with the concentration, with a minimum inhibitory concentration (MIC) of 0.4 percent. The antimicrobial effect was sustained for more than three days. The oligosaccharides species had reactive double bonds and is optimal at a certain size. This research is part of a broad study on oligosaccharides to be used as natural antimicrobial agents, as probiotics, and in other nutraceutical applications. 3c. Polyhydroxyalkanoates (PHA) are biologically-produced polyesters that are of great commercial interest due to both their inherent biodegradability and sustainability, since they can be derived from agriculturally-derived biomass feedstocks. Efforts to commercialize PHA have achieved limited success because producers generally focused on utilizing gram negative bacteria for their production, which has proven expensive, requiring sugars as feedstocks. ARS researchers in Albany, California, have been focusing on PHA production from gram positive bacteria, specifically Bacillus megaterium, and several methanotrophic (methane-consuming) bacteria. While utilizing cheaper feedstocks and requiring less sterilization than gram positive microbes, these bacteria generally only produce the homopolymer, poly-3-hydroxybutyrate (P3HB), a polymer with a limited range of applications. In a recent breakthrough, however, we have been genetically modifying B. megaterium to produce increased amounts of other co-polymers within the PHA family. By characterizing and expanding the useful range of polymer properties, this team hopes to broaden the commercial viability of this interesting bacterially-produced family of polyesters. 3d. Steam autoclaving of solid wastes has long been used to sterilize medical wastes, but ARS researchers in Albany, California, have shown that it is also an efficient method for the separation and near complete recovery of organics from traditional �curbside� municipal solid wastes (MSW). In a pilot-scale study, we established that autoclaving can thus be the basis of a �biorefinery� whereby autoclaved solid wastes are converted into both ethanol and/or methane-rich biogas. Material produced by the autoclave contains a high concentration of solubilized food waste absorbed onto a lignocellulosic matrix which was converted into biogas in a 1,500 gallon or 5,677 liter (L) high solids anaerobic digester operated on-site at a Northern California landfill. Total solids (TS) reductions were high, 56 percent, and volatile solids (VS) and biodegradable volatile solids (BVS) reductions were 63 and 79 percent, respectively. Gas yields were also high, producing 248 L of methane (CH4) per kilogram (kg) VS fed or 393 L CH4/kg VS destroyed at a methane content of 60 percent. Unique design elements such as hydraulic conveyance of material, in situ classification, and in-place buffering to maintain pH stability were tested and confirmed. The digestate passed all criteria for land application of biosolids in the U.S. Accomplishments 01 A multi-enzyme scaffolding system to convert crop residues to green chemicals. Crop residues such as straw and bagasse (excess plant remaining after a product has been extracted) represent a potentially large feedstock to supply the world�s fuel and chemical needs; however, for biochemical conversion, multiple different enzymes need to work together to convert complex sugars into commercially viable products. ARS researchers in Albany, California, created a way for enzymes to work synergistically by mounting them on large, multi-enzyme complexes. An artificial enzyme scaffold, a Rosettazyme, that tethers up to eighteen different active enzymes onto a single platform was developed and these Rosettazymes were utilized to convert lignocellulosic material into value-added products. In one example, multiple enzymes were used to release sugars from the lignocellulosic component found in most crops. Several more tethered enzymes were employed to further convert the released sugars into their corresponding acids, called aldaric acids, which can be used as building blocks for nylon plastics. Four different types of enzymes were activated onto the same enzyme scaffold to highlight its synergy, demonstrating that tethering of multiple enzymes in a complex resulted in 71 percent more activity than using the same amount of enzymes free in solution.

