B12 Production Research Articles

Lettuce fortification through vitamin B12-producing bacteria - proof of concept study.

Vitamin B12 (cobalamin) can be produced de novo only by certain bacteria and archaea. It plays a crucial role in the health of animals and humans, which obtain it only through diet, mainly from animal products. This study aimed to identify endophytic bacterial strains capable of synthesizing vitamin B12 and enriching edible plants with it as a potential solution for vitamin B12 deficiency in vegetarians, vegans, and people with poor diets. An in silico genome analysis was performed on 66 bacterial genomes, including the reference strain Pseudomonas denitrificans ATCC 13867, a known vitamin B12 producer. The genomes were analyzed using the Rapid Annotations using Subsystems Technology (RAST) server and the MetaCyc database to verify the presence and completeness of the vitamin B12 metabolic pathway. The ability of the strains to produce vitamin B12 was confirmed with a high-performance liquid chromatography with diode-array detection (HPLC-DAD) analysis of pure culture extracts. Eleven strains produced detectable amounts of vitamin B12 under tested conditions. The best performing candidates were further tested for their efficacy in producing vitamin B12 in lettuce grown under sterile conditions on Murashige and Skoog (MS) medium with or without CoCl2 supplementation. Methylobacterium sp. strain P1-11 produced detectable amounts of vitamin B12 in planta: 1.654 and 2.559 μg per g of dry weight without and with CoCl2 supplementation, respectively. This is the first time a bacterial endophyte was used to produce vitamin B12 in planta, suggesting that bacterial endophytes could be utilized to enhance the nutraceutical values of plant-based foods. © 2025 The Author(s). Journal of the Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Complete genome sequence of Limosilactobacillus reuteri LU150, a potential vitamin B12 producer from the NORDBIOTIC collection.

We performed whole-genome sequencing of Limosilactobacillus reuteri LU150, a human fecal isolate from the NORDBIOTIC collection. The genome consists of 2,039,406 bp with a GC content of 38.9%. The genetic content of LU150 suggests its probiotic potential.

Strategy for Optimizing Vitamin B12 Production in Pseudomonas putida KT2440 Using Metabolic Modeling.

Background/Objectives: Vitamin B12 is very important for human health, as it is a cofactor for enzymatic activities and plays various roles in human physiology. It is highly valued in the pharmaceutical, food, and additive production industries. Some of the bacteria currently used for the vitamin production are difficult to modify with gene-editing tools and may have slow growth. We propose the use of the bacteria Pseudomonas putida KT2440 for the production of vitamin B12 because it has a robust chassis for genetic modifications. The present wok evaluates P. putida KT2440 as a host for vitamin B12 production and explore potential gene-editing optimization strategies. Methods: We curated and modified a genome-scale metabolic model of Pseudomonas putida KT2440 and evaluated different strategies to optimize vitamin B12 production using the knockin and OptGene algorithms from the COBRA Toolbox. Furthermore, we examined the presence of riboswitches as cis-regulatory elements and calculated theoretical biomass growth yields and vitamin B12 production using a flux balance analysis (FBA). Results: According to the flux balance analysis of P. putida KT2440 under culture conditions, the biomass production values could reach 1.802 gDW-1·h1·L-1, and vitamin B12 production could reach 0.359 µmol·gDW-1·h-1·L-1. The theoretical vitamin B12 synthesis rate calculated using P. putida KT2040 with two additional reactions was 14 times higher than that calculated using the control, Pseudomonas denitrificans, which has been used for the industrial production of this vitamin. Conclusions: We propose that, with the addition of aminopropanol linker genes and the modification of riboswitches, P. putida KT2440 may become a suitable host for the industrial production of vitamin B12.

Enhanced vitamin B12 production by isolated Bacillus strains with the application of response surface methodology

