Industrial Citric Acid Production: Methods, Microbiology & Industrial Applications

Table of Contents

• Introduction
• Properties of Citric Acid
• Historical Events 
• Citric Acid Biochemistry and Accumulation
• Microorganisms Used in CA Production
• Raw Materials Used in Citric Acid Fermentation
• Industrial Citric Acid Production Approach
• Limiting Factors during Citric Acid Production
• Applications of Citric Acid
• Advantages of Microbial Citric Acid Production
• Limitations and Challenges
• Conclusion
• References

Introduction

• Citric acid (CA) is an essential weak organic acid widely used in the pharmaceutical and food industries due to its presence as a common metabolite in both animals and plants.
• CA can be produced on a large scale through either synthetic or natural extraction and manufacturing methods.
• Natural production involves extracting citric acid from citrus fruits such as oranges, lemons, limes, and others.
• Synthetic production follows two main approaches:
• Chemical synthesis, utilizing enzymes to facilitate citric acid formation.
• Biological fermentation, which involves microorganisms like bacteria and yeast for CA production.
• Industrial citric acid production primarily relies on fermentation methods, with the most common being submerged fermentation, where:
• Microorganisms like Aspergillus niger (fungi) and Candida (yeast) metabolize carbon sources such as molasses and starch-based media to produce citric acid.
• Other fermentation techniques include:
• Surface fermentation, where citric acid is produced on the surface of the medium.
• Solid-state fermentation, which utilizes agro-based residues as the carbon source for large-scale citric acid production.

Properties of Citric Acid

• Citric acid (CA) is a naturally occurring weak organic acid with the molecular formula C₆H₈O₇.
• Its IUPAC name is 2-hydroxy-1,2,3-propane tricarboxylic acid.
• As a tricarboxylic acid, it has a molecular weight of 210.14 g/mol.
• Naturally present in citrus fruits such as lemons and is also a common metabolite in animals.
• Acts as a primary metabolite in the Krebs cycle (tricarboxylic acid cycle), where it is derived from carbohydrates, proteins, and fats to fulfill energy needs.
• Melting point: 153°C.
• In its pure form, citric acid is soluble in water and also dissolves in polar solvents like acetone and ethanol.
• At room temperature, it exists in a solid state.
• Produced on a large scale either through chemical biosynthesis (enzymatic process) or biological fermentation using microorganisms.

Historical Events in Citric Acid Production

• 1874: Karl Scheele was the first scientist to isolate citric acid through crystallization from lemon juice.
• 1880: Adams and Grimaux successfully synthesized citric acid from glycerol.
• 1893: Wehmer conducted fermentation using Penicillium gaucum and observed citric acid accumulation as a by-product of calcium oxalate.
• 1917: James Currie, a food chemist, demonstrated that Aspergillus niger produced higher yields of citric acid than other microorganisms when grown in a sugar-based medium.
• 1922: Millard enhanced citric acid accumulation by limiting nutrients in bioreactors where Aspergillus niger carried out fermentation.
• 1965: Yeasts were introduced for citric acid production, utilizing alkanes and carbohydrates as substrates.
• 1984: The Submerged Fermentation Technique (SmF) was introduced in the U.S. industries to enable large-scale citric acid production.

Citric Acid Biochemistry and Accumulation

• 1953: Hans Adolf Krebs was awarded the Nobel Prize in Physiology or Medicine for discovering the Citric Acid Cycle.
• Citric Acid Cycle (TCA/Krebs Cycle):
• Citrate (Citric Acid) is an intermediate metabolite in the Krebs cycle, found in bacteria, plants, and animals.
• The production of citrate occurs through the condensation of Acetyl-CoA and Oxaloacetate, catalyzed by the citrate synthase enzyme.
• Citrate acts as a substrate for downstream reactions in the TCA cycle, which regenerates oxaloacetate at the end.
• Citrate is exported from mitochondria to the cytoplasm, where it serves as a substrate and regulates various biochemical processes.
• Accumulation of Citric Acid:
• Hypothesis 1: Enzymes responsible for citric acid degradation in the Krebs cycle must be deactivated to accumulate CA. However, the Krebs cycle remains active for biomass production.
• Hypothesis 2: Tricarboxylate transporter activity competes with aconitase for binding to CA.
• The transporter has a higher affinity for CA and ejects citrate into the cytoplasm without inhibiting Krebs cycle enzymes.

