2013 KIEC - At The Interface Of Advanced Materials And Plant Biotechnology - Barbara Knutson

Research in the Commonwealth: At the Interface of Advanced Materials and Plant Biotechnology Barbara Knutson, Professor University of Kentucky Department of Chemical and Materials Engineering

Plant-Derived Products: Technology Needs Conversion of lignocellulose to fuels and chemicals Hemicellulose 20% - 40%

Lignin 15% - 25%

Cellulose 30% - 50%

Other 5% - 35%

Mechanical/ Chemical/ Biological Treatment Soluble Sugars

Agricultural Residues & Energy Crops

Fermentation/ Challenges Chemical – CelluloseFuels recalcitrance Catalysis Commodity/ – Feedstock diversity Specialty – Sugar solution Chemicals purity/concentration

Production and commercialization of therapeutics Employing the genetic machinery of plants to synthesize known and novel therapeutics

Challenges: Identification/recovery of therapeutics from plant cell cultures

Collaborators/Funding Collaboration

Dr. Stephen Rankin (UK Chemical Engineering) – Synthesis of Advanced Ceramics Dr. Sue Nokes (UK Biosystems & Agricultural Engineering) – Biomass Conversion (BRDI) Dr. John Littleton (CSO, Naprogenix, Inc) – Plant Derived Therapeutics

Graduate Research Results Presented Here:

Check out Helen Li (Knutson) – Hydrolysis of Cellulose Thin Films Suvid Joshi (Rankin) – Sugar Separation using Imprinted Silica Particles this poster Alicia Modenbach (Nokes)- Hydrolyzate Separation using Imprinted Silica Particles

Financial Support

• USDA BRDI “Separation and Recovery of Pentose Derivatives from Cellulosic Biomass using Molecular Imprinting” (Knutson) • USDA NIFA BRDI “On-Farm Bioprocessing” (Nokes) • KSEF “Engineered Porous Thin Films for Screening and Production of Therapeutics Derived from Plant Biotechnology” (Knutson) • KSEF “Interfacial Engineering of Biomass Saccharification by T. Check out this poster reesei Enzymes” (Rankin)

Synthesis of Mesoporous Silica Platforms for Separation, Catalysis, and Sensing Sol gel synthesis in the presence of a selfassembled template

Silica “Polymerization” & Extraction of Template

• Large-pore particles for protein protection & separation • Oriented silica thin film membranes • Surface-imprinted nonporous Stöber silica particles

Challenges in Lignocellulose Conversion Hemicellulose 20% - 40%

Lignin 15% - 25%

Cellulose 30% - 50%

Other 5% - 35%

Mechanical/ Chemical/ Biological Treatment

These are protective structures of the plant, meant to withstand degradation.

Soluble Sugars

C5- sugars (Pentose) + oligomers

Fermentation/ Chemical Catalysis

Fuels Commodity/ Specialty Chemicals

C6 –sugars (Hexose) + oligomers

Cellulose, the source of glucose, is recalcitrant.

Cellulose makes plant cell wall strong and difficult to breakdown in most biological system .

Intra-molecular H-bonds Inter-molecular H-bonds

Conversion of Lignocellulose: On-Farm Biomass Processing An integrated high-solids transporting/storing/processing system

Funded by USDA NIFA Biomass Research Development Initiative Award

Bioprocessing Concept University of Kentucky (PI: Sue Nokes, Biosystems & Agricultural Eng.) Objective Make useanofintegrated existing on-farm time/storage capacityconversion as a bioreactor 3 Universities/National Laboratory/Industry/Agriculture Develop material handling/biomass Produce anforenergy-dense value added productthat stream on-farm to fit 21 Investigators approach the conversion of lignocellulose is structured Reduce transportation to centralized “biorefineries�

Bioprocessing Collaboration: BAE, Chemical Eng., Chemistry, within the existing agricultural paradigm.

Horticulture, and USDA-ARS Food Animal Production Unit

On-Farm Biomass Processing Separation Challenges Bioprocessing Modified Solid Substrate Cultivation w/ Recycle: Delignify, degrade cellulose & ferment

Separate & concentrate: Products By-products Inhibitors

• Solid substrate cultivation periodically flushed & recycled – Aqueous process stream contains products (butanol), sugars, byproducts, and inhibitors • Low energy intensive technologies for aqueous based separation is required: Adsorption and Semi-Permeable Membranes

Imprinted Silica Particles as Adsorbents for Sugars Stöber Particles Imprinted for Glucose Soft-silica imprinting of Stöber Particles with a Glucose-Based Surfactant Sugar Adsorption from Pure Sugar Solutions

45

Sugar Adsorption from Biomass Hydrolyzates

Adsorbed sugar (mg sugar /g material)

40

Glucose

35

Xylose

30 25 20 15 10 5 0 Non-imprinted

Glucose-imprinted Material Type

Glucose and xylose adsorbed on glucoseimprinted silica particles

Glucose and xylose adsorbed on glucoseimprinted and non-imprinted silica particles

• Glucose had a higher affinity for glucose-imprinted particles than non-imprinted particles.

• Xylose adsorbed similarly to glucose-imprinted and non-imprinted particles.

• Evidence for successful imprinting translated to complex hydrolyzate mixtures.

