# Pharmaceuticals and fermentation: from molecule to medicine: Fermentation: microbes as chemical plants Source page: https://www.materialinsights.org/learn/chemicals/pharmaceuticals-and-fermentation/p4/ [Understanding Chemicals](https://www.materialinsights.org/learn/chemicals/) / [Module 10: Pharmaceuticals and fermentation: from molecule to medicine](https://www.materialinsights.org/learn/chemicals/pharmaceuticals-and-fermentation/) Part 4 of 5 ## Fermentation: microbes as chemical plants In fermentation a chosen microorganism does the synthesis, turning sugar into a product in water at near room temperature. The chemistry is free; the engineering is not. Air must be supplied, heat removed, contamination kept out, and a dilute product recovered from a thick broth. The same equipment makes penicillin worth tens of dollars a kilogram and ethanol or citric acid worth one or two. ### 4.1 How an industrial fermentation works Shreve and Dryden give the same list of what a fermentation needs: an organism that makes the desired product reliably, cheap raw materials, acceptable yields, a fast fermentation, and a product that is easy to recover. The work divides into stages. The strain comes first. Specialists select and mutate organisms for the highest yield and fewest by-products. A stock culture is grown up through flasks and seed tanks to an inoculum, typically a few per cent of the main fermenter volume. The medium, or broth, supplies carbon (sugar, molasses, starch), nitrogen (corn steep liquor, ammonium salts, urea) and minerals. It is sterilised by heat, usually steam at about 115 to 120°C, and cooled. Air is sterilised too; Dryden describes compressing it adiabatically to 200°C and filtering out dust and oil. Most industrial fermentations are aerobic, so the fermenter is a gas-liquid contactor. Kent gives airflows of 0.1 to more than 1 volume of air per volume of broth per minute (vvm) and mixing power of under 0.5 to over 5 kW/m3. Dryden notes that foaming worsens as tanks grow, so height-to-diameter ratios end up between 2:1 and 3:1. Heat removal is often the real limit on scale. Kent puts the metabolic heat of a vigorous aerobic fermentation above 35 kW/m3, much more than the stirring power, and hard to remove through coils at 37°C with cooling water at 18°C. Fermenters run batch, fed-batch (nutrients added during the run) or continuous. Kent and Dryden agree that batch and fed-batch dominate for antibiotics, organic acids, amino acids and enzymes, because long runs risk contamination and takeover by faster-growing, less productive mutants. Data Fermenter design numbers from the sources | Parameter | Value | Source | |---|---|---| | Aeration rate | 0.1 to over 1 vvm | Kent Ch 30 | | Mechanical power | Under 0.5 to over 5 kW/m3 | Kent Ch 30 | | Oxygen demand | 25 mmol/L/h (slow growers) to about ten times that | Kent Ch 30 | | Metabolic heat at 250 mmol O2/L/h | Over 35 kW/m3 | Kent Ch 30 | | Medium sterilisation | Steam, 115-120°C | Dryden; Shreve | | Air sterilisation | Adiabatic compression to 200°C plus fibrous filters | Dryden | | Height to diameter | 2:1 to 3:1 | Dryden | | Fermenter size (penicillin, India) | 100-200 m3, pressurised to 1 atm gauge | Dryden | | Large fermenters (fine chemicals) | Over 250 m3 | Pollak | Source: Kent (2012), Ch 30 Industrial Biotechnology: Discovery to Delivery, pdf pp. 1141-1142 Worked example #### Why recovery costs more than growing Kent gives two striking ratios. In an antibiotic plant the investment in recovery facilities is claimed to be about four times that in the fermenters and their auxiliaries. In organic and amino acid plants, up to 60% of fixed costs are for recovery. The reason is dilution: the product is a few per cent, or less, of a viscous broth full of cells and medium residues, and every kilogram