Industrial Production of Citric Acid by Aspergillus niger | Flowchart - YB Study

Industrial Production of Citric Acid by Aspergillus niger | Flowchart

Citric Acid Production- History  Stages, Process, and Techniques: 


Introduction

  • Citric acid is a weak organic acid present in fruits such as lemons, citrus, pineapple, and other fruits.
  • Citric acid can be produced by fermenting sugary substances such as sugar, molasses, starch, and grapes.
  • In 1984 Abould Zeid and Ashy introduced the “Submerged fermentation technique” for the production of citric acid.
  • To produce a citric acid large number of microorganisms are used like Yeast, bacteria, and fungi. 
  • Commercial production of citric acid is done by Aspergillus Niger (fungi) and Saccharomycopsis sp (yeasts).
  • Industrial production of citric acid can be carried out in three different ways Solid-state fermentation, Surface fermentation, and submerged fermentation.
  • Production of citric acid is completed in three steps upstream, fermentation, and downstream process.
  • citric acid is mostly produced from starch or sucrose-based media using liquid fermentation, a variety of raw materials such as molasses-like cane and beet are suitable for citric acid production.
  • The optimum temperature used for the production of citric production is between 28 to 30°C.
  • Various factors affect citric acid production like carbon and nitrogen source, Temperature, pH, Aeration and Agitation, Nutrient Concentration, Trace elements, etc.
  • Citric acid has numerous applications in the Food & drink industry, Cosmetics, Chemical industry, Detergents, Pharmaceutical, and Medical Industries.

Industrial Production of Citric Acid by Aspergillus niger | Flowchart


What is citric acid?

  • Citric acid is a weak organic acid with the chemical formula HOC(CO2H)(CH2CO2H)2 derived from the Latin word “Citrus”. 
  • Structurally, citric acid is a tricarboxylic acid and it has similar physical and chemical properties to other carboxylic acids. 
  • At room temperature, citric acid is a white crystalline powder, odorless and extremely sour.
  • Many kinds of citrus fruits and vegetables contain citric acid, especially lemons and limes.
  • Natural citric acid is widely distributed in nature, and natural citric acid is found in the bones, muscles, and blood
  • It is naturally present in fruits such as lemons, citrus, pineapple, and other fruits.
  • Synthetic citric acid is prepared by fermenting sugary substances such as sugar, molasses, starch, and grapes.


History of Citric Acid Production:

  • Citric acid was first discovered in the 8th century by Islamic alchemist Jabir Ibn Hayyan (Geber).
  • Citric acid was first isolated in 1784 by the chemist Carl Wilhelm Scheele, who crystallized it from lemon juice.
  • In 1893, C. Wehmer discovered Penicillium mold could produce citric acid from sugar.
  • In 1917, American food chemist James Currie discovered some strains of the mold Aspergillus niger that are efficient for citric acid production.
  • In 1984 Abould Zeid and Ashy introduced the “Submerged fermentation technique” for the production of citric acid.

Microorganisms used for Citric Acid Production
  • Different types of microorganisms have different abilities to produce Citric acid. Therefore, different microorganisms can be used to produce citric acid products that people need. 
  • Today, Citric acid fermentation technology plays a huge role in the food, Pharma, and Medical industry. 
  • To produce citric acid, many microorganisms are used like Yeast, bacteria, and fungi. 
  • Citric acid is mainly produced by submerged fermentation using Aspergillus niger or Candida sp. from different sources of carbohydrates, such as molasses and starch-based media. 
  • Commercial production of citric acid is done by Aspergillus Niger(fungi) and Saccharomycopsis sp (yeasts).
  • The main advantages of using this microorganism are: it is easier to obtain and easy to handle its ease of handling. It gives maximum yield and can ferment a variety of cheap raw materials like molasses and starch.
  • Fungal SpeciesAspergillus niger, A.aculeatus,  A. carbonarius, A.awamori etc.
  • Bacterial Species: Corynebacterium species, Bacillus licheniformis, Arthrobacter paraffines, etc.
  • Yeasts Species: C.guilliermondii, Candida tropicalis, C.oleophila, C.Citroformans

    Raw Materials used for Citric Acid Production
    Although citric acid is mostly produced from starch or sucrose-based media using liquid fermentation, a variety of raw materials such as molasses-like cane and beet are suitable for citric acid production.


