kilbane 2009 r&d summary

Energy Energy BiotechnologyBiotechnology

John J. Kilbane IIIllinois Institute of Technologykilbane@iit.edu

Experience in 3 Start-ups, Contract Experience in 3 Start-ups, Contract R&D, and AcademiaR&D, and Academia PhD Molecular Biology & Microbiology, Tufts

University Postdoc with Dr. Chakrabarty, U. Illinois at Chicago Petrogen, biosurfactants Gas Technology Institute, contract R&D energy

industry Energy BioSystems, petroleum biodesulfurization Qteros (SunEthanol), cellulosic ethanol Illinois Institute of Technology

Research FocusApplication of biotechnology to

various topics in the energy industry.

Biotechnology ApplicationsBiotechnology Applications

•Biofuels•Bioprospecting•Microbiologically Enhanced Oil Recovery•Biocompetitive Exclusion•Microbiologically Induced Corrosion•Biorefining of Petroleum•Environmental Remediation•Biosurfactants•Carbon Dioxide Sequestration•Recovery of Energy from Mature/Uneconomical Oil and Coal Deposits

Qteros Accomplishments

Designed & equipped startup lab Microbiological characterization Biochemical characterization Genetic analyses Sales and marketing of R&D Developed intellectual property Managed external collaborations

Qteros Accomplishments: 3 Funded SBIR Grants DOE Phase II SBIR, Genome Enabled

Advancement of Biomass to Biofuel DOE Phase I SBIR, Optimizing the

Relationship between Next-generation Pretreatment and a Unique Consolidated Bioprocessing Organism through Genomics

NSF Phase I SBIR, Production of Hydrogen from Lignocellulose using a Unique Consolidated Bioprocessing Organism

Qteros Accomplishments: 5 Invention Disclosures Plasmid vector and method for the genetic

modification of Clostridium phytofermentans Fermentation of Clostridium phytofermentans to

produce ethanol A novel process for the production of ethanol and

other products from Clostridium phytofermentans Method for the conversion of plant materials into

fuels and chemicals by sequential action of two microorganisms

Qteros Accomplishments: Microbiological Characterization Growth rate, temperature, pH, agitation Nutritional requirements/media development

(liquid and agar media) Ethanol concentration/conversion efficiency Conversion rates of lignocellulosic feedstocks Ethanol tolerance Sporulation, culture preservation, inoculum

development.

Qteros Accomplishments: Biochemical Characterization Fermentation product formation (ethanol, acetate,

formic, lactic, hydrogen) HPLC and GC Cellulase/xylanase assays and indicator media Assays with p-nitrophenyl substrates, reducing

sugar assays, NREL protocols for biomass characterization

Protein characterization (SDS and native PAGE), ethanol tolerance, specific activity, binding to substrates

Qteros Accomplishments: Business Development

USDA National Center for Agricultural Utilization Research, EPA Office of Research and Development,

US Department of Energy Office of the Biomass Program, Natural Resources Defense Council, Union of Concerned Scientists, Energy Future Coalition, United Nations Foundation, Committee on Energy and Natural

Resources US Senate, Senator Joseph I. Lieberman, Rep. Bart Gordon, Congressman Henry A. Waxman,

Christopher J. King (Professional Energy Staff, Committee on Science, US House of Representatives),

Congresswoman Rosa DeLauro, and Sen. Christopher J. Dodd.

Anaerobic Digestion of Biomass

Water hyacinths Kelp Wastewater treatment plant sludge Animal waste Recovery of methane from landfills

Anaerobic digestion of water hyacinth and kelp to make methane.

GTI pioneered the development of two-phase anaerobic digestion of biomass to make methane. The reactor pictured here is at the Woodridge, IL wastewater treatment plant.

Petrogen: Biosurfactant Production Commercialization of research from the U. of Illinois. • Technically successful: converted waste animal and

vegetable oils into biosurfactants and cleaned multiple oil storage tanks in Kuwait.

