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Time course profiles for vitamin B12 production with different propionic acid concentrations in EBAB. (A) Without controlling propionic acid concentration; (B) propionic acid concentrations controlled to 20–30 g/L; (C) propionic acid concentrations controlled to 10–20 g/L and (D) propionic acid concentrations controlled to 0–10 g/L.
Source publication
An efficient fermentation-strengthening approach was developed to improve the anaerobic production of propionic acid and vitamin B12 by co-fermentation with Propionibacterium freudenreichii. Vitamin B12 production from glucose resulted in relatively high productivity (0.35 mg/L h) but a low propionic acid yield (0.55 g/g). By contrast, glycerol gav...
Contexts in source publication
Context 1
... acid concentration on AdoCbi production were concerned in terms of carbon flux distribution (from substrates to products). Experiments on different propionic acid concentrations (0-10, 10-20, and 20-30 g/L) were conducted using the EBAB system. Time course profiles of cell growth, product biosynthesis, and substrate consumption are described in Fig. ...
Context 2
... in this study successfully pro- duced AdoCbi and propionic acid. During fermentation, propionic acid was more growth associated than AdoCbi because AdoCbi synthesis occurred later than propionic acid synthesis during cell growth. A 22 h lag phase was observed before glucose was metabolized during fermentation without propionic acid control ( Fig. 2A). The substrate was rapidly consumed, and product syn- thesis steadily increased. After 84 h of fermentation, propionic acid concentration reached 30.9 g/L and both cell growth and AdoCbi biosynthesis decreased. This result may be attributed to feed-back inhibition. The maximum AdoCbi concentration of 40.4 mg/L was achieved after 160 h ...
Context 3
... This result may be attributed to feed-back inhibition. The maximum AdoCbi concentration of 40.4 mg/L was achieved after 160 h of fermentation, with a yield of 0.67 mg/g. During fermentation at 20-30 g/L propionic acid concentration, feed-back inhibition was slightly severed, and both cell accumu- lation and product biosynthesis increased (Fig. 2B). After 160 h of fermentation, the maximum AdoCbi concentration of 48.4 mg/L was obtained. Accordingly, the glucose consumption rate was aggravated, and the lag phase was shortened to 10 h. A similar tendency of fermentation profile was also observed, and feed-back inhibition was significantly mitigated by controlling propionic acid ...
Context 4
... of fermentation, the maximum AdoCbi concentration of 48.4 mg/L was obtained. Accordingly, the glucose consumption rate was aggravated, and the lag phase was shortened to 10 h. A similar tendency of fermentation profile was also observed, and feed-back inhibition was significantly mitigated by controlling propionic acid concentration at 10-20 g/L (Fig. 2C). Interestingly, AdoCbi biosyn- thesis remarkably increased from 36 to 72 h with 10-20 g/L and 72-160 h with 20-30 g/L (Fig. 2B and C). These data indicate that propionic acid concentration should be controlled at a relatively low range (e.g., 10-20 g/L) in the early stage and at a high range (e.g., 20-30 g/L) in the late stage of ...
Context 5
... and the lag phase was shortened to 10 h. A similar tendency of fermentation profile was also observed, and feed-back inhibition was significantly mitigated by controlling propionic acid concentration at 10-20 g/L (Fig. 2C). Interestingly, AdoCbi biosyn- thesis remarkably increased from 36 to 72 h with 10-20 g/L and 72-160 h with 20-30 g/L (Fig. 2B and C). These data indicate that propionic acid concentration should be controlled at a relatively low range (e.g., 10-20 g/L) in the early stage and at a high range (e.g., 20-30 g/L) in the late stage of vitamin B12 fermentation. Exces- sively controlling propionic acid in broth (e.g., 0-10 g/L) accelerated substrate consumption and cell ...
Context 6
... at a relatively low range (e.g., 10-20 g/L) in the early stage and at a high range (e.g., 20-30 g/L) in the late stage of vitamin B12 fermentation. Exces- sively controlling propionic acid in broth (e.g., 0-10 g/L) accelerated substrate consumption and cell growth but decreased vitamin B12 biosynthesis, with a maximum AdoCbi of 42.3 mg/L (Fig. ...
