1. Wastewater Treatment Facilities: Biorefinery for Biofuels T. French, R. Hernandez, A. Mondala, J. Hall, G. Zhang, M. White, E. Alley, B. Holmes Dave C. Swalm School of Chemical Engineering Mississippi State University

2. Objectives Transform the daily disposal of industrial and domestic sewage into large quantities of lipids using microorganisms Integrate the lipid, sugar, and thermochemical platforms for producing JP-Fuels, gasoline, biodiesel and chemicals web.mit.edu

3. Evaluation of BioDiesel Production Costs Soybean Oil $4.38/gal feedstock cost $0.35/gal processing cost $0.10/gal transportation cost = $4.83/gal total cost Note: Feedstock represent ~90% of total processing costs

4. Sustainability and Logistical Barriers to Biofuels The US Consumes approximately 300,000,000,000 gal/yrThe US Consumes approximately 300,000,000,000 gal/yr –165,000,000,000 gal/yr gasoline – 75,000,000,000 gal/yr diesel Ethanol would account for 2%Ethanol would account for 2% –Must be transported via tanker –Lower energy density than gas –Requires a lot of water –Where will all the steel come from to build Fermentors Soy Bean oil accounts for 0.33%Soy Bean oil accounts for 0.33% When populations need that for food fuel will loseWhen populations need that for food fuel will lose Not Sustainable.Not Sustainable. Single-cell oil produced in a wastewater treatment facility will not have a food marketSingle-cell oil produced in a wastewater treatment facility will not have a food market Wastewater treatment will always occur and scales with population.Wastewater treatment will always occur and scales with population.

5. COMPARISON OF OIL PRODUCTION FROM FEEDSTOCKS Sample% OilTime (D) Soybean19105 Canola33255 Camelina36255 Turnip36255 Flax39240 Tallow46365 Castor55195 H. matrinalis232.5 R. glutinis16-602.5 R. opacus60-802.0

6. WASTEWATER AS CARBON SOURCE Wastewater contains: –Carbon –Nitrogen –Phosphorous –Water –Micronutrients Approximately 10 million gallons* of wastewater are produced daily in Starkville –Residential waste –Industrial waste

7. 35 Billion Gallons of Municipal Wastewater Generated Daily

8. Forced Main Primary Clarification Air Aeration Tank Finished Effluent to Discharge Waste Sludge Biogas Anaerobic Digestor Grit Dewatering & Thickening Influent blending Grit Removal Headworks Influent Secondary Clarification Recycled Biomass Solids Effluent Wastewater Primary Solids Thickening Dewatering Biosolids Thickened Waste Sludge Primary Sludge Influent

9. COMPARISON OF GROWTH CURVES Oleaginous Yeast Grown on Wastewater with 0.1g/L of Dextrose 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 020406080 Time (hr) Optical Density (600nm) R. glutinis C. curvatus Pos. Control: R. glutinis Pos. Control: C. curvatus Negative control: wastewater Oleaginous Yeast Grown on Wastewater with 1g/L of Dextrose 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 020406080 time (hr) optical density (600nm) R. glutinis C. curvatus Pos. control: R. glutinis Pos. control: C. curvatus Negative control: wastewater

10. CHEMICAL OXYGEN DEMAND 10% Oil Content

11. Carbon Sources Supporting Growth of Oleaginous Microorganisms Sugars –Glucose –Xylose –Ribose –N-acetyl glucosamine Glycerol Flour

12. Lignocellulosic Biomass switchgrass Composition

13. Conversion of Lignocellulosic Biomass to Microbial Oil Sugar Cell mass concentrationTotal Lipid production. in culture g/L% dry wt. Xylose1530 Dextrose2050 Artificial acid hydrolysate1635 Switch Grass Hydrolysates2.6326

14. Conversion of 30 g/L Acid Hydrolysate by Oleaginous Microbes

15. Conversion of 45 g/L Acid Hydrolysate by Oleaginous Microbes

16. Fatty Acid Composition Caproic (6:0) 0.0 0.00.00.00.00.0 Myristic (14:0) 0.00.00.03.00.0 Palmitic (16:0) 14.02.24.023.39.9 Stearic (18:0) 7.0 7.00.92.417.93.6 Oleic (18:1) 32.012.665.038.019.1 Linoleic (18:2) 20.012.117.30.055.6 Linolenic (18:3) 3.08.07.80.010.2 Eicosatrienoic (20:3) 8.07.41.30.00.2 Behenic (22:0) 0.00.70.40.00.3 Erucic (22:1) 0.049.90.10.00.0 YeastRapeCanolaTallowSoy Fatty Acid

17. Envisioned Process: The New Biorefineries Lignocellulose 3 B G/Yr

18. Example Biorefinery Locations

19. ACKNOWLEDGEMENTS Department of Energy Environmental Protection Agency MS/AL Sea Grant 23 students

20. Thank You