of water must be pumped, filtered and often evaporated away. - Shreve (1984), Ch 31 Fermentation Industries · pdf pp. 598-599 - Dryden (1997), IIID Carbohydrates and Fermentation Industries · pdf pp. 337-339 - Kent (2012), Ch 30 Industrial Biotechnology: Discovery to Delivery · pdf pp. 1139-1143 - Pollak (2011), Fine Chemicals · pdf p. 65 ### 4.2 Penicillin: from mould to crystals Penicillin is made by the mould Penicillium chrysogenum in deep, aerated tanks. To steer the mould towards penicillin G, the plant feeds a precursor, phenylacetic acid or a derivative, which the mould builds into the side chain of the molecule. Feeding phenoxyacetic acid instead gives penicillin V. Kent's typical medium contains, per litre, 30 g corn steep liquor, 30 g lactose, 5 g glucose, 3 g sodium nitrate, 3 g calcium carbonate, trace salts and 0.05 g phenyl acetamide as precursor. It is sterilised, cooled to 24°C and seeded from a 24-hour seed culture. Sterile air is sparged at about 1 vvm, and production runs 60 to 200 hours. Shreve gives 100 to 150 h at 25 to 27°C and pH kept below about 6.5 to 7. Recovery exploits the fact that penicillin is an acid. At low pH it is uncharged and moves into an organic solvent; at neutral pH it is a salt and moves back into water. The mycelium is filtered off on rotary vacuum filters, the filtrate is chilled (Dryden says to 1 to 2°C, since penicillin is unstable) and acidified to pH 2 to 2.5, and the penicillin is extracted into amyl acetate or methyl isobutyl ketone in a Podbielniak centrifugal contactor. It is then back-extracted into a buffer at pH 6.5 to 7.5, with about 90% recovery at this step. Shreve notes that purity rises from 5 to 10% in the broth to 75 to 80% after the first extraction. Further purification and sterile filtration give the final salt. Shreve's typical 45,000 L batch yields about 300 kg of procaine penicillin G, or about 6.7 g per litre of fermenter, consistent with his broth concentration of 5 to 10 mg/mL. Kent's later figure of 20 to 30 g/L shows how much strain improvement has added since. Process flowchart Penicillin G by fermentation (typical 45,000 L batch, Shreve 1984) Inputs **Water**37,850 L **Sugar**3,990 kg **Corn-steep liquor**500 kg **Precursor (phenylacetic acid)**500 kg **Minerals**50 kg **Sterile air**about 1 vvm (Kent) **Filter aid**1,800 kg **Amyl acetate (solvent)**3,785 L **Procaine hydrochloride**150 kg **Sulphuric or phosphoric acid, buffers, activated carbon**for pH swing and decolourising Process steps 1 **Culture build-up** Slant to flasks to seed tanks; Penicillium chrysogenum 2 **Medium sterilisation** Steam at 120°C; cool 3 **Fermentation** Aerated, stirred; 25-27°C; pH preferably below 6.5 and not above 7; 100-150 h 4 **Mycelium removal** Rotary vacuum filter; broth at 5-10 mg/mL penicillin, 5-10% pure 5 **Acid extraction** Chill; acidify to pH 2.0; extract into amyl acetate (1:10 volume) in Podbielniak contactor; purity 75-80% 6 **Back-extraction** Into water at pH 7.5; carbon decolourise; centrifuge off slime 7 **Crystallisation** Intermediate crystals; mother liquor re-extracted 8 **Sterile finishing** Seitz filtration; precipitate with procaine HCl or as K/Na salt; dry under sterile conditions Outputs **Procaine benzylpenicillin (procaine penicillin G)**about 300 kg per batch **Mycelium**dried and sold as fertiliser **Spent solvent**to solvent recovery Batch quantities read from the page image of Fig. 40.5. Yield works out to about 6.7 g product per litre and about 13 kg sugar per kg product (course arithmetic). Dryden's older basis per kg of penicillin II (G): 135 kg broth (dry basis), 100 kg lactose, 35 kg CaCO3, 3 t process water and 7,000 Nm3 air, in plants of 10-50 kg/day. Source: Shreve (1984), Ch 40 Pharmaceutical Industry, Shreve pdf pp. 836-840 (Fig. 40.5); Dryden pdf p. 576 