Impacts
(N/A)

Publications

• Wagschal, K.C., Stoller, J.R., Chan, V.J., Lee, C.C., Grigorescu, A.A., Jordan, D.B. 2016. Expression and characterization of hyperthermostable exo-polygalacturonase TtGH28 from Thermotoga thermophilus. Molecular Biotechnology. 58(7):509-519. doi: 10.1007/s12033-016-9948-8.
• Wagschal, K.C., Stoller, J.R., Chan, V.J., Jordan, D.B. 2017. Expression and characterization of hyperthermostable exo-polygalacturonase RmGH28 from Rhodothermus marinus. Applied Biochemistry and Biotechnology. doi: 10. 1007/s12010-017-2518-0.
• Lee, C.C., Kibblewhite, R.E., Paavola, C., Orts, W.J., Wagschal, K.C. 2017. Production of D-xylonic acid from hemicellulose using artificial enzyme complexes. Journal of Microbiology and Biotechnology. 27(1):77�83 doi: 10. 4014/jmb.1606.06041.
• Orts, W.J., McMahan, C.M. 2016. Biorefinery developments for advanced biofuels from a widening array of biomass feedstocks. BioEnergy Research. 9(2):430-446. doi: 10.1007/s12155-016-9732-4.
• Wong, D., Rafique, N., Tabassum, R., Awan, S., Orts, W.J. 2016. Cloning and expression of Pectobacterium carotovorum endo-polygalacturonase gene in Pichia pastoris for production of oligogalacturonates. BioResources. 11(2):5204-5214.
• Kim, J.H., Hart-Cooper, W.M., Chan, K.L., Cheng, L.W., Orts, W.J., Johnson, K. 2016. Antifungal efficacy of octylgallate and 4-isopropyl-3- methylphenol for control of Aspergillus. Microbiology Discovery. 4:2.
• Bilbao-Sainz, C., Chiou, B., Valenzuela-Medina, D., Imam, S.H., Vega- Galvez, A., Orts, W.J. 2016. Biopolymer films to control fusarium dry rot and their application to preserve potato tubers. Journal of Applied Polymer Science. doi:10.1002/app.44017.
• Arantes, A.C., Almeida, C.G., Dauzacker, L.C., Bianchi, M., Wood, D.F., Williams, T.G., Orts, W.J., Tonoli, G.H. 2017. Renewable hybrid nanocatalyst from magnetite and cellulose fortreatment of textile effluents. Carbohydrate Polymers. 163:101-107. doi: 10.1016/j.carbpol.2017. 01.007.
• Buckley, H.L., Hart-Cooper, W.M., Kim, J.H., Faulkner, D.N., Cheng, L.W., Chan, K.L., Vulpe, C.D., Orts, W.J., Amrose, S.E., Mulvihill, M.J. 2017. Design and testing of safer, more effective preservatives for consumer products. ACS Sustainable Chemistry & Engineering. 5(5):4320-4331. doi: 10. 1021/acssuschemeng.7b00374.
• Chiou, B., Valenzuela-Medina, D., Bilbao-Sainz, C., Klamczynski, A., Avena- Bustillos, R.D., Milczarek, R.R., Du, W., Glenn, G.M., Orts, W.J. 2016. Torrefaction of almond shells: Effects of torrefaction conditions on properties of solid and condensate products. Industrial Crops and Products. 86:40-48.
• Dong, N., Dong, C., Ponciano, G.P., Holtman, K.M., Placido, D.F., Coffelt, T.A., Whalen, M.C., McMahan, C.M. 2017. Fructan reduction by downregulation of 1-SST in guayule. Industrial Crops and Products. doi: 10. 1016/j.indcrop.2017.04.034.
• Torres, L., McMahan, C.M., Ramadan, L.E., Holtman, K.M., Tonoli, G.H., Flynn, A., Orts, W.J. 2015. Effect of multi-branched PDLA additives on the mechanical and thermomechanical properties of blends with PLLA. Journal of Applied Polymer Science. doi: 10.1002/app.42858.
• Holtman, K.M., Bozzi, D.V., Franqui-Villanueva, D.M., Offeman, R.D., Orts, W.J. 2016. A pilot-scale steam autoclave system for treating municipal solid waste for recovery of renewable organic content: Operational results and energy usage. Waste Management and Research. 34(5):457-464. doi: 10. 1177/0734242x16636677.
• Reza, M., Coronella, C., Holtman, K.M., Franqui-Villanueva, D.M., Poulson, S.R. 2016. Hydrothermal carbonization of autoclaved municipal solid waste pulp and anaerobically treated pulp digestate. ACS Sustainable Chemistry & Engineering. 4(7):3649-3658. doi: 10.1021/acssuschemeng.6b00160.
• Offeman, R.D., Holtman, K.M., Covello, K.M., Orts, W.J. 2014. Almond hulls as a biofuels feedstock: Variations in carbohydrates by variety and location in California. Industrial Crops and Products. 54(54):109-114.

