Fermentation, in chemistry, is a metabolic process where microorganisms like bacteria or yeast break down carbohydrates, such as sugars, into simpler molecules, like ethanol or lactic acid, often in the absence of oxygen

1. FERMENTATION

2. Fermentation is a biochemical process in which microorganisms, such as bacteria, yeast, or
fungi, convert sugars and other carbohydrates into acids, gases, or alcohol under anaerobic
(oxygen-free) conditions.
• both anaerobic and aerobic fermentation are processes by which microorganisms
convert substrates into energy, they differ primarily in the presence or absence of
oxygen and the byproducts produced

3. Anaerobic Fermentation:
Oxygen Requirement: Anaerobic fermentation occurs in the absence of oxygen.
Microorganisms Involved: Commonly involves yeasts (such as Saccharomyces cerevisiae) and
certain bacteria (like Lactobacillus).
Energy Production: The process generates energy through the partial breakdown of glucose or
other carbohydrates.
By-products: The by-products can include:
Ethanol and Carbon Dioxide: In alcoholic fermentation (e.g., beer and wine production).
Lactic Acid: In lactic acid fermentation (e.g., yogurt and pickles).
Other Compounds: Such as butyric acid, acetic acid, or hydrogen gas, depending on the type of
bacteria involved.

4. Aerobic Fermentation:
Oxygen Requirement: Aerobic fermentation occurs in the presence of oxygen.
Microorganisms Involved: Typically involves bacteria and yeasts that can utilize oxygen, such
as certain strains of yeast and acetic acid bacteria.
Energy Production: The process is more efficient at producing energy compared to anaerobic
fermentation and often leads to complete oxidation of substrates.
Byproducts: The primary byproducts are carbon dioxide and water, alongside biochemical
compounds like acetic acid (in case of some fermentation processes, like vinegar production).

5. Outline of a Typical Fermentation Process
1. Preparation Stage
• Selection of Microorganism:
Choose the appropriate strain (bacteria, yeast, or fungi) that produces the desired product
(e.g., antibiotics, alcohol, organic acids).
• Medium Formulation:
Prepare a nutrient-rich growth medium containing essential substrates (e.g., sugars, amino
acids, minerals) tailored to the needs of the selected microorganism.

6. 2. Inoculation Stage
• Inoculum Preparation:
Grow a small-scale culture (seed culture) in a defined medium to the desired cell density,
ensuring the microorganism is healthy and active.
• Inoculation of Fermentor:
Transfer the inoculum into the fermentation vessel (bioreactor) containing the bulk growth
medium.

7. 3. Fermentation Stage
• Environmental Control:
Maintain optimal conditions for growth (temperature, pH, dissolved oxygen, pressure) using
automated control systems.
• Scheduled Nutrient Addition:
Implement feeding strategies (e.g., batch, fed-batch, or continuous fermentation) to provide
nutrients over time and maintain optimal growth conditions.
• Monitoring:
Continuously monitor parameters such as pH, temperature, dissolved oxygen levels, substrate
concentration, and microbial growth (cell density, metabolite production) for maintaining ideal
fermentation conditions.

8. 4. Harvesting Stage
• Fermentation Termination:
Determine the optimal time to terminate the fermentation based on maximum production
of the desired product (e.g., antibiotic concentration).
• Separation:
Separate microorganisms from the fermentation broth using techniques such as
centrifugation, filtration, or sedimentation.

9. 5. Recovery and Purification Stage
• Product Isolation:
Extract the target product from the fermentation broth. This can involve liquid-liquid
extraction, adsorption, or precipitation methods depending on the nature of the product.
• Purification:
Purify the isolated product further using techniques such as chromatography, crystallization, or
distillation to remove impurities and byproducts.
• Concentration:
Concentrate the purified product to achieve the desired concentration for formulation.

10. 6. Formulation and Packaging
• Formulation:
Mix the purified product with suitable excipients to create the final product form (e.g.,
tablets, liquids, powders).
• Quality Control:
Conduct quality control tests to ensure the product meets specified standards (potency,
purity, stability, and safety).
• Packaging:
Package the final product in appropriate containers for distribution, considering factors
such as stability and shelf-life.