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The dairy propionibacteria, which are traditionally used for the production of Swiss cheeses, are able to synthesize valuable biomolecules, e.g. B group vitamins, propionic acid, and trehalose with unique chemical and physical properties. Both, dairy propionibacteria cells and trehalose, have found many applications as attractive and effective comp...
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Background
Propionibacterium freudenreichii is an industrially important bacterium granted the Generally Recognized as Safe (the GRAS) status, due to its long safe use in food bioprocesses. Despite the recognized role in the food industry and in the production of vitamin B12, as well as its documented health-promoting potential, P. freudenreichii r...
The fermented dairy products produced by various microorganism’s activity provide valuable nutrients for human. Fermentation affects the physicochemical and organoleptic characteristics of foods as well as human health. In the present review, we describe the production of vitamin B12 in a fermented dairy product by Propionibacterium species. The ef...
Citations
... Furthermore, the production of high amounts of other organic acids as by-products adds to the challenge. To address this issue, several approaches have been explored, such as increasing the initial cell density through either cell immobilization [13,[35][36][37][38][39] or recycling [14,[40][41][42][43][44], in situ product removal [9,15,22,[45][46][47][48], increasing N-source concentration [43,49], using propionic acid-tolerant mutants [17,35,[50][51][52], continuous removal of the resulting acid via continuous operation [13,40], and genetic engineering to reduce the acidic by-products and increase carbon flux towards PA [17,24,[53][54][55]. ...
... Fermentation of the glycerol/glucose mixture (90 g/L total C-sources) was somewhat similar to that of 85 g/L glycerol with regards to cell density (20 g CDW /L), and PA-, SA-, and AA concentrations but with a lower PA volumetric productivity (0.48 vs. 0.77), [14]. The addition of glycerol as a feed after 24 h or 48 h of glucose fermentation rather than at the beginning of the fermentation was reported to enhance the yield of both PA (0.7 g/g C-sources) and vitamin B12 (0.72 mg/g C-sources) by Propionibacterium freudenreichii [48]. ...
Background
Propionic acid fermentation from renewable feedstock suffers from low volumetric productivity and final product concentration, which limits the industrial feasibility of the microbial route. High cell density fermentation techniques overcome these limitations. Here, propionic acid (PA) production from glucose and a crude glycerol/glucose mixture was evaluated using Acidipropionibacterium acidipropionici, in high cell density (HCD) batch fermentations with cell recycle. The agro-industrial by-product, heat-treated potato juice, was used as N-source.
Results
Using 40 g/L glucose for nine consecutive batches yielded an average of 18.76 ± 1.34 g/L of PA per batch (0.59 gPA/gGlu) at a maximum rate of 1.15 gPA/L.h, and a maximum biomass of 39.89 gCDW/L. Succinic acid (SA) and acetic acid (AA) were obtained as major by-products and the mass ratio of PA:SA:AA was 100:23:25. When a crude glycerol/glucose mixture (60 g/L:30 g/L) was used for 6 consecutive batches with cell recycle, an average of 35.36 ± 2.17 g/L of PA was obtained per batch (0.51 gPA/gC-source) at a maximum rate of 0.35 g/L.h, and reaching a maximum biomass concentration of 12.66 gCDW/L. The PA:SA:AA mass ratio was 100:29:3. Further addition of 0.75 mg/L biotin as a supplement to the culture medium enhanced the cell growth reaching 21.89 gCDW/L, and PA productivity to 0.48 g/L.h, but also doubled AA concentration.
Conclusion
This is the highest reported productivity from glycerol/glucose co-fermentation where majority of the culture medium components comprised industrial by-products (crude glycerol and HTPJ). HCD batch fermentations with cell recycling are promising approaches towards industrialization of the bioprocess.
... To reduce this latter limitation, main industrial P. freudenreichii producers have been obtained through random mutagenesis processes where strains with high tolerance to propionic acid were selected [25]. Other approaches, like genetic engineering [26,27], several strategies to decrease propionic acid concentration, such as expanded bed adsorption bioreactors [28,29], co-culture with propionic consuming strains [30] and supplementation with different Cbl precursors [23,31], were tested in the past, although results are often very strain dependent and further characterization and optimization is still needed. ...