    Stages of Citric Acid Production

    Industrial fermentation of citric acid requires three most important stages to complete product (Citric Acid) fermentation. These are the upstream process, actual fermentation and last one is a downstream process.

    1. Upstream  process of fermentation:
    • The upstream process includes the initial steps of a fermentation process.
    • The Up-Stream process of citric acid fermentation comprises initial tasks to screen and identification of suitable microorganisms, and their improvement, Substrate selection, and Culture media preparation to enhance productivity and yield. 
    • It also includes the maintenance of strain purity, preparation of inoculum, sterilization of media, and bioreactor to improve the economic efficiency of the process.

    2. Actual Fermentation Process:
    • Fermentation is a process in which complex substances are converted into simple substances with the help of microorganisms.
    • In this process, all the materials are added together in a bioreactor to produce the desired product.
    • Maintaining optimal conditions required for citric acid production like nutrients, are supplied for the growth and multiplication of microorganisms inside a bioreactor, Temp, pH, Oxygen concentration, etc.

    3. Down-stream process:
    • The downstream process of fermentation involves the recovery of products from the bioreactor by different methods like extraction, separation, purification, and packaging of the product.


    Citric acid production techniques
    Industrial citric acid production can be carried out in three different ways:
    1. Solid-state fermentation
    2. Surface fermentation
    3. Submerged fermentation

    1. Solid State Fermentation:
    • A solid-state fermentation (SSF) method was used to produce citric acid by Aspergillus niger using agro-industrial wastes such as sugarcane bagasse, coffee husk, etc.
    • Solid-state fermentation is an important technique for the production of citric acid.
    • The substrate used for Citric Acid Production is Pineapple waste, Banana peels, Apple pomace, Coffee husk, corn cob, Grape pomace, etc.
    • The solid-state fermentation of citric acid requires low energy and cost requirements and gives a higher yield.
    • The SSF method requires low-cost natural media and better oxygen circulation.
    • It has a low risk of bacterial contamination and less amount of post-recovery waste.
    • But sometimes  Solid state fermentation is Difficult to scale up, difficult to control process parameters higher recovery product costs.

    2. Surface Fermentation
    • The material used in surface fermentation is Sweet potato starch hydrolysate, Turnip whey (supplemented with molasses), Brewery waste, etc.
    • This method requires a large amount of heat generation, is time-consuming, and needs a large area/space, 
    • In this process, there is a chance of occurring contamination by Penicillium, other Asperigilli, yeasts, and lactic acid bacteria.

    3. Submerged Fermentation:
    • Substrate use as Beet molasses, Cane molasses, Coconut oil, soybean oil, etc.
    • The submerged fermentation method of citric acid production has easy control mechanisms and it lowers labor costs, 
    • This technique gives higher productivity and yield. 
    • The only disadvantage of this technique is it requires high media cost, sensitivity to trace metal inhibition, and high wastewater generation.
    • According to statistics, 80% of the citric acid in the world comes from the liquid-submerged fermentation of Aspergillus niger, and the medium is starch or raw materials containing glucose and sucrose. 
    • In addition, some agricultural processing wastes and their by-products can be used as carbon sources in the submerged fermentation of citric acid. 
    • Compared with other fermentation types, submerged fermentation has significant advantages, such as high productivity and low labor costs. 
    • Both batch fermentation and fed-batch fermentation can be used in submerged fermentation, but batch fermentation is still the main method. 
    • As the main source of citric acid fermentation, liquid-submerged fermentation still needs to develop the application of renewable raw materials in line with sustainable development, to meet the huge market demand for citric acid.