11. 7. Post-Production Step
• Waste Treatment:
Treat and dispose of waste generated during the fermentation process responsibly,
including spent media and microbial culture residues.
• Documentation and Compliance:
Document the entire process for regulatory compliance and quality assurance, ensuring
traceability and adherence to good manufacturing practices (GMP).

13. General features of an ideal fermenter
• Material used in the construction of a fermenter must be able to bear high pressure and
temperature conditions mediated by the fermentation medium.
• the material used for the construction of a fermenter must be selected according to the
nature of fermentation which has to be carried out in it .
• The fermenter material must be resistant to corrosion, It must not have any toxigenecity on
the microbial culture and it must not affect the purity of product.
• There must be an inlet present in the fermenter to provide easy and aseptic inoculation
• If aerobic fermentation has to be carried out, proper exposure to oxygen is necessary hence
an aerating device must be present in the fermenter.

14. • There must be a stirring device in fermenter for equal distribution of air, microbes and
nutrients
• In order to avoid vortex formation, baffles must be present in fermenter
• There must be a way to control temperature and pH of the fermenting medium.
• There must be a sampling valve resent in fermenter to withdraw media and product
time by time for laboratory analysis
• A draining outlet should be present for complete removal of medium from the
fermenter and for the recovery of product
• A large hole should be present at the top of fermenter in order to get access to the
inside of fermenter for various purposes such as repairing, cleaning etc

15. Types of Bioreactors/Fermenters
• Mechanically Agitated Fermenter, Non-mechanically agitated fermenter, Non-agitated
fermenter
Continuous stirred tank fermenter, Tower fermenter,
Deep jet fermenter, Batch fermenter,
Cyclone column fermenter, Gas lift fermenters,
Air lift bioreactor, Fluidized bed bioreactor,
Bubble column bioreactors, Wave bioreactors, Sparged tank fermenters, Photo bioreactor,
Membrane bioreactor, Novel see saw fermenter, Rotary drum bioreactor, Mist bioreactor.

17. PRODUCTION OF PENICILLIN
Penicillin is primarily produced by the mold Penicillium chrysogenum (formerly known as
Penicillium notatum) under controlled conditions in a fermentation vessel.
1. Inoculum Preparation:
Strain Selection: The process begins with selecting a high-yielding strain of Penicillium
chrysogenum that has been genetically selected or mutated for maximum penicillin
production.
Inoculum Culture: The inoculum is prepared by growing the selected Penicillium strain in a
small culture medium (usually a broth containing glucose, salts, and other nutrients) in a
laboratory flask or small-scale fermenter. The inoculum is cultured until the fungal growth is
ready for transfer to the main fermenter.(SURFACE CULTURE AND SUBMERGED
CULTURE METHOD-with agitation)

18. 2. Fermentation Medium:
•The fermentation medium is carefully formulated to provide the nutrients required for the
growth of Penicillium chrysogenum and the production of penicillin. The key components of
the medium include:
• Carbon Source: Typically, glucose or lactose is used as the carbon source.
• Nitrogen Source: Ammonium salts (like ammonium nitrate) or other nitrogen
compounds are included to support fungal growth.
• Minerals and Vitamins: Essential minerals (like phosphorus, magnesium, potassium)
and vitamins are added to promote fungal growth and penicillin production.
• Trace Elements: Micronutrients such as iron, copper, and zinc are required for enzyme
function and overall metabolism.

19. 3. Inoculation of the Fermenter:
•The prepared inoculum is transferred into a large-scale fermentation vessel, often
called a fermenter or bioreactor.
•The fermenter is usually equipped with a variety of sensors to control and monitor
conditions such as temperature, pH, oxygen concentration, and agitation speed.

20. 4. Fermentation Conditions:
•Temperature: The optimal temperature for Penicillium chrysogenum growth and penicillin
production is typically around 24–26°C (75–78°F).
•pH: The pH of the medium is maintained at slightly acidic levels (around 6.5 to 7.0) to
optimize the growth and metabolism of the microorganism.
•Oxygen Supply: Penicillin production is aerobic, requiring adequate oxygen supply. The
fermenter is equipped with an aeration system (often with spargers (AERATION/GAS
EXCHANGE) or diffusers(ENHANCE OXYGEN TRANSFER)) and agitation (via stirrers or
impellers) to ensure the microorganism gets sufficient oxygen for optimal metabolism.
•Agitation: The fermenter is continuously stirred to mix the contents, ensuring even
distribution of nutrients and oxygen to the fungal cells.

