BIOCONVERSION OF AGRICULTURAL RESIDUES TO BIOPRODUCTS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1017399
• Project Start Date: Oct 1, 2018
• Project End Date: Sep 30, 2023
• Program Code: [(N/A)]- (N/A)

Recipient Organization
UNIVERSITY OF CALIFORNIA, DAVIS
410 MRAK HALL
DAVIS,CA 95616-8671

Performing Department
Biological and Agricultural Engineering

Non Technical Summary
This project supports the mission of the Agricultural Experiment Station by addressing the Hatch Act area(s) of: sustainable agriculture; biotechnology.California agriculture produces an abundance of residual lignocellulosic biomass. For example, the almond industry alone produces over 2.5 million dry tons of waste biomass in the form of prunings, hulls and shells each year. Other residues unique to CA include rice straw. The availability of agricultural residues presents an opportunity to develop processes to produce high-value, renewable bioproducts such as essential amino acid enriched animal feed, specialty chemicals and liquid fuels. The biggest technological barrier to establishing cost-effective bioconversion processes is the lack of fundamental mechanistic understanding of the enzymatic hydrolysis of biomass to soluble, fermentable sugars (i.e. saccharification). In this project, we tackle the challenge of solving the enzymatic saccharification mechanisms and apply new fundamental knowledge towards developing processes to valorize California's agricultural residues.'Biomass recalcitrance' refers to the fact that lignocellulosic biomass (such as agricultural residues) are highly resistant to being broken down into its constituent sugars. The origin of biomass recalcitrance is poorly understood and consequently effective strategies to overcome biomass recalcitrance is lacking. Lignocellulosic biomass is typically subjected to a pretreatment step at high temperatures with corrosive chemicals prior to the addition of enzymes that break internal bonds to release sugars. Experimental evidence demonstrate the effectiveness of pretreatment for accelerating enzyme access but why this works is not well understood. Moreover, in many circumstances, enzymatic hydrolysis rates will slowdown and halt prematurely even in the presence of excess substrate. To address these questions, we conduct fundamental mechanistic studies that focus on the study of biomass surface chemical and structural changes that occur as a result of thermochemical pretreatment and during enzymatic hydrolysis. Enzymes must locate 'productive binding sites', i.e. sites on the substrate surface where then can bind and hydrolyze bonds. Effective pretreatments must maximize enzyme access to the substrate surfaces by altering surface and internal structures, while also maximizing the availability of productive binding sites for enzymes. Our research results have shown that accessible productive binding sites is a significant limiting factor and further suggest that higher reaction rates are maintained when substrates maintain higher concentrations of productive binding sites during enzymatic hydrolysis. Still unanswered questions are where the productive binding sites are located on the surfaces of the biomass substrates, what structural and chemical modifications by pretreatments lead to increased productive binding sites, and what surface and structural changes during hydrolysis are associated with maintaining higher productive binding site concentrations. These are questions that we are addressing with high resolution imaging techniques including atomic force microscopy.Complementary to the fundamental studies to understand how enzymes breakdown biomass to release sugars, we apply current knowledge to tackle the bioconversion of regional agricultural residues. In one example, we work with lignocellulosic residues from the production of almonds. Almond hulls contain high amounts of soluble sugars but the sugars need to be released into a liquid medium for yeasts to convert them to products. In collaboration with Dr. Boundy-Mills, curator of the UC Davis Phaff Yeast Collection, we are developing a process to release and convert the sugars into an essential amino acid enriched animal feed using high-protein producing yeasts. Almond shells and orchard prunings are considerably more recalcitrant than the hulls and contain larger fractions of the non-carbohydrate polymer, lignin. Lignin has historically been considered to be a nuisance in the biomass conversion because it does not contribute sugars and it can inhibit enzymes. Also in collaboration with Dr. Boundy-Mills, with these feedstock, we are developing processes to release lignin into yeast growth media and are screening the vast Phaff yeast collection for yeasts that can convert solubilized lignins to specialty chemicals (in particular a surfactant). This project also involves Dr. Peter Hernes (LAWR) whose expertise in lignin analysis will facilitate the analysis of the solubilized lignin in the yeast growth media.Overall, this project will generate fundamental understanding of 'upstream' pretreatment and enzymatic saccharification reactions that heavily influence the economics of converting agricultural residues to value-added products. Currently, attempted commercial-scale biomass conversion plants are struggling because the lack of fundamental understanding of the underlying reactions have led to unexpected challenges in scale-up. Solving the mechanisms of cellulose hydrolysis by cellulases and understanding the biomass chemical structural contributions to the reaction rates will enable rational engineering design of the cost-effective bioconversion processes. As part of this project, while the fundamental studies are on-going, we are also working to apply current knowledge to valorize CA-specific agricultural residues.