BackgroundVitamin B12 is a crucial B-group vitamin, first isolated from the liver due to its role in combating pernicious anemia. It is distinguished by its unique and complex structure, which makes its chemical synthesis challenging and expensive. Consequently, vitamin B12 is alternatively obtained through microbial fermentations. Molasses, an affordable and safe agro-industrial waste, can be used as a carbon source for vitamin B12 production, offering a cost-effective alternative to expensive sugars in the production medium.ResultsA total of 87 yeast, actinomycete, and bacterial isolates were screened for vitamin B12 production, with 15 isolates showing high productivity. Bacillus isolates were selected for further analysis using MALDI-TOF and molecular identification. These isolates were identified as four strains of Bacillus subtilis (MZ08, JT10, BY11, and JT17), one strains of Bacillus sp. (CB09), and one strain of Peribacillus acanthi (MZ01). Genetic circuits associated with vitamin B12 production were demonstrated in a closely related strain of Peribacillus acanthi MZ01 strain. Three strains (MZ01, MZ08, and JT17) were selected for further evaluation of vitamin B12 productivity under different sugar types (glucose, sucrose, fructose, lactose, and galactose) and varying inoculum sizes. The inoculum size significantly impacted vitamin B12 production, with an increase from 5 to 10% enhancing yields. The ability of the strains to produce vitamin B12 varied depending on the type of sugar used. Peribacillus acanthi MZ01 strain showed the highest productivity and subsequently, selected for optimizing vitamin B12 production conditions using response surface methodology. Furthermore, the optimized conditions were then applied to molasses-based medium to achieve high vitamin B12 yields by MZ01 strain.ConclusionIn this study, Peribacillus acanthi was characterized for the first time as a vitamin B12 producer, demonstrating high productivity among various tested strains. The optimization of production conditions using response surface methodology, further enhanced vitamin B12 yields, showcasing the strain’s efficiency in microbial fermentations. This research also highlights the potential of using molasses as a cost-effective alternative carbon source, significantly reducing production costs.

Lactic and propionic acid bacteria starter cultures for improved nutritional properties of pea, faba bean and lentil

Increasing plant-based food consumption as a sustainable and health-oriented alternative to meat is pivotal. Pulses are rich in proteins, minerals, and vitamins; however, they also contain antinutritional compounds, impairing their nutritional value. This study addresses this challenge through the development and application of four distinct microbial consortia in pulse-based fermentations, featuring lactic acid bacteria or a combination of lactic and propionic acid bacteria. Microbial starters significantly reduced galacto-oligosaccharides in all pulse materials, concurrently degrading vicine and convicine in faba beans, while the impact on tannins in faba beans and lentil was moderate. Fermentation with lactic acid and propionic acid bacteria consortia exhibited notable vitamin B12 production, and the effect on the content of phenolic compounds of the studied pulses was also evidenced. Additionally, genomic analyses discerned distinctive profiles among the samples, elucidating the microbial community dynamics shaping fermentation outcomes. The results of this study proved how fermentation can advance the development of pulse-based products with improved nutritional and sustainability attributes.

In situ fortification of cereal by-products with vitamin B12: An eco-sustainable approach for food fortification

Vitamin B12 deficiency poses significant health risks, especially among populations with limited access to animal-based foods. This study explores the utilisation of cereal bran by-products, wheat (WB) and oat bran (OB), as substrates for in situ vitamin B12 fortification through solid-state fermentation (SSF) using Propionibacterium freudenreichii. The impact of various precursors addition, including riboflavin, cobalt, nicotinamide and DMBI on vitamin B12 production, along with changes in microbial growth, chemical profiles, and vitamin B12 yields during fermentation was evaluated. Results showed that WB and OB possess favourable constituents for microbial growth and vitamin B12 synthesis. The substrates supplemented with riboflavin, cobalt, and DMBI demonstrated enhanced B12 production. In addition, pH levels are essential in microbial viability and cobalamin biosynthesis. Monosaccharides and organic acids play a crucial role, with maltose showing a strong positive association with B12 production in OB, while in WB, citric acid exhibits significant correlations with various factors.

Source of Vitamin B12 in plants of the Lemnaceae family and its production by duckweed-associated bacteria

While most plants are not known to contain significant levels of the essential corrinoid cobalamin (vitamin B12), considerable levels of vitamin B12 were found in the duckweed clone Wolffia globosa Mankai. Although endophytes were speculated to be the source of this vitamin B12, it has not been tested yet. In this study, we carried out comparative analysis of vitamin B12 content in 15 duckweed accessions across 11 species. Significant but variable levels of vitamin B12 were found in all unsterile duckweed cultures tested. In contrast, disinfected duckweed cultures with lower levels of bacteria, as monitored by PCR-based methods, contained little vitamin B12. Importantly, two strains of duckweed-associated bacteria were shown to produce vitamin B12. This work provides evidence for bacteria as the source of vitamin B12 in duckweeds. Bioinformatics-aided identification of vitamin B12 producers among duckweed-associated bacteria could facilitate their systematic incorporation into duckweed-based foods to support a planetary health diet.