Progress 10/01/15 to 09/30/16

Outputs
Progress Report Objectives (from AD-416): 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 (from AD-416): 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. Objective 1A. Biorefinery strategies for feedstocks prevalent in the Western United States. In a study of biorefinery options, ARS researchers in Albany, California, investigated biomass feedstock options and published a review entitled �Biorefinery Developments for Advanced Biofuels from a Sustainable Array of Biomass Feedstocks�. Biomass feedstock costs play the largest role in the economics of a biorefinery so it is important that research into biorefinery strategies be closely coupled to advances in crop science that account for crop yield and crop quality. Accordingly, the ARS team continues to make stepwise progress in biorefinery technology that couples the properties of individual feedstocks with output targets as the industry moves from corn ethanol toward utilizing a wider array of lignocellulose-based biomass feedstocks, including sorghum. Objective 1A. Converting solid waste to bioenergy. In a another study of biorefinery options, ARS researchers in Albany, California, developed and ran a pilot-scale steam autoclave system for treating municipal solid waste for recovery of renewable organic content for energy usage. This study provides the evaluation of autoclave operation, including mass and energy balances for the purpose of integration into organic diversion systems for biorefinery operation. Several methods of cooking municipal solid waste were explored from indirect oil heating only, a combination of oil and direct steam during the same cooking cycle, and steam only. Gross energy requirements averaged 1290 kJ/kg showing the municipal solid waste and food wastes are viable feedstocks for bioenergy production. Steam recycle from one vessel to the next can reduce gross energy requirements to an average of 790 kJ/kg, roughly halving the energy costs. Objective 1B. Optimizing commercial copolymers from methane-consuming microbes. ARS researchers in Albany, California, continue to study biopolymer production from methane and recently published a report on methanotrophic production of polyhydroxybutyrate-co-hydroxyvalerate with high hydroxyvalerate content. This copolymer has been commercialized as a promising biodegradable plastics with significant market potential to replace commodity plastics in many applications including packaging, cups, bowls, utensils and other single-use items. This team showed that Type II methanotrophic bacteria are a promising production platform for such biopolymers. Type II methanotrophic bacteria are known to produce pure poly-3-hydroxybutyrate homopolymer (PHB) but this newly isolated strain, Methylocystis sp. WRRC1, was capable of producing a wide range of polyhydroxy-butyrate-co-hydroxyvalerate copolymers (PHB-co-HV) when co- fed methane and valerate or n-pentanol. The ratio of HB to HV monomer was directly related to the concentration of valeric acid in the PHA accumulation media. The PHB-co-HV copolymers produced had decreased melting temperatures and crystallinity compared with methanotroph- produced PHB. Objective 1C. Using enzyme scaffolds for multi-enzyme reactions. Rosettasomes are artificial engineered ring scaffolds designed to mimic the bacterial cellulosome in which enzymes are tethered to a larger structure for use in biomass-to-biofuel conversion. ARS researchers in Albany, California, have developed rosettasomes, showing that enzymes can be successfully tethered to a larger structure for complex multi-step reactions. They showed that they facilitate much higher rates of biomass hydrolysis compared to using the same enzymes free in solution. A paper was published investigating whether tethering enzymes involved in both biomass hydrolysis and oxidative transformation to glucaric acid onto a rosettasome scaffold would result in an analogous production enhancement in a combined hydrolysis and bioconversion metabolic pathway. Three different enzymes were used to hydrolyze birchwood hemicellulose and convert the substituents to glucaric acid, a top-12 DOE value added chemical feedstock derived from biomass. It was demonstrated that colocalizing the three different enzymes to the synthetic scaffold resulted in up to a 40% improvement in acid production compared to enzymes that were free in solution, thus highlighting the advantage of applying this novel scaffolding system. Objective 2A. Adding value to almond coproducts. The United States has almost doubled its production of almonds over the last seven years resulting in an increase in generation of byproducts, such as almond shells and hulls. Shells have little value and are