Production of Citric Acid using Aspergillus niger by Submerged Fermentation
Aim: To produce citric acid from sucrose using Aspergillus niger through submerged fermentation and estimate the yield by titration with sodium hydroxide.
Principle: Aspergillus niger is a black mold and one of the most studied fungi for organic acid production. Under specific conditions, it accumulates citric acid in the culture medium instead of using it fully in the TCA cycle. This happens when the manganese concentration in the medium is very low. Manganese deficiency actually disturbs nitrogen metabolism in the fungus, and this leads to ammonia accumulation inside the cell, which in turn blocks isocitrate dehydrogenase. So the TCA cycle stalls at isocitrate, and citrate keeps building up. The fungus then pushes citrate out. High sugar concentration and low pH, roughly below 3.5, also favor citric acid accumulation over other metabolites. Simple fermentation setup. Big yield if conditions are right.
Requirements:
Organism:
- Aspergillus niger culture
Fermentation Medium (Production Medium) for 1000 ml:
- Sucrose: 100 g/L
- Ammonium nitrate (NH4NO3): 2.5 g/L
- Potassium dihydrogen phosphate (KH2PO4): 1.0 g/L
- Magnesium sulphate heptahydrate (MgSO4.7H2O): 0.25 g/L
- Zinc sulphate (ZnSO4.7H2O): 0.001 g/L
- Distilled water to make 1000 ml
- pH adjusted to 3.5 before autoclaving using dilute HCl
- Note: Do not add any manganese source. This is deliberate.
Spore Suspension Medium (Potato Dextrose Broth for spore harvesting):
- Potato extract (boiled and filtered): 200 g/L equivalent
- Dextrose: 20 g/L
Other Requirements:
- 250 ml Erlenmeyer flasks (at least 6)
- Orbital shaker incubator set at 30°C, 150 rpm
- Autoclave
- pH meter or wide-range pH paper
- Haemocytometer or spore counting chamber (optional but useful)
- Whatman no. 1 filter paper and funnel
- Conical flasks for titration
- Burette, pipettes (1 ml, 5 ml, 10 ml)
- 0.1 N NaOH solution (freshly standardized)
- Phenolphthalein indicator
- 0.1% bromothymol blue (optional, for a rough color check)
- Distilled water
Procedure:
Day 1: Preparation of Spore Suspension
- Take one colony of well-sporulated Aspergillus niger culture (7 to 10 days old, showing good black sporulation).
- Add 5 ml of sterile distilled water to the slant and scrape the spores gently using a sterile inoculation loop.
- Transfer this spore suspension into a sterile tube. Mix well.
- Count spores using a haemocytometer if possible. Aim for approximately 10^6 spores per ml. If not counting, use the suspension as is.
Day 1: Preparation of Production Medium
- Dissolve sucrose and all salts except zinc sulphate in approximately 900 ml distilled water. Dissolve zinc sulphate separately in 10 ml water and add it to the bulk. Adjust volume to 1000 ml.
- Adjust pH to 3.5 using dilute HCl. Check with pH meter or pH paper carefully.
- Dispense 50 ml of medium into each 250 ml Erlenmeyer flask.
- Cover flasks with cotton plugs and aluminum foil. Autoclave at 121°C for 15 minutes.
- Allow flasks to cool to room temperature before inoculation.
Day 1: Inoculation
- Inoculate each production flask with 1 ml of the spore suspension under aseptic conditions near a flame or in a laminar airflow hood if available.
- Label flasks. Keep one flask uninoculated as control.
- Transfer all flasks to the orbital shaker at 30°C, 150 rpm.
Days 2 to 6: Incubation
- Incubate with continuous shaking for 5 to 6 days. Do not disturb unnecessarily.
- On Day 3, carefully observe one flask without sacrificing it if possible. Check for mycelial growth and any change in the smell of the medium. A sharp, faintly sour smell indicates citric acid accumulation, which is honestly quite satisfying to notice for the first time.
Day 6 or 7: Harvesting and Estimation
- After incubation, filter the fermented broth through Whatman No. 1 filter paper to remove mycelial biomass. Collect the clear filtrate in a conical flask.
- Take 10 ml of the filtrate into a clean conical flask.
- Add 2 to 3 drops of phenolphthalein indicator.
- Titrate against 0.1 N NaOH from the burette. Swirl gently and continuously.
- Note the volume of NaOH used when the solution turns faint pink and the color persists for 30 seconds.
- Repeat titration two more times for concordant readings.
Calculation:
Equivalent weight of citric acid = 64 g/equivalent (since citric acid is triprotic, molecular weight 192, divided by 3)
Observation: After 5 to 6 days of shaking incubation, we have noticed dense white to yellowish-brown mycelial mat floating or loosely dispersed in the medium, and the filtered broth will be clear and noticeably more acidic in smell compared to the uninoculated control. During titration, the broth changes from colourless to faint pink at the endpoint, and the volume of NaOH consumed will be higher than what you'd expect from the control flask.
Result: The titration confirms the presence and approximate quantity of citric acid in the fermentation broth, and the yield typically ranges between 20 and 45 g/L, depending on incubation duration and how carefully the medium composition was maintained, particularly the absence of manganese.
Conclusion: Aspergillus niger efficiently accumulates citric acid under manganese-deficient, low-pH, high-sugar submerged fermentation conditions, which directly block normal TCA cycle progression and divert carbon toward citrate. This experiment gives a clear, hands-on understanding of how industrial fermentation is not just about growing an organism but about stressing it in a very specific, controlled way to redirect its metabolism.
Note: This protocol is for educational use in standard teaching laboratories. Small variations may occur depending on lab conditions and handling. Results may vary. biologynotes.in cannot take responsibility for any errors, misuse, or results obtained. Always follow proper lab safety and instructor guidance.
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