H.K.U.S.T CENG 361 INTRODUCTION TO BIOCHEMICAL ENGINEERING AND BIOPROCESSSING

1. H.K.U.S.TCENG 361 INTRODUCTION TO BIOCHEMICAL ENGINEERING AND BIOPROCESSSING BIOCHEMICAL ENGINEERING PRODUCTS LECTURE SLIDES PRESENTED TO BIEN STUDENTS PREPARED BY C.K. YEUNG

2. References: • Atkinson, B., and M. Ferda, Biochemical Engineering and Biotechnology Handbook, 2nd ed., Stockton Press, New York, NY (1991). • Bailey, J. E., and D. F. Ollis, Biochemical Engineering Fundamentals, 2nd ed., McGraw Hill, New York, NY (1986). • Blanch, H. W., and D. S. Clark, Biochemical Engineering, Marcel Dekker, New York, NY (1996). • Buerk, D. G.,Biosensors: Theory and Applications, Technomic Publishing Company, Inc., Lancaster, PA (1993). • Gebelein, C. G., ed.,Biotechnological Polymers, Technomic Publishing Company, Inc., Lancaster, PA (1993). • Hall, E. A. H.,Biosensors, Prentice Hall, Inc., Englewood Cliffs, NJ (1991). • Harsanyi, G.,Sensors in Biomedical Applications: Fundamentals, Technology and Applications, Chapter 7, Technomic Publishing Company, Inc., Lancaster, PA (2000). • Ouellette, R. P., and P. N. Cheremisinoff, Applications of Biotechnology, Technomic Publishing Company, Inc., Lancaster, PA (1985). • Rho, J. P., and S. G. Louie,Handbook of Pharmaceutical Biotechnology, Haworth Press, Inc., Binghamton, NY (2003).

3. Major classes of bioproducts, such as chemical, biochemical, biopharmaceutical and bio- engineered products will be introduced. The significant impacts of these bioproducts will also be discussed. This course covers the processes and production of certain bioproducts and the methods that can be used for their separation, purification and identification. Some current approaches to the bioproduct productions and applications including recombinant DNA technology, cell/tissue engineering, product forms and bio-devices will also be introduced.

4. Major Classes of Bioproducts (Products derived from bio-sources or used in bio-applications) • Basic Chemicals • Biochemicals • Biopharmaceuticals • Engineered Bioproducts

5. 1. Basic Chemicals Organic Acids (Citric Acid, Lactic Acid) Alcohols (1,3 Propanediol) Amino Acids (Glutamic Acid, Lysine) 2. Biochemicals Enzymes (Proteolytic Enzymes) Surfactants (Lecithin, Esters)

6. 3. Biopharmaceuticals Antibiotics (Penicillin) Monoclonal/Polyclonal Antibodies Hormones (Growth Hormones) Vaccines (Hep B Vaccine) Therapeutic Proteins (tPA) 4. Engineered Products Bio-devices (Bio-devices, Microorganisms DNA microarray chips, tissue/cell based)

7. Major Classes of Bioproducts (Products derived from bio-sources or used in bio-applications) • Basic Chemicals • Biochemicals • Biopharmaceuticals • Engineered Bioproducts

8. 1. Basic Chemicals Organic Acids Citric Acid 2-hydroxy-1,2,3-propane-tricarboxylic or beta-hydroxytricarballylic acid. As part of the tricarboxylic acid (TCA) cycle Citrate synthase catalyses the reaction between acetyl-CoA and oxaloacatate to form citric acid

9. The TCA cycle

10. (Citric Acid) HO2CCH2C(OH)(CO2H)CH2CO2H, an organic carboxylic acid containing three carboxyl groups; Citric acid, anhydrous, crystallizes from hot aqueous solutions as colorless translucent crystals or white crystalline powder. Citric acid is deliquescent in moist air and is optically inactive.