21. 5. Penicillin Production:
•Initially, the fungus grows rapidly, consuming glucose and other nutrients in the medium.
During the early phase of growth (lag and exponential phases), the fungus focuses on cell
division and biomass formation.
•Once the cells have sufficiently grown, the fungus shifts its metabolism to produce
penicillin. This usually occurs in the stationary phase of growth, when the fungus starts to
secrete penicillin into the surrounding culture medium.
•Penicillin biosynthesis is a secondary metabolic process that requires the precursor
molecule phenylacetic acid (which is often added to the medium to enhance penicillin
production). Penicillin is produced from this precursor through a series of enzyme-catalyzed
reactions.

22. 6. Nutrient Control and Feeding Strategy:
•Fed-Batch Process: To maintain optimal conditions for penicillin production, the fed-
batch fermentation method is often used. This involves controlling the feeding of glucose
to the fermenter. Initially, a rich glucose medium is added, and then, during fermentation,
glucose is continuously or intermittently fed to the culture to avoid excess nutrients that
could inhibit penicillin production.
•Carbon Source Limitation: After the fungal cells have grown sufficiently, the glucose
concentration is limited. This forces the fungus to switch to penicillin production as the
primary metabolic activity.

23. 7. Harvesting and Penicillin Extraction:
•Once the fermentation reaches its maximum penicillin production (typically after 4-7
days), the culture broth is harvested.
•Filtration: The fungal biomass (mycelium) is removed from the broth through filtration
or centrifugation.
•Penicillin Extraction: The penicillin is present in the liquid portion of the fermentation
broth. It is extracted using various methods, often involving solvent extraction (e.g.,
using ethyl acetate) to separate penicillin from the aqueous phase.
•The solvent is then evaporated, and the penicillin is further purified, typically using
crystallization or chromatography.

24. 8. Purification and Formulation:
•After extraction, penicillin undergoes several purification steps to remove impurities. The
final product is typically purified penicillin, which may be in the form of penicillin G or
other forms, depending on the desired type of penicillin.
•The purified penicillin is formulated into its final medicinal form, such as tablets,
injections, or oral solutions, for use as an antibiotic.

27. Industrial production of riboflavin can be performed by both chemical synthesis and
fermentation.
The fermentation route allows the
production of vitamin B2 in a single step,
which is cost-effective.
chemical processes are multistage and expensive.
Thus, nowadays, the fermentative production of riboflavin is economically and ecologically
more feasible and has completely replaced chemical synthesis.
Global producers, such as BASF (Germany), DSM (formerly Roche; Netherlands), Hubei
Guangji Pharmaceuticals, and Shanghai Acebright Pharmaceuticals (formerly Desano; China),
derive riboflavin from the cells of industrial microbial strains of Ashbya gossypii, Candida
famata var. flareri, and Bacillus subtilis, reaching a titer of up to 15, 20, and 14 g/L,
respectively (Lim et al., 2001; Revuelta et al., 2016).

28. • The first commercial microbiological production of riboflavin using bacteria was
performed with Clostridium acetobutylicum by acetone-butanol fermentation, where
riboflavin was formed as a by product (Leviton, 1946).

29. Later, several species of fungi, such as
• Eremothecium ashbyii,
• Pichia guilliermondii (asporogenic Candida guilliermondii),
• Candida boidinii,
• Schwanniomyces occidentalis,
• Pichia caribbica,
• Candida oleophila, Aspergillus terreus, and methanol-utilizing Hansenula polymorpha
However, these microorganisms accumulated riboflavin slowly and at a low concentration,
which were not satisfactory for commercial production of riboflavin.
Precursors
ribulose 5- phosphate
(Ribu5P; pentose
phosphate pathway) and
guanosine triphosphate
(purine pathway)

30. The fermentative production of riboflavin is naturally carried out by the wild-type
flavinogenic ascomycetes, such as E. ashbyii and A. gossypii, with the accumulation of
riboflavin in mycelia at the end of the growth phase, which provides the fungi with a bright
yellow color (Aguiar et al., 2015)
Among them, A. gossypii is commercially preferred
as it maintains a steady high-producing capacity of
riboflavin