Biofortification, metabolomic profiling and quantitative analysis of vitamin B12 enrichment in guava juice via lactic acid fermentation using Levilactobacillus brevis strain KU15152.

Chemical fortification and dose supplementation of vitamin B12 are widely implemented to combat deficiency symptoms. However, in situ, fortification of vitamin B12 in food matrixes can be a promising alternative to chemical fortification. The present study aimed to produce vitamin B12-rich, probiotic guava juice fermented with Levilactobacillus brevis strain KU15152. Pasteurized fresh guava juice was inoculated with 7.2 log CFU mL-1 L. brevis strain KU15152 and incubated for 72 h at 37 °C anaerobically. The antioxidants, total phenolic compounds, vitamin B12 production, sugars, organic acids, pH and viable count were analyzed at 24, 48 and 72 h of incubation. The fermented juice was stored at 4 °C, and the changes in its functional properties were analyzed at 7-day intervals up to 28 days of storage. During fermentation, the bacteria cell count was increased from 7.01 ± 0.06 to 9.76 ± 0.42 log CFU mL-1 after 72 h of fermentation and was decreased to 6.94 ± 0.34 CFU mL-1 during storage at 4 °C after 28 days. The pH, total soluble solids, crude fiber, citric acid and total sugars decreased, while titratable acidity, total protein, antioxidants, phenolic compounds and lactic acid contents increased during fermentation. The fermented guava juice exhibited higher 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid (ABTS)) radical scavenging activities (85.97% and 75.97%, respectively) at 48 h of fermentation. The concentration of active vitamin B12 in the sample reached 109.5 μg L-1 at 72 h of fermentation. However, this concentration gradually decreased to 70.2 μg L-1 during the storage period. During storage for 28 days at 4 °C, both the fermented and control guava juices exhibited a decline in antioxidant and phenolic compound concentrations. Furthermore, the addition of 20% honey and guava flavor enhanced the organoleptic properties and acceptability of fermented guava juice. The value-added fermented guava juice could be a novel functional food product to combat vitamin B12 deficiency. © 2024 The Author(s). Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Obtaining Novel Vitamin B12 Production Strains Acetobacter malorum HFD 3141 and Acetobacter orientalis HFD 3031 from Home-Fermented Sourdough

Vitamin B12 is a critical nutrient in vegan and vegetarian lifestyles as plant-based vitamin sources are rare. Traditional fermented foods could be enriched by adding vitamin B12-producing bacteria to offer non-animal vitamin sources. The aim was to isolate a vitamin B12 producer that is capable of producing the human-active vitamin even at low pH values so that it can be used in fruit juice fortification. Therefore, fermented foods (homemade and industrial) and probiotics were screened for vitamin B12 production strains. A modified microbiological vitamin B12 assay based on Lactobacillus delbrueckii subsp. lactis DSM 20355 was used to identify vitamin B12-containing samples and the presence of vitamin B12-producing strains. The screening resulted in isolating several positive strains for vitamin B12 formation derived from sourdough. Mass spectrometry confirmed the biosynthesis of solely the human physiologically active form. Species identification carried out by the German Strain Collection of Microorganisms and Cell Cultures resulted in two species: Acetobacter orientalis and Acetobacter malorum, of which two isolates were further characterised. The potential for cobalamin biosynthesises in food matrixes was demonstrated for A. malorum HFD 3141 and A. orientalis HFD 3031 in apple juice at different pH values (2.85–3.80). The isolates synthesised up to 18.89 µg/L and 7.97 µg/L vitamin B12 at pH 3.80. The results of this study suggest that acetic acid bacteria (AAB) and fermented acetic acid foods are promising resources for vitamin B12 and its producers, which might have been overlooked in the past.

Ligand cross-feeding resolves bacterial vitamin B12 auxotrophies.

