Production of Citric Acid using Aspergillus niger by Submerged Fermentation
Aim: To produce citric acid using Aspergillus niger by submerged fermentation under controlled laboratory conditions.
Principle: Citric acid production by Aspergillus niger relies on a deliberate metabolic imbalance high carbon excess, limited nitrogen, trace metal restriction, and acidic pH all push the tricarboxylic acid cycle toward citrate overflow. Looking at it this way, the fungus is stressed but productive. Sucrose or glucose is rapidly taken up, glycolysis accelerates, oxaloacetate accumulates, and citrate synthase keeps firing. Here is where it gets tricky iron and manganese must be nearly absent, or the carbon flux shifts to biomass. Aeration matters; oxygen starvation kills yield fast. Temperature stays boringly constant. Deviate, and the organism rebels. Follow the chemistry. Respect the fungus.
Requirements:
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Aspergillus niger pure culture
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Fermentation medium (sucrose 150 g/L, NH₄NO₃ 2 g/L, KH₂PO₄ 1 g/L, MgSO₄·7H₂O 0.5 g/L)
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Distilled water
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250 ml Erlenmeyer flasks
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Autoclave
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pH meter or pH paper
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Incubator shaker
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Filter paper and funnel
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Calcium carbonate
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Standard NaOH solution
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Phenolphthalein indicator
Procedure:
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Prepare 100 ml of fermentation medium in a 250 ml flask and adjust pH to 3.0 using dilute HCl.
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Autoclave the medium at 121°C for 15 minutes and cool it to room temperature without shaking.
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Inoculate the sterile medium with 2 ml of Aspergillus niger spore suspension under aseptic conditions.
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Incubate the flask at 30°C on a rotary shaker at 150 rpm for 5–7 days.
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Observe daily for mycelial growth and avoid disturbing the pellet structure—do not swirl.
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After incubation, filter the fermented broth to remove fungal biomass completely.
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Add excess calcium carbonate slowly to the filtrate until effervescence stops.
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Filter again to collect calcium citrate precipitate and wash it with distilled water.
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Dissolve the precipitate in dilute sulfuric acid to release free citric acid.
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Titrate the final solution against standard NaOH using phenolphthalein to quantify citric acid.
Observation: White to light-brown mycelial pellets form within 48 hours, and the broth becomes clear and acidic with time.
Results: The experiment is successfully performed, and citric acid is produced by Aspergillus niger, confirmed by titration showing significant acid concentration in the fermented broth.
Conclusion: Citric acid can be efficiently produced by submerged fermentation using Aspergillus niger when carbon excess, low pH, and metal limitation are strictly maintained—miss one factor, and yield drops.
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