    Citric Acid Production Medium
    1. Sucrose - 150 gm
    2. Ammonium nitrate - 2.5 gm
    3. Potassium Dihydrogen Orthophosphate - 1.0 gm
    4. Magnesium sulphate heptahydrate - 0.25 gm
    5. Distilled water - 1 Lit.
    6. pH - 3.5

    Process for citric acid Production by Aspergillus niger:
    1. Prepare the citric acid medium & dispense about 100ml in a 250ml conical flask.
    2. Autoclave and allow it cool.
    3. Inoculate the medium with spores of Aspergillus niger & incubate it in a shaker water bath at 25°C with gentle shaking for 3-5 days.
    4. After Incubation, filter the mycelium using double-layered muslin cloth & measure the amount of citric acid in the filtrate by colorimetric and titrimetric methods.

    Estimation of Citric Acid by Titrimetric Method:
    The filtrate obtained is titrated against an alkali of known strength using phenolphthalein as an indicator. The endpoint is the formation of pale pink color. The volume of alkali used for neutralization is used to find the normality and the percentage of acid in the sample.

    • 100ml of the filtrate is pipetted into a conical flask and 2-3 drops of phenolphthalein indicator are added to it.
    • This is titrated against 0.1N NaOH taken in the burette till a pale pink color is formed.
    • The titration is repeated till concordant values are obtained.


    Applications of Citric Acid : 
    • It is a natural preservative
    • Citric acid is used as a buffer. 
    • Citric acid is mainly used in the food industry. Because citric acid has a mild and refreshing sour taste, it is mostly used in various beverages, soft drinks, canned juices, dairy products, etc. 
    • Citric acid as a color-retaining agent for fruit and vegetable raw materials
    • Citric acid can also be used as an antioxidant in edible oils in addition to being a flavoring agent.
    • Citric acid is used as a pH adjuster in canned food, jam, jelly, and other products and can reduce the pH value and inhibit the reproduction of spoilage microorganisms.
    • Citric acid can be added to soft drinks, cold drinks, baked goods, candies, and gummies.
    • In chemical technology, citric acid can be used as a reagent for chemical analysis, experimental reagent, and chromatographic analysis reagent.
    • In dairy products, it acts as an emulsifier in ice creams and processed cheese and; acidifying agent in many cheese products.


    Factors Affecting Citric acid Production:

    1. Temperature : 
    • The effect of temperature on citric acid production is numerous. 
    • The optimum temperature used for the production of citric acid by submerged fermentation of Aspergillus niger and other fungi is between 28 to 30°C.
    • Temperature can affect the growth of Aspergillus niger activity. In the optimum temperature range, as the temperature increases the growth and metabolism of Aspergillus niger accelerate, and the rate of the fermentation reaction increases. 
    • When the temperature exceeds the optimal temperature range as the temperature rises the Aspergillus niger activity is quickly inactivated and the fermentation rate is reduced. 
    • Therefore, to ensure a normal fermentation process, it is necessary to maintain the optimum temperature.

    2. pH : 
    • pH can affect the activity of microorganisms, enzymes, and fermentation rate. 
    • Based on the type of substrate and media and microorganisms the pH is vary. 
    • In defined media, the initial pH of the medium is usually adjusted to 2.5–3.5, while in the case of molasses, the pH must be neutral or slightly acidic. 
    • When yeasts are used for the production of citric acid, the pH is often adjusted to 6.0–7.0 by the addition of calcium carbonate, sodium hydroxide, and lime.
    • The pH of the medium is adjusted with NaOH, H2SO4, and HCl. 
    • The pH of the fermentation broth decreases during fermentation (from 7.0 to 2.0) due to the production of citric acid and other acids generated in the TCA cycle. 
    • In addition, pH will also affect the decomposition of nutrients in the medium. Therefore, the pH of the fermentation broth should be controlled.

