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KIEC 08.29.2013, session two, 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
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
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
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
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.
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
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
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
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)
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.
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
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.
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
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