Microorganisms Involved in Citric Acid Production

Microbial fermentation is the most efficient and widely adopted method for citric acid production. The primary microorganism used in this process is Aspergillus niger, a filamentous fungus that has a high yield of citric acid under optimized conditions. Other microorganisms such as Candida lipolytica, Yarrowia lipolytica, and Penicillium species can also produce citric acid but are less commonly used in industrial applications.

Raw Materials Used in Citric Acid Fermentation

Citric acid production requires carbon and nitrogen sources, along with essential minerals. Common carbon sources include:

• Molasses (sugarcane or beet-based)

• Glucose or sucrose

• Starch hydrolysates

• Agricultural residues like corn steep liquor or fruit waste

Nitrogen sources include ammonium salts, peptones, yeast extract, and corn steep liquor. The choice of raw materials significantly affects the yield and efficiency of citric acid production.

Industrial Citric Acid Production Approach

Approximately 99% of global citric acid (CA) production is achieved through microbial fermentation using surface or submerged culture techniques. The final product is either marketed as citric acid monohydrate or in its anhydrous form.

Usage of Citric Acid:

• Food & Beverage Industry (70%): Used as an acidifier and antioxidant to enhance flavors and preserve products such as fruit juices, marmalades, ice cream, and other food items.
• Pharmaceutical Industry (20%): Utilized as a blood preservative, antioxidant, and vitamin preservant, and in the formulation of effervescent tablets, iron citrate supplements, ointments, and cosmetic products.
• Chemical Industry: Functions as a foaming agent for textiles, a cement hardening agent, and a phosphate substitute.

a. Surface Culture Method

Surface culture fermentation is categorized into solid-state and liquid fermentation, with the liquid approach being more economically viable.

Process of Surface Culture Fermentation:

1. Culture Medium Preparation:

• The medium is poured into shallow aluminum trays (5–20 cm deep) and adjusted to a pH of 5–6.
• Temperature, humidity, and air circulation are maintained in the fermentation chamber.

2. Inoculation with Aspergillus niger:

• A. niger spores are blown onto the culture surface for 5–6 days, with controlled dry air passage.
• The medium pH is adjusted to 1.5–2 to promote citric acid synthesis.
• After 24 hours, the spores germinate, forming white mycelial growth.
• The mold begins consuming sugar from the medium, and once the sugar is fully utilized, the remaining liquid is extracted.
• A. niger produces citric acid as its primary metabolite in this method.

Inoculum Preparation:

• A high-yielding strain of A. niger is selected from stock cultures.
• Glass vials are incubated for 10–14 days, after which sporulating media are inoculated with the selected strain.
• Trace elements (zinc, iron, manganese) are added to the sporulating media.
• The mature spores are suspended in a diluent containing sodium lauryl phosphate (a wetting agent).

Media Preparation:

• The fermentation medium requires a carbon source (sucrose or beet molasses) and inorganic salts.
• Beet molasses are the preferred commercial-scale carbon source due to their rich inorganic salt content.
• To remove excess inorganic salts, ferricyanide or ferrocyanide treatment is performed.
• Additional nutrients include nitrogen, phosphorus, potassium, and magnesium.
• Salts such as ammonium nitrate, magnesium sulfate, and potassium mono-hydrogen phosphate are added in small quantities.
• The pH is adjusted to 3.4–3.5 using HCl, which helps in sterilization, contamination control, oxalic acid suppression, and citric acid yield enhancement.