Background
Vitamin B12 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. Besides, the use of Generally Recognized as Safe (GRAS) and Qualified Presumption of Safety (QPS) microorganisms, like Propionibacterium freudenreichii, especially non-GMO wild-type producers, are becoming an interesting alternative in markets where many final consumers have high health and ecological awareness. In this study, the production of vitamin B12 using the Propionibacterium freudenreichii NBRC 12391 wild-type strain was characterized and optimized in shake flasks before assessing several scale-up strategies.
Results
Initial results established that: (i) agitation during the early stages of the culture had an inhibitory effect on the volumetric production, (ii) 5,6-dimethylbenzimidazole (DMBI) addition was necessary for vitamin B12 production, and (iii) kinetics of vitamin B12 accumulation were dependent on the induction time when DMBI was added. When scaling up in a bioreactor, both batch and fed-batch bioprocesses proved unsuitable for obtaining high volumetric productivities mainly due to carbon source limitation and propionic acid inhibition, respectively. To overcome these drawbacks, an anaerobic single-phase continuous bioprocess strategy was developed. This culture strategy was maintained stable during more than 5 residence times in two independent cultures, resulting in 5.7-fold increase in terms of volumetric productivity compared to other scale-up strategies.
Conclusion
Overall, compared to previously reported strategies aimed to reduce propionic acid inhibition, a less complex anaerobic single-phase continuous and more scalable bioprocess was achieved.
... Among ISPR techniques, the use of expanded-bed adsorption bioreactors (EBABs) with high biocompatibility resins, such as ZGA330, has been reported to support vitamin B 12 volumetric production levels between 40 mg/L and 60 mg/L [52]. In EBABs, adsorption occurs when the column is expanded, allowing the culture to pass through the chromatographic column without clogging, while propionic acid is retained in the resin [54]. Different culture conditions [54], carbon and nitrogen sources [51] and the addition of media supplements, such as DMBI [53], have been tested for the simultaneous improvement of propionic acid and vitamin B 12 production. ...
... In EBABs, adsorption occurs when the column is expanded, allowing the culture to pass through the chromatographic column without clogging, while propionic acid is retained in the resin [54]. Different culture conditions [54], carbon and nitrogen sources [51] and the addition of media supplements, such as DMBI [53], have been tested for the simultaneous improvement of propionic acid and vitamin B 12 production. In an EBAB system, the combination of glucose and glycerol [54] and corn stalk hydrosylates [51] have proven to be efficient carbon sources, with reported volumetric CNCbl production levels of 43.2 mg/L and 47.6 mg/L, respectively. ...
... Different culture conditions [54], carbon and nitrogen sources [51] and the addition of media supplements, such as DMBI [53], have been tested for the simultaneous improvement of propionic acid and vitamin B 12 production. In an EBAB system, the combination of glucose and glycerol [54] and corn stalk hydrosylates [51] have proven to be efficient carbon sources, with reported volumetric CNCbl production levels of 43.2 mg/L and 47.6 mg/L, respectively. ...
Vitamin B12 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 B12 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 B12 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 B12 in different markets are discussed.
... Dairy Propionibacterium sp. and Acidipropionibacterium sp. (dairy PAB) comprise the most relevant genera to the food industry due to their role in Swiss cheese ripening, vitamin B 12 biosynthesis , propionic acid production, as well as their potential probiotic proprieties when added to foods (Chamlagain, 2016;Rabah et al., 2017;Wang et al., 2014;Yang et al., 2018). ...
... Dairy PAB are versatile microorganisms presenting few nutritional requirements, capable to metabolize several carbon sources such as glucose, xylose, molasses, and residual glycerol (Coral et al., 2008;Dishisha et al., 2013;Wang et al., 2014;Yang et al., 2018). However, the growth of these bacteria under batch process systems has some performance limitations due to strong end-product inhibition, slow-growing cells, low product titer, yields and productivities, which reduces the bioprocess outcomes, thus limiting their application at industrial scale (Ahmadi et al., 2017a;Coral et al., 2008;Liang et al., 2012;Ozadali et al., 1996). ...
... PAB can metabolize several carbon sources, such as: sugars (sucrose, glucose, fructose, lactose, galactose, and xylose), molasses (from various sources such as sugarcane and soybeans), organic acids (lactic and glucuronic acids), fatty acids (linolenic, oleic, and palmitic), and other organic compounds such as glycerol (Coral et al., 2008;Goswami & Srivastava, 2000;Hedayati et al., 2020;Wang et al., 2014;Yang et al., 2018). Energy (ATP) and reduced co-factors are initially obtained in pyruvate production throughout glycolysis or the pentose phosphate pathway. ...