11. (Citric Acid) It is a solid at room temperature, Melts at 153°C, Taste of various fruits in which it occurs, e.g., lemons, limes, oranges, Citric acid loses water at 175 °C to form aconitic acid, HOOCCH=C(COOH) (CH2COOH), which loses carbon dioxide to yield citraconic anhydride

12. (Citric Acid) Itaconic anhydride rearranges to citraconic anhydride(see Fig)or adds water to form itaconic acid , (HOOCCH2 ) (HOOC)C=CH2 Add water to Citraconic anhydride: gives citraconic acid, cis-HOOCCH=C(CH3) (COOH). Evaporation of a citraconic acid solution in the presence of nitric acid yields mesaconic acid, the trans isomer of citraconic acid.

13. +H2O +H2O Itaconic Acid Citraconic Acid

14. Citric Acid – Source and Production Can be extracted from the juice of citrus fruits by adding calcium oxide (lime) to form calcium citrate, Precipitate can be collected by filtration, Citric acid can be recovered from its calcium salt by adding sulfuric acid. It is obtained also by fermentation of glucose with the aid of the mold Aspergillus

15. The most economical method for producing citric acid since the 1930s has been fermentation, which employs a strain of Aspergillus niger to convert sugar to citric acid. Both surface fermentation and submerged fermentation have been used.

16. Citric Acid – Surface Fermentation (1) A. niger is grown on a liquid substrate in pans stacked vertically in a chamber The chamber and pans are sterilised either before or after introduction of the substrate The pans are filled manually or automatically. The chamber is warmed by the introduction of moist, sterile air at a controlled temperature.

17. Citric Acid – Surface Fermentation (2) The liquid and the surface microorganisms are removed manually or automatically from the pans The pans are cleaned before the next batch is introduced. The substrate for the fermentation is a carbohydrate, usually a sugar, such as raw beet, refined beet, or cane sugars, or a syrup.

18. Citric Acid – Surface Fermentation (3) Glucose syrups can be prepared from wheat, corn, potato, or other starch. The sugar content of the syrup can vary from about 10 to 25 wt %. Certain inorganic nutrients, such as (1)ammonium nitrate, (2)potassium phosphate, (3)magnesium sulfate, (4)zinc sulfate, and (5)potassium ferrocyanide, are added.

19. Citric Acid – Surface Fermentation (4) The pH is adjusted to between 3 and 7, depending on the carbohydrate source. Sterilisation may be batchwise or continuous; the latter uses less energy and is usually faster. After sterilisation, the temperature is adjusted as required.

20. Citric Acid – Surface Fermentation (5) The surface of the sterile substrate in the pans is inoculated with A. niger spores, which germinate and cover the surface of the liquid with a matt of mold. After two to three days the surface is completely covered and citric acid production begins, continuing at almost a constant rate until 80 –90 % of the sugar is consumed. Fermentation then continues more slowly for an additional six to ten days.

21. Citric Acid – Surface Fermentation (6) The theoretical yield from 100 kg of sucrose is 123 kg of citric acid monohydrate or 112 kg of anhydrous acid. However, the A. niger uses some sugar for growth and respiration, and the actual yield varies between 57 and 77 % of theoretical value, depending on such factors as: (1) Substrate purity, (2) Particular strain of organism, and (3) Control of fermentation

22. Citric Acid – Submerged Fermentation (1) Submerged fermentation is similar to surface fermentation, but takes place in large fermentation tanks. This method is used more frequently because labour costs are lower with large tanks than with small pans; Equipment costs are also lower.

23. Citric Acid – Submerged Fermentation (2) The fermentation vessel can be short and wide or tall and narrow, and equipped with mixing devices, such as top-entering or side-enteringagitators of the turbine or propeller type. Agitation can be increased by use of a draft tube, a re-circulation loop, or a nozzle through which air and re-circulated substrate is pumped.

24. Citric Acid – Submerged Fermentation (3) Spargers (agitation by means of compressed air) located at the bottom of the vessel or under the stirrer supply air, which may be enriched with oxygen. Oxygen is usually recovered from the exhaust gas. The air is supplied by a compressor and passes through a sterile filter; if necessary, the air is cooled.

