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ARS Home » Pacific West Area » Albany, California » Western Regional Research Center » Bioproducts Research » Research » Research Project #427427

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

Location: Bioproducts Research

2017 Annual Report

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.

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
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.

1. 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.

Review 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. 183(4):1503-1515.
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.