The dairy bacteria Propionibacterium sp. and Acidipropionibacterium sp. are versatile and potentially probiotic microorganisms showing outstanding functionalities for the food industry, such as the production of propionic acid and vitamin B12 biosynthesis. They are the only food grade microorganisms able to produce vitamin B12. However, the fermentation batch process using these bacteria present some bioprocess limitations due to strong end-product inhibition, cells slow-growing rates, low product titer, yields and productivities, which reduces the bioprocess prospects for industrial applications. The high cell density culture (HCDC) bioprocess system is known as an efficient approach to overcome most of those problems. The main techniques applied to achieve HCDC of dairy Propionibacterium are the fed-batch cultivation, cell recycling, perfusion, extractive fermentation, and immobilization. In this review, the techniques available and reported to achieve HCDC of Propionibacterium sp. and Acidipropionibacterium sp. are discussed, and the advantages and drawbacks of this system of cultivation in relation to biomass formation, vitamin B12 biosynthesis, and propionic acid production are evaluated.
... Over 70 g/L propionic acid, equivalent to 0.42 g/g yield Whey as source of lactose mainly (Anderson et al. 1986;Boyaval and Corre 1987;Champagne et al. 1989;Colomban et al. 1993;Jiang et al. 2015;Teles et al. 2019;Woskow and Glatz 1991) Various propionibacteria One of the highest propionate productivities ever reported (14 g/L h) (Boyaval and Corre 1987). One of the highest propionate concentrations ever reported (135 g/L) (Jiang et al. 2015) Corn steep liquor mainly as source of nitrogen and vitamins (Quesada-Chanto et al. 1997;Quesada-Chanto et al. 1998;Wang et al. 2014;Wang and Yang 2013;Yang et al. 2018), and occasionally as source of carbon too (Ozadali et al. 1996;Paik and Glatz 1994;Teles et al. 2019) Mostly A. acidipropionici and P. freudenreichii Up to 45.6 g/L concentration using corn steep liquor as rich medium (Paik and Glatz 1994) of 0.54 g/g (Kagliwal et al. 2013). Wheat flour is rich in gluten, which can be enzymatically hydrolyzed to serve as a source of both carbon and nitrogen, thus further improving the economics and sustainability of the process. ...
... Corn steep liquor, a byproduct of wet corn milling processes, has been widely investigated, mostly as inexpensive nitrogen and vitamin source that can replace more expensive sources, such as yeast extract (Quesada-Chanto et al. 1997;Quesada-Chanto et al. 1998;Wang et al. 2014;Wang and Yang 2013;Yang et al. 2018). Still, relatively few studies have been conducted on propionic acid production from corn steep liquor as a rich feedstock providing carbon (mainly lactic acid) and nitrogen, vitamins, and minerals (Ozadali et al. 1996;Paik and Glatz 1994;Teles et al. 2019). ...
Propionic acid is an important organic acid with wide industrial applications, especially in the food industry. It is currently produced from petrochemicals via chemical routes. Increasing concerns about greenhouse gas emissions from fossil fuels and a growing consumer preference for bio-based products have led to interest in fermentative production of propionic acid, but it is not yet competitive with chemical production. To improve the economic feasibility and sustainability of bio-propionic acid, fermentation performance in terms of concentration, yield, and productivity must be improved and the cost of raw materials must be reduced. These goals require robust microbial producers and inexpensive renewable feedstocks, so the present review focuses on bacterial producers of propionic acid and promising sources of substrates as carbon sources. Emphasis is placed on assessing the capacity of propionibacteria and the various approaches pursued in an effort to improve their performance through metabolic engineering. A wide range of substrates employed in propionic acid fermentation is analyzed with particular interest in the prospects of inexpensive renewable feedstocks, such as cellulosic biomass and industrial residues, to produce cost-competitive bio-propionic acid.
Key points
• Fermentative propionic acid production emerges as competitor to chemical synthesis.
• Various bacteria synthesize propionic acid, but propionibacteria are the best producers.
• Biomass substrates hold promise to reduce propionic acid fermentation cost.
