Kilbane 2009 R&D Summary

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Kilbane 2009 R&D Summary

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  • Three main areas of current and past research Biorefining of Petroleum = Upgrading of petroleum quality using microbes Expression systems using unconventional hosts = unconventional meaning not E. coli, Different host are required in applications of biotechnology to the energy industry. MIC = Little is known about the microorganisms that survive in the harsh environment of an internal natural gas pipeline and can be causative agents of corrosion.
  • I gave you an overview of some of the projects that I research and manage. I wanted briefly to describe other interactions at GTI that go into developing these projects and commercialization of any technologies that result.
  • Kilbane 2009 R&D Summary

    1. 1. Energy Biotechnology John J. Kilbane II Illinois Institute of Technology [email_address]
    2. 2. Experience in 3 Start-ups, Contract R&D, and Academia <ul><li>PhD Molecular Biology & Microbiology, Tufts University </li></ul><ul><li>Postdoc with Dr. Chakrabarty, U. Illinois at Chicago </li></ul><ul><li>Petrogen, biosurfactants </li></ul><ul><li>Gas Technology Institute, contract R&D energy industry </li></ul><ul><li>Energy BioSystems, petroleum biodesulfurization </li></ul><ul><li>Qteros (SunEthanol), cellulosic ethanol </li></ul><ul><li>Illinois Institute of Technology </li></ul>
    3. 3. Research Focus <ul><li>Application of biotechnology to various topics in the energy industry. </li></ul>
    4. 4. Biotechnology Applications <ul><li>Biofuels </li></ul><ul><li>Bioprospecting </li></ul><ul><li>Microbiologically Enhanced Oil Recovery </li></ul><ul><li>Biocompetitive Exclusion </li></ul><ul><li>Microbiologically Induced Corrosion </li></ul><ul><li>Biorefining of Petroleum </li></ul><ul><li>Environmental Remediation </li></ul><ul><li>Biosurfactants </li></ul><ul><li>Carbon Dioxide Sequestration </li></ul><ul><li>Recovery of Energy from Mature/Uneconomical Oil and Coal Deposits </li></ul>
    5. 5. Qteros Accomplishments <ul><li>Designed & equipped startup lab </li></ul><ul><li>Microbiological characterization </li></ul><ul><li>Biochemical characterization </li></ul><ul><li>Genetic analyses </li></ul><ul><li>Sales and marketing of R&D </li></ul><ul><li>Developed intellectual property </li></ul><ul><li>Managed external collaborations </li></ul>
    6. 6. Qteros Accomplishments: 3 Funded SBIR Grants <ul><li>DOE Phase II SBIR, Genome Enabled Advancement of Biomass to Biofuel </li></ul><ul><li>DOE Phase I SBIR, Optimizing the Relationship between Next-generation Pretreatment and a Unique Consolidated Bioprocessing Organism through Genomics </li></ul><ul><li>NSF Phase I SBIR, Production of Hydrogen from Lignocellulose using a Unique Consolidated Bioprocessing Organism </li></ul>
    7. 7. Qteros Accomplishments: 5 Invention Disclosures <ul><li>Plasmid vector and method for the genetic modification of Clostridium phytofermentans </li></ul><ul><li>Fermentation of Clostridium phytofermentans to produce ethanol </li></ul><ul><li>A novel process for the production of ethanol and other products from Clostridium phytofermentans </li></ul><ul><li>Method for the conversion of plant materials into fuels and chemicals by sequential action of t wo microorganisms </li></ul>
    8. 8. Qteros Accomplishments: Microbiological Characterization <ul><li>Growth rate, temperature, pH, agitation </li></ul><ul><li>Nutritional requirements/media development (liquid and agar media) </li></ul><ul><li>Ethanol concentration/conversion efficiency </li></ul><ul><li>Conversion rates of lignocellulosic feedstocks </li></ul><ul><li>Ethanol tolerance </li></ul><ul><li>Sporulation, culture preservation, inoculum development. </li></ul>
    9. 9. Qteros Accomplishments: Biochemical Characterization <ul><li>Fermentation product formation (ethanol, acetate, formic, lactic, hydrogen) HPLC and GC </li></ul><ul><li>Cellulase/xylanase assays and indicator media </li></ul><ul><li>Assays with p-nitrophenyl substrates, reducing sugar assays, NREL protocols for biomass characterization </li></ul><ul><li>Protein characterization (SDS and native PAGE), ethanol tolerance, specific activity, binding to substrates </li></ul>
    10. 10. Qteros Accomplishments: Business Development <ul><li>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. </li></ul>
    11. 11. Anaerobic Digestion of Biomass <ul><li>Water hyacinths </li></ul><ul><li>Kelp </li></ul><ul><li>Wastewater treatment plant sludge </li></ul><ul><li>Animal waste </li></ul><ul><li>Recovery of methane from landfills </li></ul>
    12. 12. Anaerobic digestion of water hyacinth and kelp to make methane.