• Commercially unsuccessful: Inadequate business expertise, particularly in market analysis (chemical surfactants are commodity chemicals, targeting a specialty application like cleaning contact lenses might have been successful).

Petrogen: Biosurfactant Production

• Commercialization of research from the U. of Illinois.• Technically successful: converted waste animal and vegetable oils into biosurfactants and cleaned multiple oil storage tanks in Kuwait.•Commercially unsuccessful: Inadequate business expertise, particularly in market analysis (chemical surfactants are commodity chemicals, targeting a specialty application like cleaning contact lenses might have been successful).

Microbial CorrosionMicrobial Corrosion

A faster, more accurate, and less expensive method to detect microorganisms associated with corrosion was developed from concept through commercialization.

Corrosion Damage Costs U. S. Corrosion Damage Costs U. S. Industries Billions AnnuallyIndustries Billions Annually

• Natural Gas Industry Annual Cost (48 states) - $840,000,000

All USA Industries CorrosionAll USA Industries Corrosion

• Annual Cost (1995 est.) - $300 Billion

Genetic Techniques Were Used to Genetic Techniques Were Used to Characterize the Microbial Ecology Characterize the Microbial Ecology

of Gas Pipelinesof Gas Pipelines

A Quantitative PCR Method for A Quantitative PCR Method for Corrosion-Associated Microbes Was Corrosion-Associated Microbes Was

DevelopedDeveloped “Quantifying the Contribution of Various Bacterial Groups

to Microbiologically Influenced Corrosion,” Kilbane II, J. J., B. Bogan, and B. Lamb, Corrosion2005, Paper 05491, NACE International, Houston, TX pp. 1-9 (2005).

“Faster and More Accurate Data Collection for Microbiologically Influenced Corrosion,” Zhu, X. Y., and J. J. Kilbane II, Society of Petroleum Engineers SPE-93089-PP, pp. 1-9 (2005).

“Improved Method for Monitoring Microbial Communities in Gas Pipelines,” X. Zhu, J. Lubeck, K. Lowe, A. Daram, and J. J. Kilbane II, Corrosion2004 Paper No. 04592, NACE Houston, TX, pp. 1-13 (2004).

Quantifying Corrosion-Associated Microbes was Successfully Commercialized Separate cost center within GTI. 2 full-time employees (1 PhD, 1 BS) plus part

time support for administrative personnel. Technology validation, patents, QA/QC,

marketing, pricing, equipment, personnel, etc. Led to unexpected new technology:

biodiversity exploration.

New Techniques to Investigate New Techniques to Investigate Microbial Diversity Were DevelopedMicrobial Diversity Were Developed

Demonstration of Flow Cytometry to Characterize Untreated and Stained Cell Populations

Analysis of 16S RNA Genes by DGGE Reveals that FACS Allows the Fractionation of Complex Microbial Communities and the Cultivation of Species not Detectable Using an Unfractionated Inoculum

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Expertise in Developing Molecular Tools for Expertise in Developing Molecular Tools for Unique and Understudied MicroorganismsUnique and Understudied Microorganisms

1. Rhodococcus

2. Clostridium

3. Methanotrophic organisms

4. Thermus

Little is known about the genetics of the organisms and next to nothing was known about the control of gene expression

– Scientific publications issued or patents issued, filed Scientific publications issued or patents issued, filed or disclosed on each of these molecular systems or disclosed on each of these molecular systems

Biorefining of Petroleum

Use biotechnology to remove sulfur and nitrogen from petroleum.

The selective cleavage of carbon-sulfur or carbon-nitrogen bonds in molecules typically found in petroleum.

Petroleum Upgrading Research Needs Sulfur removal Metals removal Nitrogen removal Viscosity reduction Molecular weight reduction

Limitations of Conventional Petroleum Refining Technologies Existing hydrotreatment capacity is

insufficient to meet demand. An increased consumption of hydrogen in

refining competes with the use of hydrogen as a fuel.

