Location: Bioproducts Research
2018 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
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
1. 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.
2. 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.
3. 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.
Review Publications
Jordan, D.B., Stoller, J.R., Lee, C.C., Chan, V.J., Wagschal, K.C. 2016. Biochemical characterization of a GH43 ß-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. 5:669-675. https://doi.org/10.1016/j.mex.2018.06.011.
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. 10:3125-3142. https://doi.org/10.1007/s12649-018-0295-9.
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. https://doi.org/10.1021/acs.energyfuels.6b03185.
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. https://doi.org/10.1016/j.ijbiomac.2018.06.124.