31. Among Candida strains, the mutant C. famata ATCC 20849
demonstrates the highest flavinogenic potential, but its
extreme sensitivity to the presence of iron makes the
fermentation process complicated (Heefner et al., 1992,
1993

32. PRODUCTION OF VITAMIN B2
1. Microorganism Selection
• Key Strains:
• Fungal: Ashbya gossypii (most commonly used due to high riboflavin yields).
• Bacterial: Genetically modified strains of Bacillus subtilis or Candida famata.
• 2. Fermentation Process
• Media Composition:
• Carbon Sources: Glucose ( can be replaced by sucrose /maltose), vegetable
oils (e.g., soybean oil), or molasses.
• Nitrogen Sources: Ammonium salts, corn steep liquor, or yeast extract.
• Other Nutrients: Minerals (Mg² , Fe² ), vitamins, and purine precursors (e.g.,
⁺ ⁺
guanine) to boost riboflavin synthesis.

33. •Fermentation Conditions:
• Mode: Fed-batch fermentation to maintain optimal nutrient levels and prevent substrate
inhibition.
• Temperature: 28–32°C (for Ashbya gossypii).
• pH: Slightly acidic (pH 6.0–7.0).
• Aeration: High oxygen supply (riboflavin production is aerobic)-0.3vvm.
• Process for 5-7 days by submerged aerated fermentation in stirred tank fermentor.
•Production Phase:
Riboflavin is a primary metabolite, synthesized during the exponential growth phase.
Overproduction is triggered by nutrient limitations (e.g., iron depletion).

34. Phase 1:
• rapid growth of organism utilizing glucose.
• Pyruvic acid accumulation, pH becomes acidic
• As glucose gets exhausted, growth of the organism stops
• No riboflavin production in phase 1
Phase 2:
• Sporulation
• Pyruvate concentration decreases
• Accumulation of ammonia, which makes the media alkaline
• Maximum pdn. of riboflavin

35. Phase III
• Cells get disrupted by autolysis
• This releases riboflavin, FAD, FMN into the medium

36. Harvesting and Cell Disruption
•Biomass Separation:
• The fermentation broth is centrifuged or filtered (CANDLE FILTERS-to separate solid
particles, like cell debris, from the liquid fermentation broth, which contains the
riboflavin ) to separate microbial cells (for intracellular riboflavin) from the supernatant.
• Ashbya gossypii stores riboflavin intracellularly in crystalline form, requiring cell
disruption via:
• Autolysis: Induced by pH/temperature changes.
• Mechanical Methods: Homogenization or bead milling.
•Extracellular Recovery:
Some bacterial strains (e.g., Bacillus subtilis) secrete riboflavin into the broth, simplifying
recovery.

37. Recovery: riboflavin in a bound state with cells can be released by heat treatment.
• The post-fermentation broth is heated to 45-120°C for 10 minutes to 2 hours to
eliminate microbial contaminants
• centrifugation: Following pasteurization, the broth is centrifuged to separate
riboflavin-rich components from the liquid.

38. Oxidation of Separated Supernatant: The supernatant obtained from the separation step is
then oxidized using hydrogen peroxide. This oxidation step is important for purifying the
riboflavin and preparing it for crystallization.
Neutralization and Precipitation: Following oxidation, the solution is neutralized with
hydrochloric acid. After neutralization, the solution is allowed to stand, leading to
precipitation. This step is vital for isolating riboflavin from the solution.
Filtration and Crystallization: The precipitated riboflavin is then separated and crystallized.
This is done using a plate-and-frame filter press (for solid-liquid separation, primarily to
filter out insoluble proteins and other impurities from fermentation broth), which helps in
obtaining a more refined product.

39. Dissolving and Further Purification: The crude product obtained is dissolved, followed by
another round of oxidation and filtration. The solution is then diluted with water, leading to
further precipitation and crystallization, which refines the riboflavin to a high purity level.
Final Product Quality: The final method yields food and medicine grade riboflavin with a
purity higher than 98%.

40. 5. Formulation and Quality Control
•Drying:
Crystalline riboflavin is spray-dried or lyophilized into a powder.
•Formulation:
Blended with stabilizers (e.g., starch) for use in tablets, capsules, or fortified foods.
•Quality Testing:
Compliance with pharmacopeial standards (e.g., USP, EP) for purity, microbial limits, and
heavy metals.