    13. 13. 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.
    14. 14. Petrogen: Biosurfactant Production <ul><li>Commercialization of research from the U. of Illinois. </li></ul><ul><li>Technically successful: converted waste animal and vegetable oils into biosurfactants and cleaned multiple oil storage tanks in Kuwait. </li></ul><ul><li>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). </li></ul>
    15. 15. Petrogen: Biosurfactant Production <ul><li>Commercialization of research from the U. of Illinois. </li></ul><ul><li>Technically successful: converted waste animal and vegetable oils into biosurfactants and cleaned multiple oil storage tanks in Kuwait. </li></ul><ul><li>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). </li></ul>
    16. 16. Microbial Corrosion A faster, more accurate, and less expensive method to detect microorganisms associated with corrosion was developed from concept through commercialization .
    17. 17. Corrosion Damage Costs U. S. Industries Billions Annually <ul><li>Natural Gas Industry Annual Cost (48 states) - $840,000,000 </li></ul>All USA Industries Corrosion <ul><li>Annual Cost (1995 est.) - $300 Billion </li></ul>
    18. 18. Genetic Techniques Were Used to Characterize the Microbial Ecology of Gas Pipelines
    19. 19. A Quantitative PCR Method for Corrosion-Associated Microbes Was Developed <ul><li>“ 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). </li></ul><ul><li>“ 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). </li></ul><ul><li>“ 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). </li></ul>
    20. 20. Quantifying Corrosion-Associated Microbes was Successfully Commercialized <ul><li>Separate cost center within GTI. </li></ul><ul><li>2 full-time employees (1 PhD, 1 BS) plus part time support for administrative personnel. </li></ul><ul><li>Technology validation, patents, QA/QC, marketing, pricing, equipment, personnel, etc. </li></ul><ul><li>Led to unexpected new technology: biodiversity exploration. </li></ul>
    21. 21. New Techniques to Investigate Microbial Diversity Were Developed
    22. 22. Demonstration of Flow Cytometry to Characterize Untreated and Stained Cell Populations
    23. 23. 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 4AF 7.1AF 7.2AF 8AF 9AF 10AF 1 2 3 4 5 6 7 8 9 10 11 1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8
    24. 24. Expertise in Developing Molecular Tools for Unique and Understudied Microorganisms <ul><li>Rhodococcus </li></ul><ul><li>Clostridium </li></ul><ul><li>Methanotrophic organisms </li></ul><ul><li>Thermus </li></ul><ul><li>Little is known about the genetics of the organisms and next to nothing was known about the control of gene expression </li></ul><ul><ul><li>Scientific publications issued or patents issued, filed or disclosed on each of these molecular systems </li></ul></ul>
    25. 25. Biorefining of Petroleum <ul><li>Use biotechnology to remove sulfur and nitrogen from petroleum. </li></ul><ul><li>The selective cleavage of carbon-sulfur or carbon-nitrogen bonds in molecules typically found in petroleum. </li></ul>
    26. 26. Petroleum Upgrading Research Needs <ul><li>Sulfur removal </li></ul><ul><li>Metals removal </li></ul><ul><li>Nitrogen removal </li></ul><ul><li>Viscosity reduction </li></ul><ul><li>Molecular weight reduction </li></ul>
    27. 27. Limitations of Conventional Petroleum Refining Technologies <ul><li>Existing hydrotreatment capacity is insufficient to meet demand. </li></ul><ul><li>An increased consumption of hydrogen in refining competes with the use of hydrogen as a fuel. </li></ul><ul><li>Hydrotreatment and catalytic cracking are energy intensive and produce CO 2. </li></ul><ul><li>The quality of petroleum reserves worldwide is declining. </li></ul>
    28. 28. Development of Genetic Manipulation Tools for Rhodococcus <ul><li>The desulfurization operon is from a relatively uncharacterized eubacterial genus: Rhodococcus </li></ul><ul><li>The construction of genetic tools such as </li></ul><ul><ul><ul><li>Promoter probe vectors </li></ul></ul></ul><ul><ul><ul><li>Plasmid vectors </li></ul></ul></ul><ul><ul><ul><ul><li>cloning and characterization of the dsz genes </li></ul></ul></ul></ul><ul><ul><ul><ul><li>the cloning of alternative regulatory elements </li></ul></ul></ul></ul>
    29. 29. Dsz Pathway Characterization <ul><li>The pathway of dibenzothiophene metabolism was deciphered* </li></ul><ul><li>GC/MS identification of metabolites </li></ul><ul><li>Genomic DNA library construction and dsz negative mutant complementation </li></ul>*= Publication reprints if requested
    30. 30. Purification of DszA and DszC proteins from Rhodococcus using reverse phase and ion exchange chromatography.