    3. Dissolved oxygen Concentration : 
    • The supply of oxygen is a key factor for aerobic fermentation and Aspergillus niger is an aerobic microorganism and therefore requires oxygen. 
    • Aeration and agitation meet the oxygen demand of a fermentation process of citric acid.
    • Agitation is important for heat transfer, adequate mixing, and mass transfer.
    • Aeration and agitation both maintain the homogeneous environment of culture by continuous mixing. 
    • From the perspective of oxygen demand for glucose oxidation, 1 mol of glucose is completely oxidized and decomposed it requires 6 mol of oxygen. when sugar is used to synthesize citric acid, 1 mol of glucose requires about 1.9 mol of oxygen. 
    • Therefore, a large amount of oxygen must be continuously added to the fermentation broth, and stirring can increase the solubility of oxygen in the fermentation broth. The concentration of dissolved oxygen increased with the increase in speed of agitation.

    4. Concentration of nutrients : 
    • The concentration of various nutrients in the fermentation broth, especially the ratio of carbon to nitrogen, inorganic salts, and vitamins, will directly affect the growth of bacteria and the accumulation of metabolites.
    • Such as Carbon sources Starch, glucose, sucrose, fructose, maltose, and mannose. nitrogen sources like ammonium or nitrate ions. And other nutrients like phosphate, low amounts of potassium, magnesium, sulfate, and trace metals such as iron, manganese, zinc, and copper.
    • For example, in the fermentation of glutamate, the change in NH+4 concentration will affect the metabolic pathway. 
    • Therefore, during the fermentation process, a sufficient amount of nutrients are added for fermentation.


    Frequently Asked Questions on Citric Acid Production:

    1. How to produce citric acid?
    Answer: Currently, citric acid is obtained by citric acid fermentation of sweet sugar production waste - molasses by Aspergillus niger.

    2. Does fermentation produce citric acid?
    Answer: Yes, Citric acid is exclusively produced by submerged fermentation of  Aspergillus niger. Industrial production is performed using carbohydrates or agro-industrial residues as substrates by three different types of processes: submerged, surface, and solid fermentations.

    3. Which is the most common method for citric acid production?
    Answer: Submerged fermentation is the most common method for producing citric acid. It is the most commonly employed technique and it is estimated that about 80 % of the world's production of citric acid is obtained by submerged fermentation.

    4. Which countries produce citric acid?
    Answer: About 35-40% of the world's citric acid production comes from China.

    5. Which substrate is used in the fermentation of citric acid?
    Answer: Generally sucrose is the substrate for citric acid production.

    6. What is the minimum pH required for citric acid production?
    Answer: In the control production medium, the initial pH of 6.5 is gradually reduced to 1.5 during fermentation.

    7. What microorganism is used to produce citric acid?
    Answer: Industrial citric acid is produced by submerged fermentation of starch or sucrose based on a nutrient medium (molasses) using Aspergillus niger, Aspergillus wentii, Bacillus licheniformis, Candida oleophila, etc. are used in the production of citric acid.

    8. What are the different techniques of citric acid production?
    Answer: Industrial citric acid production can be carried out in three different ways: Surface fermentation. Submerged fermentation. Solid-state fermentation.

    9. Why is Aspergillus used for citric acid production?
    Answer: Aspergillus niger is superior to other microorganisms for the commercial synthesis of citric acid because of its better production yield. It is easy to handle, can ferment various cheap raw materials, and delivers high yields.

    10. Which fungus is used in the industrial production of citric acid?
    Answer: Fungus Aspergillus niger

    11. How useful is citric acid for the body?
    Answer: The chemical characteristics of citric acid allow a person to use it in the medical field - this substance perfectly removes dangerous toxic substances from the body, including salts and toxins. In addition, it improves immunity and increases the amount of calcium in the body. 

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