1. Selected Products of Bioprocessing digging a bit deeper

2. Citric acid 1923, first commercial production by fermentation today, 99% produced via fermentation Mexico and S. America extract from lemons (7-9%) 250,000 Tons/year in 220 m 3 fermentors (about 6 m cube)

3. Producers USA: 180,000 Tons/yr Pfizer Miles Lab Inc. Sturge Ltd of Birmingham (UK)

4. Citric acid applications 60% used in food and beverage: fruit juice, candy, ice cream, marmalade enhances and preserves flavors 10% in pharmaceutical manufacture: blood preservative, iron citrate (Fe supplement), cosmetics 25% in chemical industry: antifoam, softener, textiles, metalurgical (chelator), replacement of polyphosphates in detergents

5. Citric acid biochemistry structure produced by fermentation of carbohydrates

6. glucose to pyruvate

7. Citric acid cycle key to citrate overproduction

8. Microbial culture Aspergillus niger (fungus)

9. Source of glucose? beet molasses potato starch (fungi produce amylases) sucrose

10. Types of culture stationary or surface culture submerged culture (most common) solid state, continuous, multi- stage culture

11. Surface culture 8-12 days, but mycelium can be reused

12. Submerged process 3-5 days

13. Solid state bit of a mess! porous solid materials (sugarcane bagasse, potato or beet pulp... mixed with medium and inoculated 6-7 days fermentation, then extracted with water

14. Culture conditions high initial sugar concentration important: 15-18% (A. niger likes it like that!) growth phase, followed by production phase pH from 1.4 to 3, to suppress contaminants and suppress oxalic acid production (by-product)

15. More tedious conditions? inoculum is spores or mycelium older spores (>8 days) don’t germinate very well oxygen demand often larger than supply sometimes oxygen enriched air used but $$ly if O 2 limiting, bugs can metabolize citric acid (not helpful) agitation gets the acid away from the mycelium (inhibition) T opt = 25-30 o C 7-10 days for surface, and 4-5 days for submerged cultures

17. Any genetic improvements? few mutation and selection of spores (shotgun approach) difficult as no efficient screening protocol for citrate overproducers