Case #### The price of penicillin Shreve records that penicillin fell from $25,000 per gram at first to $35/kg for procaine penicillin, with world production over 2,950 t in 1981. Kent cites a million-unit vial at $200 wholesale in 1943. Dryden, writing for India, notes that almost all antibiotics except chloramphenicol can be made in penicillin fermentation equipment, so investment in it was considered safe. Hindustan Antibiotics, set up with UNICEF and WHO help, made most of India's penicillin; Indian demand in 1993-94 was estimated at 330 t. - Shreve (1984), Ch 40 Pharmaceutical Industry · pdf pp. 836-840 - Kent (2012), Ch 30 Industrial Biotechnology: Discovery to Delivery · pdf pp. 1178-1179 - Dryden (1997), IVH Pharmaceutical Industry · pdf pp. 567-568, 574-577 ### 4.3 Bulk fermentation: ethanol from molasses Ethanol is the largest fermentation product and shows the economics of a cheap one. Yeast, Saccharomyces cerevisiae, converts sugar to ethanol and carbon dioxide without air: C6H12O6 → 2 C2H5OH + 2 CO2. By weight that is 51% ethanol on glucose; Kent notes that because some sugar feeds yeast growth, real yields are 42 to 46%. Dryden's Indian process starts from cane molasses, the syrup left after sugar crystallisation, with 50 to 55% sugar. It is diluted to 10 to 15% sugar and brought to pH 4 to 5, which favours yeast over bacteria. Nutrients such as ammonium sulphate are added if lacking. Yeast grown on sterile mash is added, and fermentation runs 30 to 70 hours at 20 to 30°C, rising to 35°C at the end, with cooling coils removing heat. The product, called beer, is only 8 to 10% ethanol, so most of the energy goes into distillation. A beer still takes off 50 to 60% alcohol; an aldehyde column removes light impurities; a rectifying column brings the alcohol to 95%, the azeotrope, a mixture that boils without changing composition. Anhydrous fuel ethanol needs a further step: azeotropic distillation with benzene or extractive distillation with ethylene glycol. The still bottoms, slops, become cattle feed or a high-strength effluent. Shreve quantifies the energy. Conventional corn plants used 13 kg of steam per litre of anhydrous alcohol, newer designs claimed 2 kg. Some processes consumed up to 42 MJ per litre, against 23 MJ released by burning it. Kent adds that continuous fermentation and yeast recycle cut capital but barely move product cost, because raw materials dominate. The "Bioethanol" tile is this process at fuel scale. Process flowchart Ethyl alcohol from cane molasses (Dryden 1997) Inputs **Molasses (50-55% total sugar)**5.6 t per t ethanol (as printed) **Sulphuric acid (60° Be)**27 kg **Ammonium sulphate**2.5 kg **Coal (for steam)**0.7-1.5 t **Process water**12 t **Cooling water**50 t **Electricity**35 kWh Process steps 1 **Mash preparation** Dilute to 10-15% sugar; pH 4-5; add nutrients 2 **Yeast propagation** Inoculate sterile mash; yeast supplies invertase and zymase 3 **Fermentation** 20-30°C rising to 35°C; 30-70 h; cooling coils; beer at 8-10% ethanol 4 **Beer still** Strips 50-60% alcohol overhead; slops at bottom 5 **Aldehyde still** Removes low-boiling impurities 6 **Rectifying column** 95% ethanol (azeotrope) side-draw; fusel oil side-streams 7 **Dehydration (if anhydrous)** Azeotropic distillation with benzene or extractive distillation with ethylene glycol Outputs **Ethanol (100% basis)**1 t (1.26 kL) **Carbon dioxide**0.76 t **Fusel oil (higher alcohols)**small **Cattle feed or fertiliser from slops**0.20-0.60 t Dryden states a basis of 90% yield on sugar, but 5.6 t of 50-55% molasses implies roughly 64-70% (course arithmetic, 0.511 t ethanol per t sugar). Typical plants: 10-100 t/day. Source: Dryden (1997), IIID Carbohydrates and Fermentation Industries, pdf pp. 341-343 (Fig. IIID-4) Case #### Why India's molasses alcohol