Cobalamin (vitamin B12, herein referred to as B12) is an essential cofactor for most marine prokaryotes and eukaryotes1,2. Synthesized by a limited number of prokaryotes, its scarcity affects microbial interactions and community dynamics2-4. Here we show that two bacterial B12 auxotrophs can salvage different B12 building blocks and cooperate to synthesize B12. A Colwellia sp. synthesizes and releases the activated lower ligand α-ribazole, which is used by another B12 auxotroph, a Roseovarius sp., to produce the corrin ring and synthesize B12. Release of B12 by Roseovarius sp. happens only in co-culture with Colwellia sp. and only coincidently with the induction of a prophage encoded in Roseovarius sp. Subsequent growth of Colwellia sp. in these conditions may be due to the provision of B12 by lysed cells of Roseovarius sp. Further evidence is required to support a causative role for prophage induction in the release of B12. These complex microbial interactions of ligand cross-feeding and joint B12 biosynthesis seem to be widespread in marine pelagic ecosystems. In the western and northern tropical Atlantic Ocean, bacteria predicted to be capable of salvaging cobinamide and synthesizing only the activated lower ligand outnumber B12 producers. These findings add new players to our understanding of B12 supply to auxotrophic microorganisms in the ocean and possibly in other ecosystems.

Whole-Genome Shotgun Metagenomic Sequencing Reveals Shifts in the Skin Microbiome and Bacteriophages of Psoriasis: An Extended Analysis of Published Data

Background Psoriasis is an immune-mediated cutaneous disease that may have shifts in the skin microbiome. Prior research on the skin microbiome in psoriasis has been limited to rRNA based approaches that lack resolution of taxonomic and functional level assessment. Objective To further illuminate strain and sub-strain level analysis of psoriatic lesions using the CosmosID-HUB Microbiome pipeline. Methods A previous study completed by Tett et al recruited patients with psoriasis who had skin microbiome samples taken from psoriatic plaques on the ear and the elbow as well as sites on the skin unaffected by psoriasis. They performed whole genome shotgun sequencing and made their dataset publicly available. We analyzed the dataset using the CosmosID-HUB Microbiome pipeline to evaluate the strain and sub-strain taxonomic analysis as well as functional gene profiling. Results When analyzed with the CosmosID pipeline, both ear and elbow sites in affected areas had decreased alpha diversity compared to unaffected areas. There was an increased relative abundance of Staphylococcus and Corynebacteria at affected sites. We identified distinguishing species and strains of the yeast Malassezia, including M. restricta. that were significantly enriched in healthy elbow samples. Vitamin B12 production genes were not present in psoriatic skin whereas it was present in healthy samples, supporting the notion of relative vitamin B12 deficiency in psoriatic plaques. Phage analysis revealed a greater diversity of Staphylococcus-related phages in unaffected elbow samples. Conclusion A greater diversity of microbial strains and their functional roles identified in this study may help to tailor treatment for psoriasis.

Developing and Optimizing Media Formulations Using Agro waste for the Production of Fungal Secondary Metabolites, Specifically Vitamin B12

Vitamin B12 is a water-soluble compound that is essential for human health, typically sourced from animal products or synthesized through microbial fermentation. This study explored the formulation of mycological media from agro waste for production of Vitamin B12. The media formulated were agro waste from sugarcane and sweet potato (SSP) and sugarcane and cassava (SC). Experimental condition variables including different percentages (1% and 2%) of carbon (Na3C6H5O7) and nitrogen (NaNO3) source, hydrogen ion concentration (pH:5,6,7and 8), different ratios of 5-6 dimethylbenzimidazole (5,6-DMB) and cobalt (Co) and temperature (30oC and 37oC) were optimized using standard procedures in order to enhance microbial production of vitamin B12. The fungi Aspergillus niger (A. niger) strains screened for production of vitamin B12 were obtained from soil sample collected from hospital dumping sites. Statistical analysis of data was done using ANOVA and results were presented as means ± standard deviations. Results obtained from optimization showed that temperature, pH, and different percentages of carbon and nitrogen have significant (p ≤ 0.05) effects on fungal growth as well as its production of secondary metabolite (vitamin B12). The result also showed that the effect of different ratios of DMB and Co on vitamin B12 production was significant (P<0.05). The optimum conditions for the growth of A. niger and synthesis of its secondary metabolite were observed at temperature 30oC (0.4µg/ml±0.006), pH 7.0 (0.92µg/ml±0.007) DMB and Co ratio 80: 0.75 mg (0.94µg/ml± 0.006) in SC medium. The outcome of this study has indicated that vitamin B12 would be effectively produced by A.niger strains using formulations of agro waste: Sugarcane and cassava media supplemented with appropriate percentage of carbon and nitrogen, adequate ratio of DMB and Co and incubation at optimum temperature and pH values. These findings have significant implications for the food, pharmaceutical and biotechnology industries

