How Is Artificial B12 Made? The Intricate Process of Microbial Fermentation

December 6, 2025 •

4 min read

Vitamin B12 biosynthesis is exclusively performed by certain bacteria and archaea, as humans and plants cannot produce it. To meet the global demand for supplements and fortified foods, artificial B12 is produced on a massive scale using a blend of controlled microbial fermentation and precise chemical engineering.

Quick Summary

Artificial B12, or cyanocobalamin, is manufactured using specific bacteria in a multi-stage process involving fermentation, extraction, purification, and chemical conversion. This method leverages microbial synthesis to create a stable, cost-effective form for supplements and fortified foods.

Key Points

Microbial Fermentation: Artificial vitamin B12 is primarily produced using selected bacteria like Propionibacterium and Pseudomonas in large, industrial fermentation tanks.
Natural Precursor Synthesis: Bacteria naturally synthesize cobalamin analogues, which are precursors to vitamin B12, through complex multi-step biosynthetic pathways.
Chemical Conversion: The bacterially produced intermediates, such as hydroxocobalamin, are converted into the more stable cyanocobalamin form through a precise chemical reaction with a small amount of cyanide.
Purification and Extraction: After fermentation, the vitamin must be carefully extracted from the microbial biomass and purified using techniques like centrifugation and chromatography to ensure a high-purity final product.
Engineered Microbes: Advancements in synthetic biology and metabolic engineering allow for the creation of more efficient bacterial strains, like engineered E. coli, to improve vitamin B12 yields and lower production costs.
Cyanocobalamin's Stability: The final cyanocobalamin form is preferred for supplements because it is more stable against heat, light, and oxidation, giving it a longer shelf life.

The Core of Artificial B12 Production: Microbial Fermentation

Artificial vitamin B12, predominantly the stable cyanocobalamin form, is not created through a purely synthetic chemical process due to its immense molecular complexity. Instead, industrial production relies on microbial fermentation, a process that harnesses specific bacteria to naturally synthesize the cobalamin molecule. These microbes are cultivated in large bioreactors under carefully controlled conditions to maximize the production yield. Common bacterial strains used include Propionibacterium freudenreichii subsp. shermanii and Pseudomonas denitrificans, with newer research exploring genetically engineered E. coli and other species for higher efficiency and yield.

Step 1: Cultivating the Microbial Culture

The process begins with preparing a starter culture of the chosen bacterial strain. A sterile growth medium is formulated to provide all the necessary nutrients, such as carbon sources (like glucose or molasses), nitrogen, and essential trace elements, including cobalt. The bacterial inoculum is added to this medium in massive fermentation tanks, often exceeding 100,000 liters, and kept under optimal conditions of temperature and pH for several days.

Step 2: Fermentation and Cobalamin Biosynthesis

During fermentation, the selected bacteria produce cobalamin analogues. Depending on the bacteria, this can be done via aerobic or anaerobic pathways. For example, P. freudenreichii often uses an anaerobic pathway, while P. denitrificans uses an aerobic one. The fermentation conditions, including oxygen levels, are tightly managed to control the metabolic processes and maximize production. For instance, in some cases, a shift from high to low oxygen transfer rates can significantly improve productivity. Throughout this stage, the bacteria synthesize the complex corrin ring structure and insert a cobalt atom, producing an intermediate form like hydroxocobalamin.

Step 3: Extracting and Purifying the Cobalamin

After the fermentation process is complete, the valuable cobalamin is extracted from the culture broth and bacterial biomass. This involves several key steps:

Cell Harvesting: The bacterial cells are separated from the liquid medium, typically through high-speed centrifugation.
Cell Disruption: The harvested cell mass is treated with heat or chemicals to rupture the cells and release the intracellular cobalamin.
Clarification: The mixture is filtered to remove solid impurities and produce a crude extract containing the target compound.
Purification: The crude extract undergoes various chromatographic and precipitation techniques to isolate the vitamin from other substances.

Step 4: Chemical Conversion to Cyanocobalamin

The cobalamin compounds extracted from the bacteria are typically in forms like hydroxocobalamin, which are less stable than cyanocobalamin. To create the stable, shelf-ready form used in supplements, a final chemical conversion is performed. This involves adding a cyanide source, such as potassium cyanide, under specific conditions to replace the hydroxyl group with a cyanide group, forming cyanocobalamin. The amount of cyanide used is minuscule, and the final product is purified to remove any trace amounts.