43. 1. Fermentation Process for Lovastatin Production
Step 1: Strain Selection and Preparation
•Microorganism: A high-yielding strain of Aspergillus terreus is selected.
Culture Maintenance: The strain is preserved on agar slants or in cryovials
at low temperatures.

44. Step 2: Inoculum Development
•Seed Culture: Spores or mycelia from the preserved strain are transferred to
a liquid medium (e.g., containing glucose, peptone, and salts) in shake flasks.
•Growth Conditions: Incubated at 25–28°C with agitation (150–200 rpm) for
48–72 hours to build biomass.

45. Step 3: Fermentation Medium Preparation
•Carbon Sources: Lactose, glucose, or glycerol
•Nitrogen Sources: Soybean meal, ammonium nitrate, or peptone.
•Minerals: MgSO , KH PO , and trace metals (Fe, Zn, Cu).
₄ ₂ ₄
•Inducers/Precursors: Methylmalonyl-CoA precursors (e.g., sodium
propionate) may enhance lovastatin biosynthesis.

46. Step 4: Fermentation Setup
•Bioreactor: A sterilized, stirred-tank fermenter is inoculated with the seed
culture (5–10% v/v).
•Operating Conditions:
• Temperature: 25–28°C.
• pH: Maintained at 5.5–6.5 using NaOH or H SO .
₂ ₄
• Aeration: 0.5–1.5 vvm (volume per volume per minute) for oxygen supply.
• Agitation: 300–500 rpm to ensure mixing and oxygen transfer.

47. Step 5: Fed-Batch Fermentation
•Nutrient Feeding: Glucose or lactose is fed incrementally to maintain
substrate availability and prevent overflow metabolism.
•Duration: 7–14 days, with lovastatin production peaking during the stationary
phase.

48. Step 6: Monitoring and Optimization
•Analytical Tools: HPLC or LC-MS tracks lovastatin yield. Dissolved oxygen,
pH, and biomass are monitored in real-time.
•Process Adjustments: Nutrient feeds or antifoam agents are added as needed.
Step 7: Harvesting the Broth
•Separation: Biomass is removed via centrifugation or filtration. Lovastatin is
secreted extracellularly but may also be intracellular.
•Cell Disruption (if needed): Ultrasonication or enzymatic lysis releases
intracellular lovastatin.

49. Step 8: Extraction and Initial Purification
•Solvent Extraction: Ethyl acetate or methanol isolates lovastatin from the
broth.
•Concentration: The solvent is evaporated under vacuum to obtain crude
lovastatin.
(Chemical modification by hydrolysis, methylation and cyclization)

50. *Biotransformation of lovastatin (Streptomyces carpaticus)
The sub cultured strains of Actinobacteria (40 different strains) were individually
propagated with 30 ml of Gause's starch medium (seed culture medium The
compositions of Gause's liquid medium - (g/L): soluble starch, NaCl, FeSO4·7H2O, K2HPO4, KNO3, MgSO4·7H2O) and kept
in a shaker incubator at 28°C for 24 h (150 rpm).
Subsequently, 5% (v/v) of each culture was inoculated into fermentation medium
(glucose-10 g/L, peptone-5 g/L, yeast extract-5 g/L, malt extract-5 g/L pH 7) and
incubated at 28°C for 24 h at 150 rpm.

51. • To this fermentation medium, lovastatin (1 g/L) was added, and fermentation
was proceeded for 5 days for each strain.
• The resulted broths were filtered, and pH was adjusted to 2–3 by adding 1 M
H2SO4.
• Eventually, the broths were extracted using ethyl acetate followed by
concentrating under normal drying, which resulted in oil-like substance.
• This was further dissolved in ethyl acetate and stored at −20°C.

52. • The purification of bio transformed product was achieved using preparative
HPLC
• Acetonitrile and acidified water were (70:30 v/v) used in the mobile phase.
*Balraj J, Murugesan T, Dhanapal AR, Kalieswaran V, Jairaman K, Archunan G,
Jayaraman A. Bioconversion of lovastatin to simvastatin by Streptomyces carpaticus
toward the inhibition of HMG CoA activity. Biotechnology and Applied Biochemistry.
‐
2023 Jun;70(3):1162-75.