Fermentation:

• The prepared media (1–2.5 cm thick) is placed in shallow trays.
• The inoculum is spread evenly, and aeration is maintained for proper spore distribution.
• The trays are incubated at 30–40°C, with constant air ventilation for efficient gas exchange.
• After 24 hours, spores germinate, forming a thin mycelial layer.
• The medium pH drops to 1.5–2.0 due to the formation of ammonium ions.
• Presence of oxalic acid and a yellow pigment after 30 hours signals high iron levels, which can halt citric acid production.
• Fermentation lasts 8–14 days, during which thick mycelial growth develops on the liquid surface.
• Citric acid yield is proportional to the surface area-to-volume ratio, meaning larger surface areas enhance production.
• Under optimal conditions, the process can produce 1.2–1.5 kg of citric acid monohydrate per square meter per hour.

Recovery Process:

1. Mycelium is separated from the fermentation broth.
2. The pressed mycelium releases any intracellular citric acid.
3. The filtered broth is treated with calcium hydroxide, forming calcium citrate precipitate.
4. The precipitate is reacted with sulfuric acid, regenerating citric acid and producing calcium sulfate (gypsum) as a byproduct.
5. The solution is filtered, demineralized, and decolorized using activated carbon.
6. The final citric acid crystals are obtained via evaporation.

b. Submerged Culture Method

This fermentation approach employs Aspergillus japonicus, a black Aspergillus species. Unlike surface fermentation, this method utilizes stainless steel bioreactors equipped with cooling systems, aeration, impellers, and monitoring equipment.

Process of Submerged Culture Fermentation:

1. Culture Medium & Carbon Sources:

• Substrates include maize starch, beet molasses, and glucose.
• Ammonia serves as the nitrogen source.
• The medium is maintained at 30°C, and A. japonicus is added to initiate fermentation.

2. Production Yield:

• Batch bioreactors using 2500 kg of glucose and 860 kg of oxygen generate:
• 1500 kg of citric acid
• 500 kg of biomass
• The type of metal used in fermenter construction, oxygenation systems, and mycelium structures significantly influences production efficiency.
• Candida lipolytica, an alkane-metabolizing fungus, can enhance citric acid yield by 45% compared to traditional methods.

Inoculum Preparation:

• A high-yielding strain of A. japonicus is selected from stock cultures.
• Mycelial mats ("pellets") serve as inoculum for fermentation.
• The seed fermenter contains 15% molasses sugar, which induces spore germination.
• Cyanide ions are introduced to stimulate high mycelial production.
• Higher cyanide concentrations are required to avoid filamentous mycelium growth, which reduces citric acid output.
• Within 24 hours at 32°C, the spores germinate, forming 0.2–0.5 mm mycelial pellets.
• The pH drops to 4.3, and these pellets serve as starter cultures for production fermenters.

Fermentation:

• Stainless steel fermenters (1000 L minimum capacity) are used to prevent heavy metal leaching.
• The iron-to-copper ratio influences mycelial pellet formation, which is crucial for optimal citric acid production.
• Oxygen concentration (20–25% saturation) is maintained for maximum yield.
• Aeration rates are set between 0.2–1 volume per minute.
• Lard oil is added as an antifoaming agent to prevent excessive foam formation.

Recovery Process:

• The same protocols as surface culture fermentation are followed for citric acid extraction, purification, and crystallization.

Limiting Factors in Citric Acid Production

1. Fungal Species

Several fungal species, including Aspergillus niger, Penicillium luteum, Ustulina vulgaris, and others, have been shown to produce citric acid. However, selecting a high-yield and quality strain is crucial for optimal CA production. Aspergillus niger is the most widely preferred species due to its high productivity, ease of cultivation, and minimal production of undesirable by-products like oxalic acid.

2. Carbon Source

For large-scale citric acid production, it is essential to use sugars that can be easily assimilated and metabolized by the microorganisms involved. Polysaccharides can serve as raw materials if the microbial strain produces hydrolytic enzymes that function effectively at the low pH required for fermentation. Sucrose is the preferred carbon source over glucose since A. niger possesses the invertase enzyme, which catalyzes the hydrolysis of sucrose into glucose and fructose.

Common industrial carbon sources include sugar beet molasses, lower-grade sugarcane by-products with cation content, and glucose syrup derived from starch hydrolysis. The highest productivity is achieved when the fermentation medium contains sugar concentrations ranging from 15% to 22%.