    31. 31. Specific Activity Improvements <ul><li>Replacement of native dsz promoter with alternative stronger promoters </li></ul><ul><ul><li>16sRNA promoter </li></ul></ul><ul><ul><li>Inducible promoters </li></ul></ul><ul><ul><li>Screen random chromosomal promoters </li></ul></ul><ul><li>Stabilized by chromosomal insertion of dsz genes </li></ul><ul><li>Use higher copy number plasmid/stabilized plasmid </li></ul><ul><li>Overexpression of dszD (cofactor generation) </li></ul>
    32. 32. Pioneering Research of Gene Expression in Rhodococcus RT-qPCR Northern Blot to Detect mRNA
    33. 33. Robotic platform used in screening of enzymatic mutants.
    34. 34. Typical results of mutant screening using different substrates.
    35. 36. Kinetic analysis of DszA enzymatic activity of wild type and two mutant derivatives.
    36. 37. Energy BioSystems Attempted to Commercialize Biodesulfurization <ul><li>Licensed biodesulfurization patents from GTI. </li></ul><ul><li>Environmental regulations regarding sulfur in diesel/fuels was the key market driver. </li></ul><ul><li>The petroleum industry relies on chemical processes and has no history with biochemical processes. </li></ul><ul><li>Improved hydrodesulfurization catalysts ultimately displaced Energy BioSystems. </li></ul>
    37. 38. Unidentified Host Factors Contribute to the Functioning of the dsz Pathway <ul><li>Temperature range </li></ul><ul><li>Substrate range </li></ul><ul><li>Specific activity </li></ul><ul><li>Biocatalyst life </li></ul><ul><li>Metabolites </li></ul>
    38. 39. Specific desulfurization activity of dsz genes in two species  , M. phlei GTIS10 ;  , R. erythropolis IGTS8.
    39. 40. Thermophiles <ul><li>Longer Shelf Life for Enzymes </li></ul><ul><li>Organic Solvent Resistance </li></ul><ul><li>Compatibility with Biorefining Processes </li></ul><ul><li>Market for Protein Reactions in Viscous or Hydrophobic Environments (i.e. Lipases) </li></ul><ul><li>Potential Protection from Contamination </li></ul><ul><li>Vessel for Directed Evolution (thermostabilization) </li></ul>
    40. 41. Gene Expression in T. thermophilus
    41. 42. Genetic Manipulation of T. thermophilus
    42. 43. GTI Interactions Beyond Scientific Project Management. <ul><li>Business Development – Need $ for development </li></ul><ul><ul><li>Strategic planning and market assessment </li></ul></ul><ul><ul><li>Identify and recruit teaming partners for collaborative proposals </li></ul></ul><ul><li>Project Management </li></ul><ul><ul><li>On-time, on-budget, quality </li></ul></ul><ul><li>Contracts </li></ul><ul><ul><li>Proposal costs and requirements </li></ul></ul><ul><li>Legal </li></ul><ul><ul><li>Patentability and ownership </li></ul></ul><ul><li>Licensing and Commercialization </li></ul><ul><ul><li>Potential market and size </li></ul></ul><ul><ul><li>Locate buyer and license technology </li></ul></ul>
    43. 44. Other Research Topics <ul><li>Treatment and Prevention of Acid Mine Drainage </li></ul><ul><li>Biodetoxification of Chemical Warfare Agents </li></ul><ul><li>Development of Biosorbents to Remove and Recover Metal Ions from Wastewater </li></ul><ul><li>Production and Use of Biosurfactants </li></ul><ul><li>Genetic Manipulation of Methanotrophs </li></ul><ul><li>Methanogenesis (Anaerobic Digestion of Organic Waste) </li></ul><ul><li>Bioprocesses to Produce Fine Chemicals </li></ul><ul><li>Biodegradation of Chlorinated Compounds </li></ul><ul><li>Development of Electroporation Method for Intra- and Inter-Species Gene Transfer </li></ul><ul><li>Characterization of Bacteriophages of P. aeruginosa </li></ul>
    44. 45. Thanks! John Kilbane Illinois Institute of Technology [email_address]

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