Materials Characterization Tools Applied to Cellulose Deconstruction QCM measurement and output QCM-D is an ultra sensitive mass sensor to measure the mass change of thin film

• Mass change of the thin films is proportional to Δf of QCM – Contributions of adsorbed species (cellulase enzymes) – Contributions of thin film loss (cellulose hydrolysis)

∆f time → Preparation of cellulose thin films Anchoring polymer

Immerse

(50 wt% polyethyleneimine ) (25⁰C, pH 10, 15 min)

Cellulose (Avicel)

solubilized

spin-coat

by NMMO (115⁰C, 2 h)

(4500 rpm, 40s)

Cellulose coated

cellulose

cellulose

Coated with NMMO-solubilized cellulose

AFM imaging 5 µm

5 µm

5 µm

9

Enzymatic hydrolysis of cellulose thin films as measured by QCM Enzyme injection

1

3

Mass change when cellulase is in contact with cellulose thin film: 1) Enzyme adsorption 2) Cellulose hydrolysis 3) Substrate depletion

2

Cellulose hydrolysis is captured by QCM in real time response of enzyme adsorption and cellulose hydrolysis.

Enzyme: Celluclast速, Sigma (celllulase from Trichoderma reesei)

No inhibitor

Enzyme injection

The inhibition of cellulase by cellobiose (a glucose-dimer formed during cellulose hydrolysis) can be quantified using QCM.

Increasing Inhibitor

5.0 g/l CB added

The effect of external variables on enzyme adsorption and cellulose hydrolysis rates can be measured using thin film analysis.

Modeling Cellulose Hydrolysis from QCM Measurements Formulate a reaction pathway and species balance in terms of species that contribute to thin film mass and unknown rate constants

E +S↔ ES → P + E k k1

k2

−1

Enzyme (E)

k3

EI + S ↔ ESI k −3

Inhibited enzyme (EI)

Inhibitor (I)

Product (P) Substrate (S) Enzyme-Substrate complex (ES)

Inhibited Enzyme-Substrate complex (ESI)

Inhibitor-substrate complex (SI) 11

Cellulose Hydrolysis Kinetics from QCM Measurements Solve for unknown rate constants by Mixed enzyme inhibition scheme fitting model to Δf data

k1

Species balance for species that contribute to thin film mass

k2

E +S↔ ES → ES + P − k −1

k3

ESI EI + S ↔ k −3

Analysis of between Cellulose Hydrolysis Kinetics using Thin Films Relationship Δf and species that contribute to thin film mass

• Complements bulk measures of cellulose degradation, which don’t QCM Apparatus Sensitivity to: provide for detailed kinetic analysis of adsorption & hydrolysis A = ∆f for enzyme binding B = ∆f for substrate lost • Provides for the testing of mechanistic models • Can be extended to a range of hydrolysis variables, thin film Enzyme adsorption Hydrolysis substrates, and enzyme/enzyme cocktails.

Plant-Derived Therapeutics Identification and Recovery • Plants synthesize bioactive small molecule metabolites that bind to target receptor proteins in other organisms (e.g. human proteins for therapeutic applications) • Plants can be genetically manipulated to express both known and novel bioactive molecules that are not readily chemically synthesized • Plant biotechnology has developed rapid screening techniques for genetically-modified plant cell cultures with therapeutic activity

Expression of green fluorescent protein is linked to bioactive molecule binding to target human receptors (here, estrogen receptor ER-β) to give a mechanism to screen plant mutant strains that overexpress known and novel ligands for that receptor.

Naprogenix, Inc.

The ability to separate the bioactive molecules (for the purpose of recovery and identification) at the plant cell culture scale lags the ability to generate plant-derived therapeutics.

Plant-Derived Therapeutics Identification and Recovery

• The current technology for the separation of bioactive ligands is affinity chromatography (desired receptor is immobilized on the surface of a nonporous particle), which is not well suited for small sample sizes

Research Approach:

+ Sensitivity of QCM to mass change

High surface area of nanoporous silica thin films with covalently bound protein receptors

Engineered Porous Thin Film Platforms for Screening Therapeutics Derived from Plant Biotechnology

Summary • The design of advanced materials to address the needs of plant biotechnology is applicable to plant-derived products that range from commodity chemicals to high-value therapeutics • The numerous successes of material design for pharmaceutical and biomedical applications indicate the potential of advanced materials aimed at plant biotechnology and natural products.

For additional information: Poster 30: Stephen Rankin*, “Interfacial Engineering of Biomass Saccharification by T. Reesei Enzymes” Poster 36: Suvid Joshi*, “Imprinting the Surface of Stöber Silica Nanoparticles with Surfactants to Create Selective Saccharide Adsorbent Materials”

Acknowledgements Collaboration

Dr. Stephen Rankin (UK Chemical Engineering) Dr. Sue Nokes (UK Biosystems & Agricultural Engineering) Dr. John Littleton (CSO, Naprogenix, Inc)

Financial Support • USDA BRDI • KSEF • NSF

Graduate Researchers

Helen Li Srivenu Seelam Dan Schlipf Shanshan Zhou Kaitlyn Wooten

Undergraduate Researchers Brianna Smith Elliott Rushing Cory Jones