generally used for bioenergy via direct combustion or as low-cost animal bedding. In a research project aimed to add value to almond shells, ARS researchers in Albany, California, torrefied ground shells in a fixed bed reactor. Their solid and condensate products were collected for analysis with data on mass and energy yields of solid products, along with the gross calorific values of condensate products, showing that these products all have value. This was the first study on condensates produced during torrefaction of almond shells. Studies continue on showing that torrefied biomass is a useful additive in plastics, adding important properties such as improved thermal stability, uniform color and increased strength. Objective 2B. Converting pectin into value-added products via cloning of a gene into yeast. ARS researchers in Albany, California, cloned a bacterial endo-polygalacturonase (endo-PGase) gene from the plant pathogen Pectobacterium carotovorum into pGAPZaA vector and constitutively expressed it in the yeast, Pichia pastoris. The recombinant endo-PGase secreted by the Pichia clone showed a 1.7 fold increase when the culture medium included glycerol in replacement of glucose as the carbon source. The mode of the enzyme action showed internal cleavage of the a-1,4 glycoside bonds found in citrus peel pectin and polygalacturonic acid. Trigalacturonate and hexagalacturonate were the main hydrolysis products. This represents the first report of a microbial endo-PGase that produced trimer and hexamer uniquely as the end products of hydrolysis, in contrast to mixtures of mono-, di-, and trigalacturonates commonly observed for the action of fungal enzymes. Pectic oligosaccharides generated from native carbohydrate polymers offer the potential application as building blocks for value-added products. Objective 2B. Converting pectin to fine chemicals: Pectin is an underutilized byproduct of the juicing industry so the industry is looking for ways to add value to pectin by, for example, creating �green� solvents such as glucaric acid. ARS researchers developed an enzyme coupled assay employing uronate dehydrogenase for assay of exo- polygalacturonase enzymes acting on pectin and similar oligogalacturonic acids from citrus peel waste. The kinetic parameters for a Thermotoga sp. enzyme were determined, measuring methanol release via a coupled alcohol oxidase reaction, especially uronate dehydrogenase. This basic assay is an important tool in confirming reaction kinetics (and ultimately product cost) for turning pectin into important industrial products such as a natural cleaning agent. Objective 2C. Applying combinatorial enzyme technology: Combinatorial enzyme technology is relatively new field that, in this project, calls for the use of enzymes to surgically remove or convert side group moieties from biomass in a very specific, individual, and sequential order. This then alters the susceptibility of the main chain polymer. ARS researchers applied combinatorial design of molecules using specific enzymes by characterizing extensively key enzymes from the ARS clone libraries for specific and surgical removal of side chain moiety. Specifically, two feruloyl esterases for mono- and di-ferulates, and two xyloglucanases for a-D-xylp-D-Glcp recognition were thoroughly investigated in their mechanistic action and end products analysis. Using a similar approach in collaboration with a corporate partner, the USDA team has created libraries of oligosaccharides, and conducted preliminary screening of their efficacy for use in cleaning products, hand creams and cosmetics. Accomplishments 01 Synergistic combinations of natural antibiotics. The use of antibiotics, which are routinely fed to livestock, poultry, and fish to promote higher yields under unsanitary conditions, is being heavily scrutinized because their persistence in the environment likely plays a role in creating antibiotic-resistant bacteria. ARS researchers in Albany, California, have developed a strategy to overcome the negative impact of residual antibiotics by creating synergistic arrays of compounds that exhibit greater antimicrobial efficacy when formulated together. For example, two amino acid type molecules that exhibit minimal antimicrobial activity when they are alone can be formulated together at high concentration, exhibiting more than a 1000-fold increase in antimicrobial activity, rivaling the efficacy of commercial antibiotics. Yet, when antimicrobial activity is no longer needed it is alleviated by lowering the concentration via dilution. The combined system falls apart into two relatively benign agents that are no more active than a typical amino acid and will thus not be a threat to promote antibiotic-resistant microbes.

