Biochemical Engineering Journal
Volume 161,
15 September 2020
, 107688
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https://doi.org/10.1016/j.bej.2020.107688Get rights and content
Abstract
Propionibacterium freudenreichii has been successfully applied to industrial anaerobic production of vitamin B12. In the traditional fed-batch fermentation process, 5,6-dimethylbenzimidazole (DMB) is used as a precursor. However, DMB can inhibit the synthesis of vitamin B12 metabolic intermediates, which is not conducive to semi-continuous fermentation of P. freudenreichii. To improve the fermentation efficiency, vitamin B12 ex-situ biosynthesis was proposed and a membrane separation coupling fermentation technology was designed with adenosylcobinamide (Ado-cbi) as the indicator signal. The results showed that P. freudenreichii cells should be partly separated online at 84 h with 0.22-μm spiral membrane. The fourfold concentrated suspension was suitable for ex-situ biosynthesis of vitamin B12 with the addition of 3.60 mg L−1 DMB at 30 ℃ and pH 7.0. In the semi-continuous coupling fermentation process, vitamin B12 concentration reached 56.76 ± 3.86 mg L−1, an improvement of 161.6 %, while reutilizing fermentation wastewater and improving the anaerobic fermentation efficiency of P. freudenreichii.
Introduction
Vitamin B12 is an essential vitamin that is widely used in medicine and nutrition [1]. At present, microbial de novo biosynthesis of vitamin B12 is used in industrial production, predominantly utilizing Pseudomonas denitrificans, Propionibacterium freudenreichii and Propionibacterium shermanii [2]. Of these organisms, P. freudenreichii is regarded as a GRAS (Generally recognized as safe) strain.
Microbial de novo biosynthesis of vitamin B12 is a very complicated process and involves about 30 enzyme-mediated steps [3]. Although the regulatory mechanism of vitamin B12 is still unclear, its vital intermediates have been identified, and include δ-aminolevulinate (ALA), uroporphyrinogen III, cobyrinic acid, cob(II)yrinate acid a,c-diamide, and adenosylcobinamide (Ado-cbi) [4]. Generally, vitamin B12 biosynthesis can be divided into four parts: synthesis of ALA, synthesis of the corrin ring component, construction of the lower axial ligand, and synthesis of cobalamin. This process can be regulated at the transcriptional level (e.g. operon organization) and the metabolic level (e.g. identification of ALA as the first metabolic intermediate) [[5], [6], [7]]. A cobalamin riboswitch is also utilized to quickly respond to the environment [8]. It was reported that a complex interdependent and interactional relationship existed among the tetrapyrrole compounds, including cobalamin, heme, and bacteriochlorophyII, which were derived from ALA [9]. Thus, to accurately investigate its regulatory mechanism, detailed studies should be carried out with the aid of systems biology and synthetic biology.
According to the proposed vitamin B12 anaerobic pathway (Fig. S1), α-pyrroleribose can be activated to form the lower ligand base (5,6-dimethylbenzimidazole, DMB), which links to Ado-cbi-GDP to synthesize vitamin B12 [10,11]. Because of its low concentration, α-pyrroleribose is a key rate-limiting factor, which leads to the accumulation of intracellular Ado-cbi. Thus, extra precursor DMB needs to be added to improve vitamin B12 productivity [12]. In the traditional fermentation process of P. freudenreichii, Ado-cbi accumulation and vitamin B12 biosynthesis were completed in the same fermenter. However, because of feedback inhibition of propionic acid or its salts (above 40 g L−1), vitamin B12 production only reached 18−20 mg L−1, which weakened the advantages of anaerobic fermentation. In addition, DMB inhibited the synthesis of vitamin B12 metabolic intermediates, and the fermentation wastewater could not be reused, which increased the burden of environmental protection measures [13].
To set up a cost-effective semi-continuous fermentation process, vitamin B12 ex-situ biosynthesis was proposed based on current knowledge. The fermentation process was artificially divided into two stages: one was for Ado-cbi accumulation and the other was for vitamin B12 biosynthesis with the addition of DMB. When Ado-cbi accumulated to its maximum concentration, some P. freudenreichii cells were separated and transferred to another reactor to biosynthesize vitamin B12, while residual unseparated cells were cultivated continuously in the fermenter.
In this study, the feasibility of vitamin B12 ex-situ biosynthesis with Ado-cbi as the indicator signal was analyzed. The impacts of different factors were investigated and the optimum process parameters were determined. Finally, a membrane separation coupling fermentation technology was applied to vitamin B12 ex-situ biosynthesis, which could realize the reutilization of wastewater and improve the anaerobic fermentation efficiency of P. freudenreichii.
Section snippets
Microorganism and medium
Propionibacterium freudenreichii CICC 10019 was obtained from the Chinese Industrial Microorganism Conservation Center. The stock culture was incubated in deep agar slants at 30 ℃, stored at 4 ℃, and transferred to a new agar monthly. For long-term preservation, the stock was stored in a freezer at -80℃.