Hydrotreatment and catalytic cracking are energy intensive and produce CO2.

The quality of petroleum reserves worldwide is declining.

Development of Genetic Manipulation Tools Development of Genetic Manipulation Tools for for RhodococcusRhodococcus

The desulfurization operon is from a relatively uncharacterized eubacterial genus: Rhodococcus

The construction of genetic tools such as• Promoter probe vectors• Plasmid vectors

– cloning and characterization of the dsz genes

– the cloning of alternative regulatory elements

Dsz Pathway Dsz Pathway CharacterizationCharacterization

The pathway of dibenzothiophene metabolism was deciphered*

•GC/MS identification of metabolites

•Genomic DNA library construction and dsz negative mutant complementation

*= Publication reprints if requested

Purification of DszA and DszC proteins from Rhodococcus using reverse phase and ion exchange chromatography.

Specific Activity ImprovementsSpecific Activity Improvements

Replacement of native dsz promoter with alternative stronger promoters – 16sRNA promoter

– Inducible promoters

– Screen random chromosomal promoters

Stabilized by chromosomal insertion of dsz genes Use higher copy number plasmid/stabilized

plasmid Overexpression of dszD (cofactor generation)

Pioneering Research of Gene Pioneering Research of Gene Expression in Expression in RhodococcusRhodococcus

RT-qPCR

Northern Blot to Detect mRNA

Robotic platform used in screening of enzymatic mutants.

Typical results of mutant screening using different substrates.

Kinetic analysis of DszA enzymatic activity of wild type and two mutant derivatives.

Energy BioSystems Attempted to Commercialize Biodesulfurization Licensed biodesulfurization patents from

GTI. Environmental regulations regarding sulfur in

diesel/fuels was the key market driver. The petroleum industry relies on chemical

processes and has no history with biochemical processes.

Improved hydrodesulfurization catalysts ultimately displaced Energy BioSystems.

Unidentified Host Factors Contribute to the Functioning of the dsz Pathway

Temperature range Substrate range Specific activity Biocatalyst life Metabolites

Specific desulfurization activity Specific desulfurization activity of of dszdsz genes in two species genes in two species , , M. phlei M. phlei GTIS10 ; GTIS10 ; , , R. erythropolis R. erythropolis IGTS8. IGTS8.

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20 30 40 50 60 70 80

Temperature (°C)

ThermophilesThermophiles Longer Shelf Life for Enzymes Organic Solvent Resistance Compatibility with Biorefining Processes Market for Protein Reactions in Viscous or

Hydrophobic Environments (i.e. Lipases) Potential Protection from Contamination Vessel for Directed Evolution

(thermostabilization)

Gene Expression in Gene Expression in T. thermophilusT. thermophilus

Genetic Manipulation of Genetic Manipulation of T. thermophilusT. thermophilus

GTI Interactions Beyond Scientific Project Management.

Business Development –Need $ for development– Strategic planning and market assessment– Identify and recruit teaming partners for collaborative proposals

Project Management– On-time, on-budget, quality

Contracts– Proposal costs and requirements

Legal– Patentability and ownership

Licensing and Commercialization– Potential market and size– Locate buyer and license technology

Other Research TopicsOther Research Topics

Treatment and Prevention of Acid Mine Drainage Biodetoxification of Chemical Warfare Agents Development of Biosorbents to Remove and Recover

Metal Ions from Wastewater Production and Use of Biosurfactants Genetic Manipulation of Methanotrophs Methanogenesis (Anaerobic Digestion of Organic Waste) Bioprocesses to Produce Fine Chemicals Biodegradation of Chlorinated Compounds Development of Electroporation Method for Intra- and

Inter-Species Gene Transfer Characterization of Bacteriophages of P. aeruginosa

Thanks!John Kilbane

Illinois Institute of Technology

kilbane@iit.edu