lost to petrochemicals Dryden records that fermentation alcohol from molasses met most of India's needs until the late 1960s, and in 1963 the synthetic rubber plant at Bareilly used nearly all the alcohol in Uttar Pradesh. Then petrochemical ethanol arrived. Small distilleries scattered through cane-growing areas could not match a large plant in a petrochemical complex. By 1990-91 India had about 127 distilleries with 744 million litres of capacity, and demand for industrial alcohol was projected at 1.5 million kL by 1994-95. The lesson: feedstock location fixes plant size, and plant size fixes cost. - Dryden (1997), IIID Carbohydrates and Fermentation Industries · pdf pp. 341-344 - Kent (2012), Ch 30 Industrial Biotechnology: Discovery to Delivery · pdf p. 1166 - Shreve (1984), Ch 31 Fermentation Industries · pdf pp. 601-604, 608 ### 4.4 Citric acid and lysine: an organic acid and an amino acid Citric acid and lysine are bulk fermentation products sold on price, and they show how strain, aeration and recovery chemistry set cost. "Citric acid" is made by the mould Aspergillus niger. Kent explains that one mole of glucose gives one mole of citric acid with no net oxygen consumed, so the theoretical yield is 112% by weight on sucrose; growth is minimal during production. In the modern submerged process the medium holds about 200 g/L sucrose with carefully balanced iron, zinc, copper, magnesium, manganese and phosphate; the medium recipe is the single most critical factor. Air is bubbled through at 0.5 to 1.5 vvm, without mechanical stirring, at 25 to 27°C for 6 to 9 days. The mould is coaxed into pellets 1 to 2 mm across, which keeps the broth thin and eases oxygen transfer and filtration. Recovery uses lime and sulphuric acid. The mycelium is filtered off. Heating with a little lime first drops out oxalic acid, a by-product. More lime at about 95°C precipitates calcium citrate, which is filtered and treated with dilute sulphuric acid, freeing citric acid and leaving gypsum. Carbon treatment, ion exchange, vacuum evaporation and crystallisation give USP-grade monohydrate. World output is about one million tonnes a year, and Bharat Starch Industries of India is among the major producers Kent names. Lysine, in the "Amino acids (lysine, methionine)" tile, is made by mutants of Corynebacterium glutamicum or Brevibacterium flavum, at 28 to 33°C and pH 6 to 8 with high aeration. Biotin above 30 mg/L must be supplied. After 48 to 72 hours the broth holds 100 to 140 g/L lysine, a yield of 40 to 50% on carbohydrate. Ion exchange isolates it, and it is sold mostly as 98% lysine hydrochloride for animal feed. Process flowchart Citric acid by submerged fermentation (Kent 2012, Shreve 1984) Inputs **Sucrose, glucose syrup or molasses**medium about 200 g/L sucrose **Mineral salts (Fe, Zn, Cu, Mg, Mn, phosphate)**balanced trace levels **Sterile air**0.5-1.5 vvm **Hydrated lime**about 0.58 t per t (stoichiometric) **Sulphuric acid**about 0.77 t per t (stoichiometric) **Activated carbon, ion-exchange resins**for purification Process steps 1 **Medium preparation** Filter syrup; cation exchange to cut trace metals; flash pasteurise 2 **Inoculum** A. niger grown from slant to seed tank, 36-48 h 3 **Fermentation** 25-27°C; air sparged, no stirrer; pellets 1-2 mm; 6-9 days 4 **Mycelium removal** Rotary vacuum filter 5 **Oxalate removal** Heat to 80-90°C with a little lime; calcium oxalate separated 6 **Calcium citrate precipitation** Lime at about 95°C; vacuum filter and wash 7 **Acidulation** Dilute H2SO4 frees citric acid; gypsum filtered off 8 **Purification and crystallisation** Carbon beds, ion exchange; multiple-effect evaporators; crystallise, centrifuge, dry, size Outputs **Citric acid (USP, usually monohydrate)**1 t **Gypsum**about 1.34 t per t (stoichiometric) **Mycelium**fertiliser