Production of vitamin B12 via microbial strains isolated from marine and food sources in Egypt

Background Vitamin B12 is a very important water-soluble vitamin, which was first isolated from the liver as an anti-pernicious anemia factor. The sole source of vitamin B12 is the animal-based food. It has a complicated structure and requires expensive multi-steps to be synthesized chemically. Intriguingly, vitamin B12 can be produced through microbial fermentation by microorganisms in a cheap and more effective manner. Objective This study aims to isolate and characterize microorganisms that have the capability to produce vitamin B12. In addition, the current work aims to optimize the vitamin B12 production conditions by isolating strains using suitable waste materials to obtain a high vitamin B12 yield. Materials and methods Different bacterial and yeast isolates were isolated from marine and food samples using the pour-plate technique. These isolates were screened for vitamin B12 production using a specific growth medium that lacked vitamin B12 and a test indicator bacterium. The content of vitamin B12 was estimated using spectrophotometer measurement and high-performance liquid chromatography (HPLC). The isolates that showed high vitamin B12 productivity were identified using MALDI-TOF technique. The identified strains were implemented for the optimization of vitamin B12 production to reveal the most proper and optimum conditions for the production. Response surface methodology (RSM) was employed to enhance the production of vitamin B12 in a flask scale. Agro-industrial wastes such as molasses were used for vitamin B12 production using the most optimum conditions as determined by RSM. Results and conclusion Eighty-seven actinomycetes, bacterial, and yeast isolates were screened for vitamin B12 production. Out of these isolates, 15 showed high vitamin B12 productivity. We found that bacilli and yeast isolates were the most productive among the tested cocci and actinomycetes isolates. The highly productive Bacillus and yeast isolates were identified using the MALDI-TOF analysis. The isolates were identified as Candida pelliculosa, Geotrichum candidum, Bacillus subtilis and Bacillus sp. One strain of Candida pelliculosa (coded BYI), three strains of Geotrichum candidum (coded as MZYC, MZYD, and MZYG) were selected for studying the effect of sugar type and inoculum size on the yield of vitamin B12 production. Strain MZYD was selected for the statistical modelling using RSM to optimize seven factors for the vitamin B12 production. These factors included temperature, fermentation time, salt concentration, pH, sugar concentration, inoculum size, and aeration. Five factors i.e., temperature, pH, sugar concentration, and inoculum size were shown to significantly improve the vitamin B12 production. A maximum yield of 64.21 μg/100 ml was obtained using the optimized RSM conditions. These optimized conditions were used to produce vitamin B12 using molasses as a raw material for the microbial growth.

Gorse (Ulex europeaus) wastes with 5,6-dimethyl benzimidazole supplementation can support growth of vitamin B12 producing commensal gut microbes.

Many commensal gut microbes are recognized for their potential to synthesize vitamin B12, offering a promising avenue to address deficiencies through probiotic supplementation. While bioinformatics tools aid in predicting B12 biosynthetic potential, empirical validation remains crucial to confirm production, identify cobalamin vitamers, and establish biosynthetic yields. This study investigates vitamin B12 production in three human colonic bacterial species: Anaerobutyricum hallii DSM 3353, Roseburia faecis DSM 16840, and Anaerostipes caccae DSM 14662, along with Propionibacterium freudenreichii DSM 4902 as a positive control. These strains were selected for their potential use as probiotics, based on speculated B12 production from prior bioinformatic analyses. Cultures were grown in M2GSC, chemically defined media (CDM), and Gorse extract medium (GEM). The composition of GEM was similar to CDM, except that the carbon and nitrogen sources were replaced with the protein-depleted liquid waste obtained after subjecting Gorse to a leaf protein extraction process. B12 yields were quantified using liquid chromatography with tandem mass spectrometry. The results suggested that the three butyrate-producing strains could indeed produce B12, although the yields were notably low and were detected only in the cell lysates. Furthermore, B12 production was higher in GEM compared to M2GSC medium. The positive control, P. freudenreichii DSM 4902 produced B12 at concentrations ranging from 7 ng mL-1 to 12 ng mL-1. Univariate-scaled Principal Component Analysis (PCA) of data from previous publications investigating B12 production in P. freudenreichii revealed that B12 yields diminished when the carbon source concentration was ≤30 g L-1. In conclusion, the protein-depleted wastes from the leaf protein extraction process from Gorse can be valorised as a viable substrate for culturing B12-producing colonic gut microbes. Furthermore, this is the first report attesting to the ability of A. hallii, R. faecis, and A. caccae to produce B12. However, these microbes seem unsuitable for industrial applications owing to low B12 yields.