Impacts
(N/A)

Publications

• Tonoli, G., Holtman, K.M., Glenn, G.M., Fonseca, A., Wood, D.F., Williams, T.G., Sa, V., Torres, L., Klamczynski, A., Orts, W.J. 2016. Properties of cellulose micro/nanofibers obtained from eucalyptus pulp fiber treated with anaerobic digestate and high shear mixing. Cellulose. doi: 10.1007/ s10570-016-0890-5.
• Wagschal, K.C., Jordan, D.B., Lee, C.C., Younger, A.R., Braker, J.D., Chan, V.J. 2014. Biochemical characterization of uronate dehydrogenases from three Pseudomonads, Chromohalobacter salixigens, and Polaromonas naphthalenivorans. Enzyme and Microbial Technology. 69:62-68.
• Jordan, D.B., Lee, C.C., Wagschal, K., Braker, J.D. 2013. Activation of a GH43 �-xylosidase by divalent metal cations: Slow binding of divalent metal and high substrate specificity. Archives of Biochemistry and Biophysics. 533:79-87.
• Jordan, D.B., Braker, J.D., Wagschal, K., Lee, C.C., Chan, V.J., Dubrovska, I., Anderson, S., Wawrzak, Z. 2015. X-ray crystal structure of divalent metal-activated �-xyloisdase, RS223BX. Applied Biochemistry and Biotechnology. 177:637-648. doi: 10.1007/s12010-015-1767-z.
• Childress, C.J., Feuerbacher, L.A., Phillips, L., Burgum, A., Kolodrubetz, D. 2013. Mlc is a transcriptional activator with a key role in integrating cyclic AMP receptor protein and integration host factor regulation of leukotoxin RNA synthesis in Aggregatibacter actinomycetemcomitans. Journal of Bacteriology. 195(10):2284-2297.
• Majeed, T., Tabassum, R., Orts, W.J., Lee, C.C. 2013. Expression and characterization of Coprothermobacter proteolyticus alkaline serine protease. The Scientific World. doi: 10.1155/2013/396156.
• Singh, S.K., Heng, C., Braker, J.D., Chan, V.J., Lee, C.C., Jordan, D.B., Yuan, L., Wagschal, K.C. 2013. Directed evolution of GH43 �-xylosidase XylBH43 thermal stability and L186 saturation. Journal of Industrial Microbiology and Biotechnology. 41(3):489-498. doi: 10.1007/s10295-013- 1377-0.

