Methods and approaches

1. The Art and Science of Cellulosic Fermentation Nicolai Panikov

2. Bioreactors

6. Mass-spectrometry

7. Example of gas monitoring with portable mass-spec: fermentation with cell recycle

8. Verification of analytical tools: full recovery of C-balance (chemostat culture of C. thermocellum

9. Conversion of cellulose to products Cellulose  CD + CB  Cells + Enzymes + Fermentation Products

10. Easy case: cellulose degradation is tightly coupled to fermentation & cell growth Cellulose CO 2 Cellulose hydrolysis rate

11. Example of uncoupling: delay in fermentation & growth (not cellulose degradation!) after prolonged cell starvation in batch culture Residual cellulose CO 2 Arrow indicates addition of fresh medium with microcrystalline cellulose

12. Other intermediates and fermentation products during temporary metabolic arrest

15. Simulation Biomass Cellulases Cellobiose and CD (cellodextrins) Residual cellulose Equation Variable

16. Fermentation Pathway 0.5 Glu Pyr Glycerol Lac EtOH Ace Acetyl-CoA Acetald H 2 CO 2 Fd(red) Fd(ox) NAD NADH 2H + ADP ATP SH-CoA NADH NAD SH-CoA NADH NAD NAD NADH NAD NADH ADP ATP SH-CoA 0.85 CD ADP ATP (C 6 H 10 O 5 ) n 6.67" 0.15 Cells ~8%

17. Unexpected results of C. thermocellum : Simulation: Multistability of Steady States Residual cellulose Cell mass

18. C. thermocellum : Residual Solids (Cellulose + Cells) vs Dilution Rate Observation Conventional chemostat model

19. Good agreement between calculation and experimental data