3. Nitrogen and Phosphate

Beet molasses, commonly used in complex fermentation media, are rich in nitrogen. In laboratory-prepared media, nitrogen is supplemented using ammonium sulfate and nitrates, which help maintain the low pH essential for fermentation. Urea is also a viable nitrogen source in media preparation.

Phosphate plays a vital role in promoting biomass growth by enhancing secondary metabolic reactions, which can indirectly influence citric acid production.

4. Culture Medium pH

pH variation occurs in two distinct stages during citric acid production. Initially, during spore germination, the pH should be maintained below 5. As fermentation progresses, the absorption of ammonium ions by germinating spores leads to the release of protons, which causes the pH to drop to 2 or lower. This pH reduction facilitates citric acid synthesis, minimizes contamination risks, and inhibits the production of unwanted organic acids such as oxalic and gluconic acids.

5. Aeration

Proper aeration is critical for efficient citric acid production, as fluctuations in oxygen supply can negatively impact output. Excessive aeration results in low carbon dioxide levels, which can affect the activity of pyruvate carboxylase, an enzyme that replenishes oxaloacetate for citrate synthase. Since oxaloacetate is a precursor for citric acid synthesis, insufficient CO₂ can hinder production. Conversely, excessively high CO₂ levels are also unfavorable for optimal citric acid yield.

6. Trace Elements

Maintaining a balanced concentration of trace elements such as manganese (Mn), iron (Fe), zinc (Zn), and copper (Cu) is essential for fungal growth and maximum citric acid production. The recommended levels of these elements are:

• Zinc – 0.3 ppm
• Iron – 1.3 ppm
• Manganese – 3 mg/L

Manganese, in particular, plays a crucial role in regulating various cellular functions, including sporulation, cell wall synthesis, and the production of secondary metabolites, all of which contribute to efficient citric acid biosynthesis.

Applications of Citric Acid

Citric acid has a broad spectrum of applications across various industries:

• Food Industry: Used as an acidulant, preservative, and flavor enhancer in beverages, candies, jams, and dairy products.
• Pharmaceutical Industry: Acts as an excipient in tablet formulation, a pH regulator, and a chelating agent in medicine.
• Cosmetic Industry: Found in skincare products, shampoos, and cosmetics for pH adjustment and preservation.
• Industrial Uses: Used in detergents, metal cleaning, and as an anticoagulant in blood transfusions.

Advantages of Microbial Citric Acid Production

• Cost-Effective: Fermentation-based production is more economical than chemical synthesis.
• Renewable Resources: Utilizes agro-industrial residues, reducing waste and promoting sustainability.
• High Purity: Microbial fermentation yields a high-purity product suitable for food and pharmaceutical applications.

Limitations and Challenges

• Substrate Availability: Dependence on sugar-based substrates can affect production costs.
• Contamination Risks: Fermentation is prone to microbial contamination, which can reduce yield.
• Process Optimization: Requires precise control of environmental factors for maximum efficiency.

Conclusion

Citric acid production through microbial fermentation, particularly using Aspergillus niger, has become an essential process in the food, pharmaceutical, and industrial sectors. Advances in bioprocess engineering and genetic modification of microorganisms continue to enhance yield, efficiency, and cost-effectiveness. With growing demand and research innovations, citric acid production will remain a crucial aspect of the biotechnology industry.

References

• "Production of Citric Acid." Biology Reader. Available at: Biology Reader
• "Citric Acid Production." Microbiology Note. Available at: Microbiology Note
• "Citric Acid." Wikipedia. Available at: Wikipedia
• "Solvents." Thermo Fisher Scientific - Qualigens Chemicals. Available at: Thermo Fisher
• Vandenberghe, Luciana P. S., et al. (1999). "Microbial Production of Citric Acid." Brazilian Archives of Biology and Technology, 42, 263-276.
• Max, Belén, et al. (2010). "Biotechnological Production of Citric Acid." Brazilian Journal of Microbiology, 41, 862-875.