2013 kiec - at the interface of advanced materials and plant biotechnology - barbara knutson

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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

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KIEC 08.29.2013, session two, Barbara Knutson

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Page 1: 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

Page 2: 2013 KIEC - At The Interface Of Advanced Materials And Plant Biotechnology - Barbara Knutson

Plant-Derived Products: Technology Needs

Conversion of lignocellulose to fuels and chemicals

Production and commercialization of therapeutics

Cellulose30% - 50%

Hemicellulose20% - 40%

Lignin15% - 25%

Other5% - 35%

Mechanical/ Chemical/ Biological Treatment

Soluble Sugars

Fermentation/ Chemical Catalysis Fuels

Commodity/ Specialty

Chemicals

Challenges – Cellulose recalcitrance – Feedstock diversity – Sugar solution

purity/concentration

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

Challenges: Identification/recovery of therapeutics from plant cell cultures

Agricultural Residues & Energy Crops

Page 3: 2013 KIEC - At The Interface Of Advanced Materials And Plant Biotechnology - Barbara Knutson

Collaborators/Funding

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.

reesei Enzymes” (Rankin)

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

Check out this poster

Check out this poster

Page 4: 2013 KIEC - At The Interface Of Advanced Materials And Plant Biotechnology - Barbara Knutson

Synthesis of Mesoporous Silica Platforms for Separation, Catalysis, and Sensing

• Large-pore particles for protein protection & separation

• Oriented silica thin film membranes

• Surface-imprinted non-porous Stöber silica particles

Silica “Polymerization” & Extraction of Template

Sol gel synthesis in the presence

of a self-assembled template

Page 5: 2013 KIEC - At The Interface Of Advanced Materials And Plant Biotechnology - Barbara Knutson

Challenges in Lignocellulose Conversion

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

Intra-molecular H-bonds

Inter-molecular H-bonds

Cellulose30% - 50%

Hemicellulose20% - 40%

Lignin15% - 25%

Other5% - 35%

Mechanical/ Chemical/ Biological Treatment Soluble

Sugars

Fermentation/ Chemical Catalysis

Fuels Commodity/

Specialty Chemicals

C5- sugars (Pentose) + oligomers

C6 –sugars (Hexose) + oligomers

Cellulose, the source of glucose, is recalcitrant.

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

Page 6: 2013 KIEC - At The Interface Of Advanced Materials And Plant Biotechnology - Barbara Knutson

Conversion of Lignocellulose: On-Farm Biomass Processing

Funded by USDA NIFA Biomass Research Development Initiative Award

An integrated high-solids transporting/storing/processing system

Bioprocessing Concept Make use of existing on-farm time/storage capacity as a bioreactor Produce an energy-dense value added product stream on-farm Reduce transportation to centralized “biorefineries”

Objective Develop an integrated material handling/biomass conversion approach for the conversion of lignocellulose that is structured to fit within the existing agricultural paradigm.

University of Kentucky (PI: Sue Nokes, Biosystems & Agricultural Eng.) 3 Universities/National Laboratory/Industry/Agriculture 21 Investigators Bioprocessing Collaboration: BAE, Chemical Eng., Chemistry, Horticulture, and USDA-ARS Food Animal Production Unit

Page 7: 2013 KIEC - At The Interface Of Advanced Materials And Plant Biotechnology - Barbara Knutson

On-Farm Biomass Processing Separation Challenges

• Solid substrate cultivation periodically flushed & recycled – Aqueous process stream contains products (butanol), sugars, by-

products, and inhibitors • Low energy intensive technologies for aqueous based separation is

required: Adsorption and Semi-Permeable Membranes

Modified Solid Substrate Cultivation w/ Recycle: Delignify, degrade cellulose & ferment

Separate & concentrate: Products By-products Inhibitors

Bioprocessing

Page 8: 2013 KIEC - At The Interface Of Advanced Materials And Plant Biotechnology - Barbara Knutson

Imprinted Silica Particles as Adsorbents for Sugars

Soft-silica imprinting of Stöber Particles Stöber Particles Imprinted for Glucose with a Glucose-Based Surfactant

0

5

10

15

20

25

30

35

40

45

Non-imprinted Glucose-imprintedA

dsor

bed

suga

r

(mg

suga

r /g

mat

eria

l) Material Type

Glucose

Xylose

• 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.

