Novel Systems for Bioremediation and Biocatalysis under Extreme Conditions

1. Novel Systems for Bioremediationand Biocatalysis under Extreme Conditions Biocatalysis and Process Design Tonya L. Peeples Chemical and Biochemical Engineering The University of Iowa

2. Evolutionary Connections • Most extremophiles belong to the domain of life known as Archaea. • Studying archaeal macromolecules (DNA, proteins, polyscaccharides, and lipids) and physiology reveal connections to bacteria and higher life forms. • The evolution and engineering of stable biocatalysts is a major area of academic and industrial interest.

3. Research Projects • Expression and Isolation of Thermophilic Enzymes for Interfacial Catalysis • NASA, IEC • Hybrid Thermal/biological Conversion of Lignocellulose to Industrial Chemicals • IEC, USDA-BBC:Iowa State • Isolation and Application of Molecular Chaperones From Thermoacidophilic Archaea for Protein-lipid Stabilization • NSF • Atrazine Degradation in a Pressure Pulsed Membrane Bioreactor for Treatment of Process Waste. • USDA-BBC: Collaboration with Professor Rodgers Needed: 1-2 graduate students for these funded research efforts

4. Saccharolytic enzymes in M. jannaschii Alpha-amylase† Endoglucanase† Gluocoamylase† Maltogenic amylase Neopullulanase †Bult, C. J, White et al. Science 273:1058-1073 Applications of Starch Processing Extremozymes high fructose corn syrup production brewing alcohol production Coupling chemical and biochemical hydrolysis steps - up to 110°C Hydrolases for Interfacial Catalysis

5. Hybrid Thermal/Biological Conversion of Lignocellulose to Industrial Chemicals • Evaluate the depolymerization of corn stover by pyrolysis (anaerobic “cracking”) • Determine the fermentation potential of pyrolysis syrups: extremely acidic and reactive including (phenols, sugars, aliphatic and aromatic hydrocarbons, levoglucosan) • Screen and select extremophilic microbes for direct fermentation of pyrolysis products • Estimate costs of integrated pyrolysis/fermentation/distillation for corn stover-to-lactic acid plant Collaboration with Robert Brown of IA State Mechanical Engineering and Tony Pometto of IA State Food Science and Nutrition

6. Inducible system for enzyme expression, optimal induction time -- 4-6 hours Heat incubation to denature E. coli proteins and activate -amylase protein Separation of denatured proteins from activated proteins intracellular proteins E. coli cells Expression and Isolation of Extremozymes • PCR Amplification of starch processing genes from M. jannaschii • Cloning and expression of sugar modification genes from M. jannaschii in E. coli • Comparison of recombinant activities to native activities

7. Analysis of hydrolysis products • Evaluate product distribution in batch • Gas Chromatography (GC) • High Performance Liquid Chromatography (HPLC) • Thin Layer Chromatography (TLC) • Colorimetric Hydrolysis Assays • Gel electrophoresis of proteins (SDS-PAGE) • Evaluate product yield and significant parameters in continuous operation

8. Biotransformation of Atrazine • Evaluate growth and atrazine degradation capacity of Pseudomonas sp. strain ADP • Determine optimal bioreactor conditions for conversion of process waste water • Validate transmembrane pressure-pulsed bioreactor design

9. The Problem of Atrazine • The most widely used s-traizine herbicide in the United States • Used to control broad-leaf weeds in corn, sorghum, and other crops • Widespread use of atrazine causes concentrations in ground and surface water to frequently exceed 3-ppb, the Environmental Protection Agency’s limit

10. Funding Sources and Industrial Contacts • National Science Foundation • CAREER Award • Genes in the Environment RTG • National Corn Growers Association • National Corn Refiners Association • Iowa Energy Center • USDA-BBC -- Iowa State University • GE Foundation • Industrial Interest (A.E.Staley, Archer Daniels Midland, Grain Processing Corporation, Cargill)