Vitamin B12 analogues from gut microbes and diet differentially impact commensal propionate producers of the human gut.

To produce the health-associated metabolite propionate, gut microbes require vitamin B12 as a cofactor to convert succinate to propionate. B12 is sourced in the human gut from the unabsorbed dietary fraction and in situ microbial production. However, experimental data for B12 production by gut microbes is scarce, especially on their produced B12-analogues. Further, the promotion of propionate production by microbially-produced and dietary B12 is not yet fully understood. Here, we demonstrated B12 production in 6 out of 8 in silico predicted B12-producing bacteria from the human gut. Next, we showed in vitro that B12 produced by Blautia hydrogenotrophica, Marvinbryantia formatexigens, and Blautia producta promoted succinate to propionate conversion of two prevalent B12-auxotrophic gut bacteria, Akkermansia muciniphila and Bacteroides thetaiotaomicron. Finally, we examined the propiogenic effect of commercially available B12-analogues present in the human diet (cyano-B12, adenosyl-B12 and hydroxy-B12) at two doses. The low dose resulted in partial conversion of succinate to propionate for A. muciniphila when grown with adenosyl-B12 (14.6 ± 2.4 mM succinate and 18.7 ± 0.6 mM propionate) and hydroxy-B12 (13.0 ± 1.1 mM and 21.9 ± 1.2 mM), in comparison to cyano-B12 (0.7 ± 0.1 mM and 34.1 ± 0.1 mM). Higher doses of adenosyl-B12 and hydroxy-B12 resulted in significantly more conversion of succinate to propionate in both propionate-producing species, compared to the low dose. B12 analogues have different potential to impact the propionate metabolism of prevalent propionate producers in the gut. These results could contribute to strategies for managing gut disorders associated with decreased propionate production.

Vitamin B12 production in solubilized protein extract of bioprocessed wheat bran with Propionibacterium freudenreichii

Wheat bran, a nutrient-rich byproduct of wheat milling, has the potential to be used for the production of vitamin B12 (B12) in order to fortify plant-based foods that are lacking in this vitamin. This study aimed to investigate B12 production in the soluble extract of bioprocessed wheat bran using Propionibacterium freudenreichii. The optimal conditions for B12 production were found to be a bioprocessing time of 24 h, along with the use of a commercial starter culture of lactic acid bacteria and yeast, with pH adjusted from 4.0 to 6.4. The highest B12 production (55 ng/mL) was achieved within three days of fermentation. Cobalt supplementation (as CoCl2 or food-grade yeast extract) resulted in up to four-fold increase in B12 production. Similar results (50–70 ng/mL) were obtained when the processes were scaled up, with bioprocessing involving up to 15 kg and B12 fermentation involving up to 5 L, demonstrating the robustness of the procedure. Microbiological safety was ensured with the absence of Enterobacteriaceae in the extracts after bioprocessing and B12 fermentation. Overall, this study highlights the potential of under-utilized wheat bran as a raw material for producing B12-enriched bran extract, which can be used to fortify plant-based products in various food applications.

Microbial communities of activated sludge: A potential contribution to the production of vitamin B12

The chemical synthesis of vitamin B12, an important nutrient for living organisms, is complex due to the asymmetric structure of the vitamin molecule. This opens up significant prospects for the development of biotechnological approaches to cyanocobalamin synthesis, which forms the basis of this work. The study examines the potential of activated sludge generated during wastewater treatment as a source of vitamin B12 products for the production of feed concentrates. As a nutrient medium, the possibility of using waste from alcohol processing production – stillage, is being considered. Together, this can make it possible to ensure the economic viability, sustainability and feasibility of biosynthesis of feed vitamin B12 on an industrial scale. The cultivation process was monitored by monitoring a set of indicators, including the pH of the medium, the amount of dry matter, dihydrogenase activity, weight gain and B12 content. The analysis of the data highlights the importance of understanding the complexity of the relationship between individual cultivation parameters to optimize vitamin B12 production processes. In general, the achieved level of vitamin B12 synthesis was 430 mcg/l, which allows to consider active sludge and distillery stillage as potentially promising components of the vitamin B12 biosynthesis process.