... Propionic acid fermentation is known to suffer from end-product inhibition and byproduct formation, mainly acetic and succinic acids, which lower the yield and productivity of propionic acid (Jin and Yang 1998). To overcome these limitations, various bioprocessing approaches have been applied (Eş et al. 2017;Liu et al. 2012a;Wang et al. 2014;Wang et al. 2012), including metabolic engineering to enhance the producer strains, but genetic manipulation of propionibacteria has proved to be difficult Ammar et al. 2014;Wang et al. 2015b;Wang et al. 2015d;Wei et al. 2016). As a result, propionic acid fermentation is still not economically competitive with petrochemical methods, and hence, further improvements are needed. ...
Propionic acid, a widely used food preservative and intermediate in the manufacture of various chemicals, is currently produced from petroleum-based chemicals, raising concerns about its long-term sustainability. A key way to make propionic acid more sustainable is through fermentation of low-cost renewable and inedible sugar sources, such as lignocellulosic biomass. To this end, we utilized the cellulosic hydrolysate of sweet sorghum bagasse (SSB), a residue from a promising biomass source that can be cultivated around the world, for fermentative propionic acid production using Propionibacterium freudenreichii. In serum bottles, SSB hydrolysate supported a higher propionic acid yield than glucose (0.51 vs. 0.44 g/g, respectively), which can be attributed to the presence of additional nutrients in the hydrolysate enhancing propionic acid biosynthesis and the pH buffering capacity of the hydrolysate. Additionally, SSB hydrolysate supported better cell growth kinetics and higher tolerance to product inhibition by P. freudenreichii. The yield was further improved by co-fermenting glycerol, a renewable byproduct of the biodiesel industry, reaching up to 0.59 g/g, whereas volumetric productivity was enhanced by running the fermentation with high cell density inoculum. In the bioreactor, although the yield was slightly lower than in serum bottles (0.45 g/g), higher final concentration and overall productivity of propionic acid were achieved. Compared to glucose (this study) and hydrolysates from other biomass species (literature), use of SSB hydrolysate as a renewable glucose source resulted in comparable or even higher propionic acid yields.
Key points
• Propionic acid yield and cell growth were higher in SSB hydrolysate than glucose.
• The yield was enhanced by co-fermenting SSB hydrolysate and glycerol.
• The productivity was enhanced under high cell density fermentation conditions.
• SSB hydrolysate is equivalent or superior to other reported hydrolysates.
... Review f reudenreichii-based cofermentation using glycerol and glucose as carbon sources to synthesize propionic acid and B 12 . 961 Butyric acid is a significant building block in the production of industrially valued chemicals. For the microbial synthesis of butyric acid, different microorganisms from various biological habitats have been isolated. ...
Bioelectrocatalysis is an interdisciplinary research field combining biocatalysis and electrocatalysis via the utilization of materials derived from biological systems as catalysts to catalyze the redox reactions occurring at an electrode. Bioelectrocatalysis synergistically couples the merits of both biocatalysis and electrocatalysis. The advantages of biocatalysis include high activity, high selectivity, wide substrate scope, and mild reaction conditions. The advantages of electrocatalysis include the possible utilization of renewable electricity as an electron source and high energy conversion efficiency. These properties are integrated to achieve selective biosensing, efficient energy conversion, and the production of diverse products. This review seeks to systematically and comprehensively detail the fundamentals, analyze the existing problems, summarize the development status and applications, and look toward the future development directions of bioelectrocatalysis. First, the structure, function, and modification of bioelectrocatalysts are discussed. Second, the essentials of bioelectrocatalytic systems, including electron transfer mechanisms, electrode materials, and reaction medium, are described. Third, the application of bioelectrocatalysis in the fields of biosensors, fuel cells, solar cells, catalytic mechanism studies, and bioelectrosyntheses of high-value chemicals are systematically summarized. Finally, future developments and a perspective on bioelectrocatalysis are suggested.
... The PA and vitamin 12 production by glucose and glycerol fermentation are in contrast to each other. Therefore, co-fermentation of glycerol and glucose is suggested to improve the productivity of both products (93). ...
During the past years, there has been a growing interest in the bioproduction of propionic acid by Propionibacterium. One of the major limitations of the existing models lies in their low productivity yield. Hence, many strategies have been proposed in order to circumvent this obstacle. This article provides a comprehensive synthesis and review of important biotechnological aspects of propionic acid production as a common ingredient in food and biotechnology industries.