after weathering Theoretical yield 112% on sucrose; surface culture achieves 90-95%, submerged slightly less. List price 60-90 cents/lb, about $1.3-2.0/kg (Kent). Source: Kent (2012), Ch 30 Industrial Biotechnology: Discovery to Delivery, pdf pp. 1157-1158 (Fig. 30.16) In the data #### The gypsum bill of the lime process Calcium citrate holds three calcium ions for every two citrate ions, so each mole of citric acid (192 g) needs 1.5 moles of lime and of sulphuric acid and leaves 1.5 moles of gypsum. Per tonne of anhydrous citric acid that is about 0.58 t hydrated lime, 0.77 t sulphuric acid and 1.34 t gypsum (CaSO4.2H2O, 172 g/mol), course arithmetic on Kent's chemistry. The waste solid outweighs the product, which is one reason producers developed solvent extraction with tri-n-butyl phosphate. - Kent (2012), Ch 30 Industrial Biotechnology: Discovery to Delivery · pdf pp. 1157-1158, 1173-1174 - Shreve (1984), Ch 31 Fermentation Industries · pdf pp. 615-617 ### See it in the table [H₂O Water](https://www.materialinsights.org/commodities/water/)[Cu Copper](https://www.materialinsights.org/commodities/copper/)[Zn Zinc](https://www.materialinsights.org/commodities/zinc/)[Mn Manganese](https://www.materialinsights.org/commodities/manganese/)[Mg Magnesium](https://www.materialinsights.org/commodities/magnesium/)[NaCl Salt](https://www.materialinsights.org/commodities/salt/)[Gyp Gypsum](https://www.materialinsights.org/commodities/gypsum/)[CaO Lime](https://www.materialinsights.org/commodities/lime/)[Su Sugar](https://www.materialinsights.org/commodities/sugar/)[H2SO4 Sulphuric acid](https://www.materialinsights.org/chemicals/sulphuric_acid/)[O2 Oxygen](https://www.materialinsights.org/chemicals/oxygen/)[CO2 Carbon dioxide](https://www.materialinsights.org/chemicals/carbon_dioxide/)[Ur Urea](https://www.materialinsights.org/chemicals/urea/)[AS Ammonium sulphate](https://www.materialinsights.org/chemicals/ammonium_sulphate/) ### Check your understanding In penicillin recovery, why is the filtered broth acidified to about pH 2 before solvent extraction? Penicillin is a weak acid. At low pH it is uncharged and partitions into amyl acetate; at pH 7-7.5 it becomes a salt and returns to water, which concentrates and purifies it. Kent says continuous fermentation and yeast recycle in ethanol plants cut capital but barely lower product cost. Why? Kent notes a significant portion of total cost comes from raw materials, so higher productivity mainly saves on the smaller capital charge. What is the main reason A. niger is grown as small pellets in citric acid fermentation? Kent states that 1-2 mm pellets reduce broth viscosity, increase oxygen transfer and simplify separation of the mycelium. [← Part 3: Formulation: turning powder into a dose](https://www.materialinsights.org/learn/chemicals/pharmaceuticals-and-fermentation/p3/)[Mark read · Part 5: Enzymes, biologics and the economics →](https://www.materialinsights.org/learn/chemicals/pharmaceuticals-and-fermentation/p5/) ## Other sections - [Module overview](https://www.materialinsights.org/learn/chemicals/pharmaceuticals-and-fermentation/index.md) - [The pharmaceutical value chain](https://www.materialinsights.org/learn/chemicals/pharmaceuticals-and-fermentation/p1/index.md) - [Making the API: multistep batch chemistry](https://www.materialinsights.org/learn/chemicals/pharmaceuticals-and-fermentation/p2/index.md) - [Formulation: turning powder into a dose](https://www.materialinsights.org/learn/chemicals/pharmaceuticals-and-fermentation/p3/index.md) - [Enzymes, biologics and the economics](https://www.materialinsights.org/learn/chemicals/pharmaceuticals-and-fermentation/p5/index.md) - [Key terms, quiz and sources](https://www.materialinsights.org/learn/chemicals/pharmaceuticals-and-fermentation/review/index.md)