Citation: Calvillo, Á.; Pellicer, T.; Carnicer, M.; Planas, A. Bioprocess Strategies for Vitamin B 12 Production by Microbial Fermentation and Its Market Applications. Bioengineering 2022, 9, 365. https://doi.org/ 10.3390/bioengineering9080365 Academic Editor: Sabine Kleinsteuber Received: 21 June 2022 Accepted: 29 July 2022 Published: 4 August 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). bioengineering Review Bioprocess Strategies for Vitamin B 12 Production by Microbial Fermentation and Its Market Applications Álvaro Calvillo 1 , Teresa Pellicer 2 , Marc Carnicer 1, * and Antoni Planas 1, * 1 Laboratory of Biochemistry, Institut Químic de Sarrià, University Ramon Llull, 08017 Barcelona, Spain 2 HealthTech Bio Actives, S.L.U., 08029 Barcelona, Spain * Correspondence: marc.carnicer@iqs.url.edu (M.C.); antoni.planas@iqs.url.edu (A.P.); Tel.: +34-93-267-2076 (M.C.); +34-93-267-2025 (A.P.) Abstract: Vitamin B 12 is a widely used compound in the feed and food, healthcare and medical industries that can only be produced by fermentation because of the complexity of its chemical synthesis. For this reason, finding better producer strains and optimizing their bioprocesses have been the main focus of industrial producers over the last few decades. In this review, we initially provide a historical overview of vitamin B 12 research and the main biosynthetic characteristics of the two microorganism families typically used for its industrial production: several strains of Propionibacterium freudenreichii and strains related to Pseudomonas denitrificans. Later, a complete summary of the current state of vitamin B 12 industrial production as well as the main advances and challenges for improving it is detailed, with a special focus on bioprocess optimization, which aims not only to increase production but also sustainability. In addition, a comprehensive list of the most important and relevant patents for the present industrial strains is provided. Finally, the potential applications of vitamin B 12 in different markets are discussed. Keywords: cobalamin; Propionibacterium freudenreichii; Pseudomonas denitrificans; cyanocobalamin production 1. Historical Overview Vitamin B 12 , also known as cobalamin, is a water-soluble molecule essential in many organisms’ metabolism. It has a complex structure and an elaborated biosynthesis, with over 30 biotransformation steps [1]. This biosynthetic pathway is only present in some bacteria and archaea, although the phyla capable of synthetizing vitamin B 12 are not necessarily interrelated, so mammals, and therefore humans, are unable to synthetize it. Investigation into vitamin B 12 began in the 1920s in connection to an illness firstly described in 1824, pernicious anemia. The main symptoms of this illness included fatigue, weight loss, headaches and, in severe cases, dementia, memory loss, muscle weakness and peripheral neuropathy, which can become lethal without treatment. In 1926, Minot and Murphy demonstrated that patients with pernicious anemia could successfully recover from the condition by a special diet with high amounts of lightly cooked liver and muscle meat [2]. They theorized that the treatment was successful because of an unknown “ex- trinsic factor” present in animal livers. For this discovery, they were awarded the Nobel Prize in Physiology or Medicine in 1934, although it was more than two decades before the so called “extrinsic factor” was identified and isolated. This occurred in 1948, when two research groups from pharmaceutical companies (Folkers at Merck, Sharp & Dohme, and Smith at Glaxo) isolated, almost at the same time, a cobalt compound from animal livers that was able to cure pernicious anemia on its own [3,4]. A year later, the same compound could also be isolated from other sources, such as milk, beef and several bacterial cultures. This red crystalline octahedral cobalt compound was called vitamin B 12 . Interestingly, years later, it was discovered that this compound was in fact one of the many isoforms of the Bioengineering 2022, 9, 365. https://doi.org/10.3390/bioengineering9080365 https://www.mdpi.com/journal/bioengineering