Utilization of propionic acid bacteria in the biotransformation of soy (tofu) whey: Growth and metabolite changes.

Soy whey, a by-product from the tofu and soy protein isolate industry was evaluated as a substrate for a biofortified beverage using several propionic acid bacteria (PAB). PAB growth and changes in sugars, organic acids, amino acids and isoflavones were investigated. Vitamin B12 and short-chain fatty acid (SCFA) production were measured over time. Acidipropionibacterium acidipropionici (DSM 20272) showed the highest growth, compared to the other three PABs (Propionibacterium freudenreichii [DSM 20271 and DSM 4902], A. jensenii [DSM 20535]). Acidipropionibacterium (DSM 20272 and DSM 20535) showed the best propionic acid and acetic acid production, while P. freudenreichii produced the most succinic acid. Propionibacterium freudenreichii exhibited significant vitamin B12 production at 4.06±0.28µg/L for DSM 20271, followed by 2.58±0.22µg/L for DSM 4902. Notably, all PAB displayed strong β-glycosidase activities evidenced by the conversion of isoflavone glycosides to isoflavone aglycones. The stark differences between Acidipropionibacterium spp. and Propionibacterium spp. indicate that the former PAB is specialized in SCFA production, while the latter PAB is better at vitamin B12 bioenrichment. This study demonstrated the possibility of employing PAB fermentation to improve SCFA and vitamin B12 content. This can open avenues for a beverage or functional ingredient development.

Functional redundancy of seasonal vitamin B12 biosynthesis pathways in coastal marine microbial communities.

Vitamin B12 (cobalamin) is a major cofactor required by most marine microbes, but only produced by a few prokaryotes in the ocean, which is globally B12 -depleted. Despite the ecological importance of B12 , the seasonality of B12 metabolisms and the organisms involved in its synthesis in the ocean remain poorly known. Here we use metagenomics to assess the monthly dynamics of B12 -related pathways and the functional diversity of associated microbial communities in the coastal NW Mediterranean Sea over 7 years. We show that genes related to potential B12 metabolisms were characterized by an annual succession of different organisms carrying distinct production pathways. During the most productive winter months, archaea (Nitrosopumilus and Nitrosopelagicus) were the main contributors to B12 synthesis potential through the anaerobic pathway (cbi genes). In turn, Alphaproteobacteria (HIMB11, UBA8309, Puniceispirillum) contributed to B12 synthesis potential in spring and summer through the aerobic pathway (cob genes). Cyanobacteria could produce pseudo-cobalamin from spring to autumn. Finally, we show that during years with environmental perturbations, the organisms usually carrying B12 synthesis genes were replaced by others having the same gene, thus maintaining the potential for B12 production. Such ecological insurance could contribute to the long-term functional resilience of marine microbial communities exposed to contrasting inter-annual environmental conditions.

Exploiting Potential Probiotic Lactic Acid Bacteria Isolated from Chlorella vulgaris Photobioreactors as Promising Vitamin B12 Producers.

Lactic acid bacteria (LAB) have been documented as potential vitamin B12 producers and may constitute an exogenous source of cobalamin for the microalga Chlorella vulgaris, which has been described as being able to perform vitamin uptake. Hence, there is an interest in discovering novel B12-producing probiotic LAB. Therefore, the purpose of the current work was to perform a phenotype-genotype analysis of the vitamin B12 biosynthesis capacity of LAB isolated from C. vulgaris bioreactors, and investigate their probiotic potential. Among the selected strains, Lactococcus lactis E32, Levilactobacillus brevis G31, and Pediococcus pentosaceus L51 demonstrated vitamin B12 biosynthesis capacity, with the latter producing the highest (28.19 ± 2.27 pg mL-1). The genomic analysis confirmed the presence of pivotal genes involved in different steps of the biosynthetic pathway (hemL, cbiT, cobC, and cobD). Notably, P. pentosaceus L51 was the only strain harboring cobA, pduU, and pduV genes, which may provide evidence for the presence of the cobalamin operon. All strains demonstrated the capability to withstand harsh gastrointestinal conditions, although P. pentosaceus L51 was more resilient. The potential for de novo cobalamin biosynthesis and remarkable probiotic features highlighted that P. pentosaceus L51 may be considered the most promising candidate strain for developing high-content vitamin B12 formulations.