Progress 10/01/14 to 09/30/15

Outputs
Progress Report Objectives (from AD-416): 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 (from AD-416): 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. Objective 1. Mass and energy balance for a biorefinery using solid-waste: ARS researchers completed a detailed mass and energy balance and presented their data to two commercial energy companies, their collaborative partners, for the purposes of procuring funding for full- scale demonstration of a 25 T/batch steam autoclave. Currently these two companies (licensee of a former Cooperative Research and Development Agreement [CRADA] partner) are in final negotiations to begin engineering design and construction planned to begin in October of 2015. Further testing and development of methane production in a collaboration between the USDA and the Salinas Valley Solid Waste Authority continued, successfully demonstrating a pilot scale 1500 gal, high solids anaerobic reactor designed specifically by ARS researchers to convert the lignocellulosic feedstock produced at a landfill into biogas. ARS researchers are consulting with engineering design firms for scale up details and costs. Improved methane-using bacteria via directed evolution: ARS researchers mutagenized plastics-producing bacteria using chemicals and high intensity, ultraviolet radiation with the goal of optimally converting methane into the bioplastic, polyhydroxybutyrate (PHB). Thousands of these bacteria were subjected to high throughput fluorescence activated cell sorting (FACS), and bacteria demonstrating high signal intensity (i. e. elevated levels of PHB) were collected. Some of these bacteria showed upwards of 80% increase in PHB bioplastic accumulation. These lines are being evaluated by a corporate partner for scale-up. Preliminary genomic DNA analysis has been conducted on these bacteria. Converting methane into green chemicals via bacteria: ARS researchers collected multiple bacterial strains that have methane monooxygenase (MMO) activity with the goal of improving methane uptake and conversion to (bio)chemicals. There are two types of MMO proteins, membrane-bound) and soluble. ARS scientists determined that the type of bioconversions utilized here requires soluble MMO so that the enzyme can be tethered to nanoassemblies (rosettazymes). Therefore, strains are being cultured in specialized media that will select for those bacteria that have soluble MMO. Two strains have been identified that clearly encode soluble MMO, as screening and strain optimization continues. Objective 2. Optimizing the value of almond by-products: ARS researchers are applying hot water extraction and hot water digestion to almond byproducts to optimize their value in new applications. In extraction, the time required for almond hulls to come to equilibrium with the contacting liquid is an important consideration for commercial extraction processes. Whole hulls were milled and screened to yield particle sizes in the following ranges: 3.35�6.35mm, 2.36�3.35mm, 2.00�2.36mm, and 1. 70�2.00mm. These fractions, as well as whole, unmilled hulls, were equilibrated with water at different temperatures and the time for Brix, % dry matter and total sugar (glucose, fructose, sucrose, xylose, inositol, sorbitol) to reach 90% of the plateau value (t90) was calculated for each sample. Milling had a stronger effect than extraction temperature, over the ranges studied. Research continues on milling parameters. Converting pectin to fine chemicals: ARS researchers developed an enzyme coupled assay employing uronate dehydrogenase for assay of exo- polygalacturonase enzymes acting on pectin and similar oligogalacturonic acids from citrus peel waste. The kinetic parameters for a Thermotoga sp. enzyme were determined, measuring methanol release via a coupled alcohol oxidase reaction, especially uronate dehydrogenase (UDH). The corresponding gene from a Thermotoga sp. (NCBI#ACB08857) was successfully cloned; i.e., UDH genes with NCBI#�s yp_003898474 and wp_004580342 were successfully cloned. Applying combinatorial enzyme technology: Combinatorial enzyme technology is relatively new field that, in this project, calls for the use of enzymes to surgically remove or convert side group moieties from biomass in a very specific, individual, and sequential order. This then alters the susceptibility of the main chain polymer. ARS researchers applied combinatorial design of molecules using specific enzymes by characterizing extensively key enzymes from the ARS clone libraries for specific and surgical removal of side chain moiety. Specifically, two feruloyl esterases for mono- and di-ferulates, and two xyloglucanases for a-D-xylp-D-Glcp recognition were thoroughly investigated in their mechanistic action and end products analysis. Using a similar approach in collaboration with a corporate partner, the USDA team has created libraries of oligosaccharides, and conducted preliminary screening of their efficacy for use in cleaning products, hand creams and cosmetics. Accomplishments 01 Improved enzymes for converting citrus processing waste into value- added products. Pectins and other similar polysaccharides derived from citrus processing represent a significant untapped biomass resource which can be used for the development of fine chemicals such as adipic acid (used to make nylon). Through genomic mining of bacterial strains, ARS Researchers at the Western Regional Research Center in Albany, California, have identified a highly active enzyme, exo- polygalacturonase from Thermotoga sp., which converts peel waste into glucaric and/or galacturonic acids. This enzyme is hyperthermostable, possessing a melting temperature of 86 �C after a 1 hour incubation. In combination with a previously identified hyperthermostable pectin methylesterase, these enzymes allow the processing of pectin-rich citrus waste residue at elevated temperatures, making the process, cleaner, faster and well-suited for �green� production of adipic acid, a main constituent of nylon. This represents a multi-billion dollar market.

Impacts
(N/A)

Publications

• Offeman, R.D., Dao, G.T., Holtman, K.M., Orts, W.J. 2015. Leaching behavior of water-soluble carbohydrates from almond hulls. Industrial Crops and Products. 65:488-495.
• Wong, D., Takeoka, G.R., Chan, V.J., Liao, H., Marakami, M. 2015. A novel feruloyl esterase from rumen microbial metagenome: Gene cloning and enzyme characterization in the release of mono- and diferulic acids. Protein and Peptide Letters. 22(2):681-688.
• Santos, C.R., Cordeiro, R.L., Wong, D., Murakami, M.T. 2015. Structural basis for xyloglucan specificity within GH5 family and the molecular determinants for a-D-Xylp(1�6)-D-Glcp recognition at the -1 subsite. Biochemistry. 54:1930-1942.