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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

2016 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
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.

1. 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.


Review 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. 23(2):1239-1256. 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 ß-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 ß-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 ß-xylosidase XylBH43 thermal stability and L186 saturation. Journal of Industrial Microbiology and Biotechnology. 41(3):489-498. doi: 10.1007/s10295-013-1377-0.