Advancements in B12 Production

Advances in metabolic engineering and synthetic biology are constantly improving B12 production. These innovations include:

Optimizing Fermentation: Research focuses on fine-tuning fermentation conditions like temperature, pH, and nutrient supply to increase yields.
Engineering Strains: Scientists genetically engineer bacteria like E. coli to improve their ability to produce cobalamin, making them more efficient than traditional producers.
Cell-Free Systems: Newer research explores using cell-free enzymatic systems that don't rely on living microbes, offering higher yields and faster production times.
Affinity Chromatography: Novel purification techniques are being developed to streamline the extraction process, potentially reducing purification time significantly.

Comparison of Key Vitamin B12 Production Strains


Feature	Propionibacterium freudenreichii	Pseudomonas denitrificans	Engineered Escherichia coli
Production Pathway	Primarily anaerobic pathway	Primarily aerobic pathway	Salvage pathway (engineered for de novo synthesis)
Media Requirements	Can use cheaper substrates like molasses and needs specific conditions	Historically used more complex media, but now optimized for cost-effective sources	Versatile, can use cheap carbon sources like glucose, but needs specific genes added
Cobalamin Form Produced	Can produce a variety of cobalamin analogues	Produces cobalamin analogues, converted to cyanocobalamin	Can be engineered to produce different cobalamin types
Key Advantages	Efficient anaerobic process; long-established industrial use	Historically high-yielding aerobic strain; well-understood pathway	Rapid growth; highly customizable via genetic engineering
Challenges	Potentially long fermentation cycle; requires specific media	Can produce potentially harmful waste products; costly handling of materials	Pathway can be complex to engineer; potential metabolic burden on host

Conclusion: A Blend of Nature and Technology

The industrial production of artificial vitamin B12 represents a sophisticated marriage of natural biological processes and advanced chemical and genetic engineering. By leveraging the inherent biosynthetic capabilities of microorganisms like Propionibacterium and Pseudomonas and refining the process with modern techniques, manufacturers can reliably produce large quantities of stable, high-purity cyanocobalamin for global demand. This multi-step process, from fermentation to final chemical conversion, ensures a safe and cost-effective product for use in dietary supplements and food fortification, addressing a crucial nutritional need for millions worldwide. As research continues to advance, the efficiency and sustainability of B12 manufacturing are expected to improve further. More information on the pathways involved can be found on the National Institutes of Health website.

Note: A detailed review of the microbial production of vitamin B12 can be found here: Microbial production of vitamin B12: a review and future perspectives.

Frequently Asked Questions

No, the minuscule amount of cyanide used in the final chemical conversion is safely and efficiently detoxified by the body. The resulting cyanocobalamin is considered safe and is converted to other active forms of B12 within the body.

The ability to produce vitamin B12 is confined to specific microorganisms, such as bacteria and archaea. Animals obtain B12 through their diet or gut flora, while plants typically lack this synthesis pathway entirely.

The main difference is in the final chemical form. Artificial B12 is typically cyanocobalamin, a synthetic and highly stable form. Natural B12 found in animal products is predominantly methylcobalamin and adenosylcobalamin. The body efficiently converts cyanocobalamin into these active forms.

Bacteria are the 'factories' that perform the complex biological synthesis of the basic cobalamin structure. They are cultivated on a large scale to produce the precursor molecules that are later modified and purified to create the final artificial B12.

The process involves several stages: culturing specific bacteria, fermenting them to produce cobalamin, extracting the vitamin from the bacterial biomass, purifying the crude extract, and finally, chemically converting the cobalamin into the stable cyanocobalamin form.

Cyanocobalamin is favored for supplements due to its superior stability and cost-effectiveness. It is less susceptible to degradation from light, heat, and oxygen compared to other, more sensitive, B12 forms, ensuring a longer shelf life.

A purely synthetic chemical synthesis of vitamin B12 is theoretically possible but is too complex, expensive, and inefficient for industrial-scale production. Thus, leveraging microbial biosynthesis remains the standard and most economically viable method.