25. Citric Acid – Submerged Fermentation (4) Because the process is exothermic, the vessel must be equipped with heat-exchange surfaces, which can be the outside walls or internal coils. Ports are provided for introducing substrate, inoculum, and steam or other sterilising agents; sampling and exhaust ports are also provided.

26. Citric Acid – Submerged Fermentation (5) The substrate is prepared in a separate tank and its pH adjusted; The micronutrients may be added to this tank or directly to the fermenter. The substrate is sterilised by a batchwise or, more commonly, by a continuous operation.

27. Citric Acid – Submerged Fermentation (6) The fermenter is sterilised, charged with substrate, and inoculated. Fermentation requires 3 –14 days. After it is completed, the air supply is stopped to prevent the microorganisms from consuming the citric acid.

28. Citric Acid – Recovery (1) The citric acid broth from the surface or submerged fermentation processes must be purified. First, biological solids usually are removed by filtration using a rotary vacuum filter or the more recent belt-press filter, or by centrifugation. The solids are washed to improve recovery of citric acid.

29. Citric Acid – Recovery (2) The dissolved citric acid must then be separated from residual sugars, proteins generated by the fermentation, and other soluble impurities. This has traditionally been accomplished by precipitation and crystallisation. Addition of lime precipitates calcium citrate, which is filtered and stirred in dilute sulfuric acid to form a precipitate of calcium sulfate; filtration yields a purified citric acid solution

30. Dissolved Citric Acid + Lime  calcium citrate (ppt)  filtered and stirred in dilute sulfuric acid  calcium sulfate (ppt)  filtration  purified citric acid solution

31. Citric Acid – Recovery (3) • Control of pH and temperature in these operations helps to optimise the results. • Citric acid is then crystallised from solution and recrystallised from water; • The mother liquors are recycled to remove accumulated impurities

32. Citric Acid – Its use Can be obtained synthetically from acetone or glycerol. Citric acid is used in soft drinks (45%) and in laxatives and cathartics. Its salts, the citrates, have many uses, e.g., ferric ammonium citrate is used in making blueprint paper. Sour salt, used in cooking, is citric acid

33. Citric Acid • Reference • The Merck Index, 11th ed., Merck & Co., Rahway, N.J. 1989. • R. C. Weast, CRC Handbook of Chemistry and Physics, 69th ed., CRC Press, Boca Raton, Fla., 1988 CRC Handbook of Chemistry and Physics, 1989, p. 163. • A. Seidell, Solubilities of Inorganic and Organic Compounds, 3rd ed., Vol. 2, D. Van Nostrand Co., Inc., New York, 1941, 427–429. • Ethyl Corp., DE-OS 2 240 723, 1972. • M. Rohr, C. P. Kubicek, J. Kominek: “Citric Acid” in H. Dellweg (ed.): Biotechnology Microbiology Products, Biomass, and Primary Products, vol. 3, Verlag Chemie, Weinheim 1983, pp. 456 – 465. • G. T. Austin, Shreve's Chemical Process Industries, 5th ed., McGraw-Hill Book Co., Inc., New York, 1984.

34. Lactic acid It was first discovered in 1780 by the Swedish chemist Scheele. CH3CHOHCO2H, is the most widely occurring hydroxycarboxylic acid A colorless liquid organic acid. Miscible with water or ethanol.

35. (Lactic acid) Lactic acid is a naturally occurring organic acid that can be produced by fermentation or chemical synthesis. Lactic acid is also a principal metabolic intermediate in most living organisms, from anaerobic prokaryotes to humans Anhydrous lactic acid is a white, crystalline solid with a low melting point. Generally, it is available as a dilute or concentrated aqueous solution.

36. Lactic acid – Its use (1) A fermentation product of lactose (milk sugar) Is produced in muscles during intense activity. Calcium lactate, a soluble lactic acid salt, is used as a source of calcium in the die Present in sour milk, yogurt, and cottage cheese(in situ microbial fermentation).

37. Lactic acid – Its use (2) The protein in milk is coagulated (curdled = go bad!) by lactic acid. Lactic acid is produced commercially for use in pharmaceuticals and foods, in leather tanning and textile dyeing, and in making plastics, solvents, inks, and lacquers (paint / natural varnishes a solution of cellulose derivative).

