Location: Bioproducts Research
2015 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
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
1. 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.
Review Publications
Offeman, R.D., Dao, G.T., Holtman, K.M., Orts, W.J. 2014. Leaching behavior of water-soluble carbohydrates from almond hulls. Industrial Crops and Products. 65(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.