The inoculum consisted of glucose (3.5 %), corn steep liquor (2.1 %), (NH4)2SO4 (0.5 %), KH2PO4 (0.4 %), CoCl2 (0.005‰), and distilled water (pH 6.8–7.0). The fermentation medium was composed of
Effect of DMB addition time on vitamin B12 ex-situ biosynthesis
In the batch fermentation process of P. freudenreichii, vitamin B12 concentration was only 4.96 mg L−1 without DMB addition. In contrast, when 0.90 mg L-1 DMB was added in the initial stage of fermentation, vitamin B12 concentration could reach 7.87 mg L−1, an increase of 58.7 %. Therefore, it was deemed necessary to add DMB to improve vitamin B12 production. Ado-cbi is an essential compound for vitamin B12 biosynthesis and continually accumulates intracellularly. After DMB addition, Ado-cbi
Discussions
Given the complexity of metabolic regulation of vitamin B12 biosynthesis, many strategies have been applied such as random mutagenesis, promoter engineering, protein engineering, riboswitch engineering, chromosome engineering and synthetic biology. A highly efficient vitamin B12 producing strain was constructed by random mutagenesis using ultraviolet light, ethyleneimine, nitrosomethyluretane or nitrosoguanidine at Rhone-Poulenc Rorer (RPR, France) [18]. Some related genes were overexpressed,
Conclusion
Vitamin B12 ex-situ biosynthesis using a membrane separation coupling technology was designed with Ado-cbi as the indicator signal. This technique has many advantages, such as achieving wastewater reutilization, reducing sewage discharge, and improving equipment utilization and vitamin B12 fermentation efficiency. In addition, the process requires no complicated in situ product removal (ISPR) operation and is amenable to industrial amplification. After optimization, a 0.22-μm spiral membrane
CRediT authorship contribution statement
Ziqiang Wang: Conceptualization, Methodology, Formal analysis, Writing - original draft, Writing - review & editing, Funding acquisition. Guoxia Xu: Validation, Investigation, Writing - original draft, Visualization. Wei Du: Validation, Writing - original draft, Visualization. Yuhan Zhang: Validation, Writing - review & editing. Yunshan Wang: Conceptualization, Resources, Supervision, Project administration. Zhiguo Su: Conceptualization, Resources, Supervision, Funding acquisition.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgments
We thank Austin Schultz, PhD, from Liwen Bianji, Edanz Editing China (www.liwenbianji.cn/ac), for editing the English text of a draft of this manuscript. This work was financially supported by the National Natural Science Foundation of China (Grant No. 21506227).
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Citation Excerpt :
Propionibacterium was the second most abundant in this study AFBR-NP biofilm (12.6% - Fig. 6). This genus is highly desirable in acidogenic reactors whose purpose is propionic acid and vitamin B12 production and it is commonly applied as pure culture in metabolic engineered anaerobic reactors [67–70]. Furthermore, propionic acid was the third most present acid (Table 2) both in operational Stage I – 0.99 ± 0.54 g L-1 and Stage II – 0.27 ± 0.05 g L-1.
This study is the first report that describes Iron (16.7mg Fe0 g-1) and Nickel (0.04mg Ni0 g-1) nanoparticles (NPs) immobilization on the support material of an Anaerobic Fluidized Bed Reactor (AFBR-NP) and the assessment of its influence on fermentation pathway when compared with a reactor without NPs (AFBR-Control). AFBRs were operated at initial pH 6.8, 37°C, 3.0h Hydraulic Retention Time. Natural fermentation along with bioaugmentation with Clostridium butyricum were used as inoculation strategy. Both reactors were fed with glucose as main substrate (56±6 and 21±2g COD L-1 d-1 at operational Stages I and II, respectively). 78–98% higher Volatile Fatty Acid (VFA) was observed on AFBR-NP. Metals higher availability specifically favored butyric (1.04±0.37gL-1) and acetic (1.22±0.33gL-1) acid production metabolic route on AFBR-NP. Under the same operational conditions, lactic acid was observed as main metabolite on AFBR-Control (1.23±0.25gL-1). Enzymes related to butyric (butyryl-CoA dehydrogenase, phosphate butyryltransferase, butyrate kinase) and propionic acid (pyruvate-ferredoxin oxidoreductase) metabolic routes were identified on AFBR-NP reactor biofilm. Through these results, it is possible to infer that Fe0 and Ni0 NPs could effectively improve VFA production and the fermentation process.
Advances in the biosynthesis of tetrapyrrole compounds
2022, Shengwu Gongcheng Xuebao/Chinese Journal of Biotechnology
Metabolic Flux Analysis of Simultaneous Production of Vitamin B<inf>12</inf> and Propionic Acid in a Coupled Fermentation Process by Propionibacterium freudenreichii
2021, Applied Biochemistry and Biotechnology
Efficient co-production of propionic acid and succinic acid by Propionibacterium acidipropionici using membrane separation coupled technology
2021, Engineering in Life Sciences
Metabolic flux analysis of simultaneous production of vitamin B<inf>12</inf> and propionic acid in a coupled fermentation process by Propionibacterium freudenreichii
2021, ResearchSquare
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