38. Lactic acid – Production (1) Although it can be prepared by chemical synthesis, production of lactic acid by fermentation of glucose and other substances is a less expensive method. Chemically, lactic acid occurs as two optical isomers, a dextro and a levo form; only the levo form takes part in animal metabolism. The lactic acid of commerce is usually an optically inactive racemic mixture of the two isomers.

39. Lactic acid is the simplest hydroxy acid that is optically active. L-Lactic acid (1) occurs naturally in blood and in many fermentation products. The chemically produced lactic acid is a racemic mixture and some fermentations also produce the racemic mixture or an enantiomeric excess of D-lactic acid (2)

40. Lactic acid – Production (2) The commercial process is based on lactonitrile which used to be a by-product of acrylonitrile synthesis. It involves base-catalysed addition of hydrogen cyanide to acetaldehyde to produce lactonitrile This is a liquid-phase reaction and occurs at atmospheric pressures

41. Lactic acid – Production (3) The crude lactonitrile is then recovered and purified by distillation and is hydrolysed to lactic acid using either concentrated hydrochloric or sulphuric acid,producing the corresponding ammonium salt as a by-product. This crude lactic acid is esterified with methanol, producing methyl lactate(see next slide)

42. Lactic acid – Production (4) The latter is recovered and purified by distillation andhydrolysed by water under acid catalysts to produce lactic acid, which is further concentrated, purified, and shipped under different product classifications, and methanol, which is recycled.

43. Lactic acid – Fermentation (1) The existing commercial production processes use homolactic organisms such as Lactobacillus delbrueckii, L. bulgaricus, and L. leichmanii. A wide variety of carbohydrate sources, eg, molasses, corn syrup, whey, dextrose, and cane or beet sugar, can be used. Other complex nutrients required by the organisms are provided by corn steep liquor, yeast extract, soy hydrolysate, etc.

44. Lactic acid – Fermentation (2) Excess calcium carbonate/hydroxide is added to the fermenters to neutralise the acid produced and produce a calcium salt of the acid in the broth. The fermentation is conducted batchwise, taking 4–6 days to complete, and lactate yields of approximately 90wt% from a dextrose equivalent of carbohydrate are obtained.

45. Lactic acid – Fermentation (3) It is usually desired to keep the calcium lactate in solution so that it can be easily separated from the cell biomass and other insolubles, This limits the concentration of carbohydrates that can be fed in the fermentation and the concentration lactate in the fermentation broth, which is usually around 10 wt%.

46. Lactic acid – Fermentation (4) The calcium lactate-containing broth is filtered to remove cells, carbon-treated, evaporated, andacidified with sulfuric acidto convert the salt into lactic acid and insoluble calcium sulfate, which is removed by filtration(See Figure) The filtrate is further purified by carbon columns and ion exchange and evaporated to produce technical- and food-grade lactic acid, but not a heat-stable product, which is required for the stearoyl lactylates, polymers, and other value-added applications.

47. Lactic acid – Fermentation (5)

49. Lactic Acid • References • C. H. Holten, A. Muller, and D. Rehbinder, Lactic Acid, International Research Association, Verlag Chemie, Copenhagen, Denmark, 1971. • S. C. Prescott and C. G. Dunn, Industrial Microbiology, 3rd ed., McGraw-Hill Book Co., Inc., New York, 1959. • R. C. Schulz and J. Schwaab, Makromol. Chem. 87, 90–102 (1965). • Biomass Process Handbook, Technical Insights, Inc., Fort Lee, NJ., 1982, 96–103.

50. Alcohols 1,3 Propanediol (PDO) – Properties 1,3-Propanediol, trimethylene glycol, HOCH2CH2CH2OH, is a clear, colorless, odorless liquid that is miscible with water, alcohols, ethers, and formamide. It is sparingly soluble in benzene and chloroform. The chemical properties of 1,3-propanediol are typical of alcohols.