We first discuss some of the most important production processes, mainly focusing on biological production. Then, we provide a summary of important propionic acid producers, including Propionibacterium freudenreichii and Propionibacterium acidipropionici, as well as a wide range of reported growth/production media. Furthermore, we describe bioprocess variables that can have impact on the production yield. Finally, we propose methods for the extraction and analysis of propionic acid and put forward strategies for overcoming the limitations of competitive microbial production from the economical point of view.
Several factors influence the propionic acid concentration and productivity such as culture conditions, type and bioreactor scale; however, the pH value and temperature are the most important ones. Given that there are many reports about propionic acid production from glucose, whey permeate, glycerol, lactic acid, hemicelluloses, hydrolyzed corn meal, lactose, sugarcane molasses and enzymatically hydrolyzed whole wheat flour, only few review articles evaluate biotechnological aspects, i.e. bioprocess variables.
... However, co-fermentation of glucose and glycerol resulted in 0.71 g/g yield and 0.36 g/L/h productivity values. It was also reported that productions in expanded bed adsorption bioreactor reached up to 91.4 g/L propionic acid production with 0.75 g/g yield, and 0.35 g/L/h productivity [168]. Addition to its antimicrobial properties, 3-hydroxypropionic acid has a tremendous potential for the production of 3-carbon intermediates such as acrylamide, acrylonitrile, malonic acid, 1,3-propanediol, and ethyl 3-hydroxypropionic acid. ...
Food safety is a global health and socioeconomic concern since many people still suffer from various acute and life-long diseases, which are caused by consumption of unsafe food. Therefore, ensuring safety of the food is one of the most essential issues in the food industry, which needs to be considered during not only food composition formulation but also handling and storage. For safety purpose, various chemical preservatives have been used so far in the foods. Recently, there has been renewed interest in replacing chemically originated food safety compounds with natural ones in the industry, which can also serve as antimicrobial agents. Among these natural compounds, organic acids possess the major portion. Therefore, in this paper, it is aimed to review and compile the applications, effectiveness, and microbial productions of various widely used organic acids as antimicrobial agents in the food industry.
... In Fig. 2a and b clearly the extremum point is visible while in Fig. 2c monotonic profile reflects the effect of amino acids on the growth and metabolic regulation in the pfre. Growth-associated cobalamin synthesis has been proved in Propionibacterium genus (Wang et al., 2014). Different carbon sources, as well as the effect of the combination of nutrients were studied on the growth of pfre. ...
Vitamin B12 contributes many substantial metabolic cycles in the living organisms. Since human beings cannot produce such co-factor by their metabolism, they have to receive this vitamin from foods and supplements. Dimethylbenzimidazole (DMBI) distinguishes the active form of vitamin B12 from pseudo-vitamin B12. De Novo total biosynthesis of vitamin B12 in the bacteria should include DMBI biosynthesis through riboflavin pathway. Propionibacterium freudenreichii can produce vitamin B12 through anaerobic biosynthesis pathway. As vitamin B12 production by P. freudenreichii is the growth-associated phenomena, the effect of different carbon sources (rice bran oil, argan oil), nutrients (DMBI) and amino acids (L-Serin, L-Tryptophan, l-cysteine, l-Methionine) on the growth of Propionibacterium freudenreichii PTCC1674 (pfre) were investigated. Through the statistical analysis of vitamin B12 production, rice bran oil (RBO) was selected as the sole carbon source. By applying Plackett-Burman method, significant parameters of vitamin B12 production were extracted and optimized based on Box-Behnken design of experiments. RBO, DMBI and CaCl2.2H2O concentrations and temperature were the four main effective parameters of vitamin B12 production. Via implemented response to surface methodology (RSM), the response was optimized to 2.94 mg/L, while 14% increase of vitamin B12 (cyanocobalamin) production was obtained at RBO concentration of 8.648% V/V, temperature of 38.3 (°C), DMBI concentration of 55.758 (mg/L) and elemental solution concentration of 2 (mg/L). It was concluded that pfre can grow on rice bran oil as a new carbon source while the changing of culture media composition alters the growth profile. Box-Behnken design effectively optimized parameters achieved from Plackett-Burman screening method.