Glucose and xylose adsorbed on glucose-imprinted silica particles

Sugar Adsorption from Pure Sugar Solutions

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

Sugar Adsorption from Biomass Hydrolyzates

Page 9: 2013 KIEC - At The Interface Of Advanced Materials And Plant Biotechnology - Barbara Knutson

solubilized

by NMMO (115⁰C, 2 h)

9

Materials Characterization Tools Applied to Cellulose Deconstruction

Cellulose coated

QCM measurement and output

Preparation of cellulose thin films

∆f time →

5 µm 5 µm

Cellulose (Avicel)

5 µm AFM imaging

spin-coat

(4500 rpm, 40s)

cellulose cellulose

QCM-D is an ultra sensitive mass sensor to measure the mass change of thin film

Coated with NMMO-solubilized cellulose

Anchoring polymer

(50 wt% polyethyleneimine )

Immerse

(25⁰C, pH 10, 15 min)

• 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)

Page 10: 2013 KIEC - At The Interface Of Advanced Materials And Plant Biotechnology - Barbara Knutson

Enzymatic hydrolysis of cellulose thin films as measured by QCM

Enzyme injection

1

3

2

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

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

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

Increasing Inhibitor

Enzyme injection

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.

No inhibitor The inhibition of cellulase by cellobiose (a glucose-dimer formed during cellulose hydrolysis) can be quantified using QCM.

Page 11: 2013 KIEC - At The Interface Of Advanced Materials And Plant Biotechnology - Barbara Knutson

E + S ↔ ES → P + E k1

k −1

k2

11

Modeling Cellulose Hydrolysis from QCM Measurements

Substrate (S)

Enzyme (E)

Enzyme-Substrate complex (ES)

Product (P)

Inhibited enzyme (EI)

Inhibited Enzyme-Substrate complex (ESI)

Inhibitor (I)

Inhibitor-substrate complex (SI)

ESIEI + S ↔k3

k −3

Formulate a reaction pathway and species balance in terms of species that contribute to thin film mass and unknown rate constants

Page 12: 2013 KIEC - At The Interface Of Advanced Materials And Plant Biotechnology - Barbara Knutson

Cellulose Hydrolysis Kinetics from QCM Measurements

Enzyme adsorption

QCM Apparatus Sensitivity to: A = ∆f for enzyme binding B = ∆f for substrate lost

Mixed enzyme inhibition scheme Species balance for species that contribute to thin film mass

Relationship between Δf and species that contribute to thin film mass

Hydrolysis

E + S ↔ ES → ES + P − k1

k −1

k2

ESIEI + S ↔k3

k −3

Solve for unknown rate constants by fitting model to Δf data

Analysis of Cellulose Hydrolysis Kinetics using Thin Films • Complements bulk measures of cellulose degradation, which don’t

provide for detailed kinetic analysis of adsorption & hydrolysis • Provides for the testing of mechanistic models • Can be extended to a range of hydrolysis variables, thin film

substrates, and enzyme/enzyme cocktails.

Page 13: 2013 KIEC - At The Interface Of Advanced Materials And Plant Biotechnology - Barbara Knutson

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.

Page 14: 2013 KIEC - At The Interface Of Advanced Materials And Plant Biotechnology - Barbara Knutson

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

Page 15: 2013 KIEC - At The Interface Of Advanced Materials And Plant Biotechnology - Barbara Knutson

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”

Page 16: 2013 KIEC - At The Interface Of Advanced Materials And Plant Biotechnology - Barbara Knutson

Acknowledgements

Financial Support • USDA BRDI • KSEF • NSF

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

Graduate Researchers Helen Li Srivenu Seelam Dan Schlipf Shanshan Zhou Kaitlyn Wooten Undergraduate Researchers Brianna Smith Elliott Rushing Cory Jones