Animal Health Component

30%

Research Effort Categories

Basic

70%

Applied

30%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
403	1530	1000	10%
403	1530	2020	10%
403	1212	1000	10%
403	1212	2020	10%
403	1520	1000	10%
403	1520	2020	10%
403	1549	1000	10%
403	1549	2020	10%
403	0650	1000	10%
403	0650	2020	10%

Knowledge Area
403 - Waste Disposal, Recycling, and Reuse;

Subject Of Investigation
1212 - Almond; 1549 - Wheat, general/other; 1520 - Grain sorghum; 0650 - Wood and wood products; 1530 - Rice;

Field Of Science
2020 - Engineering; 1000 - Biochemistry and biophysics;

Keywords

agricultural residues

lignocellulosic biomass

cellulosic biofuels

bioproducts

Goals / Objectives
1. To further our understanding of the mechanisms of cellulose hydrolysis in lignocellulosic biomass such as agricultural residues.2. To further our understanding of how 'upstream' pretreatment and enzymatic saccharification processes impact 'downstream' product yields.3. To develop processes for converting agricultural residues to various value-added bioproducts including fuel, feed and specialty chemicals.

Project Methods
Biomass pretreatment and fractionationBiomass pretreatment to render the material more enzymatically digestible with less inputs, and fractionation methods to partition the chemical constituents of biomass into separate stream will be pursued.- In one approach, oxidative alkali biomass pretreatment will be pursued to fractionate lignin and hemicellulose into the aqueous stream while retaining cellulose as a solid. The lignin and hemicellulose stream will be used as growth media by yeasts of the UC Davis Phaff yeast collection (in collaboration with Dr. Boundy-Mills, FST). Analysis of the lignin composition of the aqueous fraction will be conducted in collaboration with Dr. Peter Hernes (LAWR). In this approach, lignin monomers and hemicellulosic sugars (and pectic sugars if any) will be converted to biosurfactants and biodiesel. The residual cellulose fractions will utilized as a matrix component in a UC Davis patented microencapsulation process. Results of this work will be disseminated via presentations at international conferences and in peer-reviewed journals.- In another approach, high-sugar containing biomass (i.e. almond hulls) will be enzymatically liquefied to maximally release soluble sugars for conversion to an essential amino acid enriched, high-protein animal feed (also in collaboration with Dr. Boundy-Mills, FST). This work will be conducted with the Almond Board of California (ABC) and results will be shared with almond growers, processors and other stakeholders. Results will also be presented at international conferences and submitted for publication in peer-reviewed journals.Investigation of biomass substrate changes due to pretreatment and enzymatic saccharificationTowards solving the mechanisms of cellulose hydrolysis in biomass, enzyme-relevant changes to the substrate surface and internal structures and chemistry due to pretreatment and during enzymatic saccharification will be investigated. High resolution spectroscopic and microscopic techniques (atomic force microscopy, chemical force microscopy, total internal reflectance microscopy (TIRFM) and synchrotron-Fourier transform infrared (FTIR)) methods will be used to characterize surface interactions of enzymes, and surface chemical and structural modifications of the biomass substrate due to pretreatment and saccharification. Proton Nuclear Magnetic Resonance (1H-NMR) relaxometry to study changes in internal structures of lignocellulosic biomass due to pretreatment and saccharification.

Progress 10/01/19 to 09/30/20

Outputs
Target Audience:- The scientific and industrial community involved in furthering fundamental understanding of the lignocellulosic biomass bioconversion processes. - The Almond Board of California Changes/Problems:As with everyone else, our productivity took a signficant hit due to COVID-19 related shutdowns. Our ability to work in the lab is still limited but improving. What opportunities for training and professional development has the project provided?Two postdoctoral researchers trained on this project this past year: - Dr. Dana Wong worked on fractionation of almond shells for bioconversion. She took a job at Dupont Biosciences in March 2020. - Dr. Jennifer Nill worked on FTIR imaging of cellulose. She took a job at BCD Bioscience in November 2020. Two graduate students trained on this project this past year: - Alex Hitomi worked on conversion of almond hulls to protein-rich animal feed, and developing nano-FTIR methods for investigating cellulose surface properties. - Lucy Knowles joined the project to study synergistic enzymatic hydrolysis of cellulose. Three undergraduate students trained on this project this past year: - Sandra Pena purified cellulase for the project. Graduated spring 2020. - Vu Quach maintained fungal cultures for cellulase production. Graduate spring 2020. - Taylor Charlesworth fractionated almond shells. Graduated spring 2020. Currently employed at Abbott Diagnostics. How have the results been disseminated to communities of interest?With the COVID-19 pandemic, most of our plans for presenting our work were canceled. A few virtual presentations were made at technical conferences that made the switch (see presentations under 'Products'). What do you plan to do during the next reporting period to accomplish the goals?We will continue with our nanoscale experiments to elucidate substrate surface changes due to enzyme action, towards increasing knowledge in the mechanism of enzymatic cellulose hydrolysis. We are also continuing our work in valorizing lignin-rich waste biomass streams and development of bioconversion processes for agricultural residues.

Impacts
What was accomplished under these goals? 1. Towards the goal of furthering understanding of the mechanisms of cellulose hydrolysis in lignocellulosic biomass, we: - Manuscript currently in preparation for single molecule imaging experiments to study interactions of lytic polysaccharide monooxygenase (LPMO) enzymes at the surface of cellulose fibrils (in collaboration with LANL and U. of Copenhagen). - completed FTIR imaging studies of in situ cellulose enzymatic hydrolysis in buffer. Manuscript currently in preparation 2. Towards the goal of understanding how pretreatment and enzymatic saccharification processes impact downstream product yields, we: - manuscript in preparation for work fractionating almond shells into a lignin-rich soluble stream for valorization to lipids. 3. Towards the goal of developing processes to convert agricultural residues to value-added bioproducts, we: - manuscript in preparation on project outcomes for the conversion of almond hulls in to protein-rich animal feed supplement.

Publications

• Type: Journal Articles Status: Published Year Published: 2020 Citation: Nill, J.D., Jeoh, T., 2020. The Role of Evolving Interfacial Substrate Properties on Heterogeneous Cellulose Hydrolysis Kinetics. ACS Sustain. Chem. Eng. 8, 6722 -6733. https://doi.org/10.1021/acssuschemeng.0c00779
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Mudinoor, A.R., Goodwin, P.M., Rao, R.U., Karuna, N., Hitomi, A., Nill, J., Jeoh, T., 2020. Interfacial molecular interactions of cellobiohydrolase Cel7A and its variants on cellulose. Biotechnol. Biofuels 13, 10. https://doi.org/10.1186/s13068-020-1649-7
• Type: Book Chapters Status: Published Year Published: 2020 Citation: Julia S. Ribeiro, Michelle E. Kossak, Tina Jeoh, 2020. STEM for Girls: A one-day event to improve girls' attitudes toward STEM, in: Out-of-School-Time STEM Programs for Females: Implications for Research and Practice. Volume II: Short-Term Programs, Out-of-School-Time STEM Programs for Females: Implications for Research and Practice. Information Age Publishing, Inc., p. 192 pages.
• Type: Conference Papers and Presentations Status: Other Year Published: 2020 Citation: Opportunities and challenges in valorizing cellulose, UTEC GO Live Research Series (Peru), July 16, 2020.
• Type: Conference Papers and Presentations Status: Other Year Published: 2020 Citation: Understanding interfacial enzyme-substrate interactions limiting cellulose hydrolysis kinetics, 2020 Center for integrated Nanotechnologies Annual Meeting. Symposium II: Advances in Soft Matter Imaging. September 22, 2020.

Progress 10/01/18 to 09/30/19

Outputs
Target Audience:- The Almond Board of California - The scientific and industrial community involved in furthering fundamental understanding of the lignocellulosic biomass bioconversion processes. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?In the past year, this project graduated one Ph.D. student, Dr. Jennifer Nill (Ph.D., Chemical Engineering) and one B.S. student, Julia Cunniffe. Dr. Jennifer Nill is continuing research into understanding fundamental rate limiting mechanisms in enzymatic cellulose hydrolysis as a post-doc on a Department of Energy funded project. Julia Cunniffe is now working as a research assistant at the USDA ARS in Albany, CA. How have the results been disseminated to communities of interest?We have presented results at the Almond Board of California Board meetings and at the Almond Board of California's annual conference in Sacramento. Additionally, we have presented our research at technical conferences including the Symposium on Biotechnology for Fuels and Chemicals, and the Gordon Research Conference on Carbohydrate-Active Enzymes for Glycan Conversions. What do you plan to do during the next reporting period to accomplish the goals?We will continue with our nanoscale experiments to elucidate substrate surface changes due to enzyme action, towards increasing knowledge in the mechanism of enzymatic cellulose hydrolysis. We are also continuing our work in valorizing lignin-rich waste biomass streams and development of bioconversion processes for agricultural residues.

Impacts
What was accomplished under these goals? 1. Towards the goal of furthering understanding of the mechanisms of cellulose hydrolysis in lignocellulosic biomass, we: - completed single molecule imaging experiments to study interactions of lytic polysaccharide monooxygenase (LPMO) enzymes at the surface of cellulose fibrils (in collaboration with LANL and U. of Copenhagen) - installed a nano-FTIR system to enable nanoscale studies of cellulose fibril surface chemistry impacted by enzymatic hydrolysis. 2. Towards the goal of understanding how pretreatment and enzymatic saccharification processes impact downstream product yields, we: - have on-going work fractionating almond shells into a lignin-rich soluble stream for valorization to lipids. 3. Towards the goal of developing processes to convert agricultural residues to value-added bioproducts, we: - developed a process to convert waste almond hulls to a protein-enriched animal feed (in collaboration with the UC Davis Phaff Yeast Collection).

Publications

• Type: Journal Articles Status: Submitted Year Published: 2019 Citation: Nill, J., & Jeoh, T. (2019). The role of evolving interfacial substrate properties on heterogeneous cellulose hydrolysis kinetics. BioRxiv, 691071. https://doi.org/10.1101/691071
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Boundy-Mills, K., Sitepu, I., Wong, D., Hernes, P. and Jeoh, T. (2019) Conversion of lignin monomers to glycolipids by oleaginous yeasts. Oral Presentation at the 41st Symposium on Biotechnology for Fuels and Chemicals, Seattle, WA.
• Type: Theses/Dissertations Status: Published Year Published: 2019 Citation: Nill, Jennifer. Ph.D. Dissertation: Rate limitations in cellulose hydrolysis kinetics arising from the productive cellulase binding capacity. University of California, Davis, Davis, California.
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Nill, J. Bechtel, H., Holman, H.-Y., Jeoh, T. (2019) A multiscale study to elucidate the role of cellulose physicochemical properties in productive binding of cellulases. Poster Presentation at the 41st Symposium on Biotechnology for Fuels and Chemicals, Seattle, WA.
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Wong, D., Sitepu, I., Lynn, L., Hernes, P., Boundy-Mills, K., and Jeoh, T. (2019) Valorization of lignin and cellulose from California-relevant feedstocks into biosurfactants and nanocellulose. Oral Presentation at the 41st Symposium on Biotechnology for Fuels and Chemicals, Seattle, WA.
• Type: Conference Papers and Presentations Status: Other Year Published: 2019 Citation: Nill, J. (2019) The effect of LPMOs and Endoglucanases on cellulose accessibility and productive enzyme binding. 2019 Gordon Research Conference on Carbohydrate-Active Enzymes for Glycan Conversions.