Optimization of semi-anaerobic vitamin B12 (cyanocobalamin) production from rice bran oil using Propionibacterium freudenreichii PTCC1674

Journal Pre-proof Optimization of semi-anaerobic vitamin B12 (cyanocobalamin) production from rice bran oil using Propionibacterium freudenreichii PTCC1674 Rouhollah Hedayati, Morteza Hosseini, Ghasem D. Najafpour PII:

S1878-8181(19)30924-7

DOI:

https://doi.org/10.1016/j.bcab.2019.101444

Reference:

BCAB 101444

To appear in:

Biocatalysis and Agricultural Biotechnology

Received Date: 30 June 2019 Revised Date:

7 November 2019

Accepted Date: 23 November 2019

Please cite this article as: Hedayati, R., Hosseini, M., Najafpour, G.D., Optimization of semi-anaerobic vitamin B12 (cyanocobalamin) production from rice bran oil using Propionibacterium freudenreichii PTCC1674, Biocatalysis and Agricultural Biotechnology (2019), doi: https://doi.org/10.1016/ j.bcab.2019.101444. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.

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Optimization of Semi-anaerobic Vitamin B12 (Cyanocobalamin) Production from Rice

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Bran Oil using Propionibacterium freudenreichii PTCC1674

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Rouhollah Hedayati, Morteza Hosseini*, Ghasem D. Najafpour

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Biotechnology Research Laboratory, Faculty of Chemical Engineering, Babol Noshirvani University of

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Technology, Babol, Iran.

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* Corresponding Author Email: [email protected]

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Abstract

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Vitamin B12 contributes many substantial metabolic cycles in the living organisms. Since human

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beings cannot produce such co-factor by their metabolism, they have to receive this vitamin from

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foods and supplements. Dimethylbenzimidazole (DMBI) distinguishes the active form of vitamin

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B12 from pseudo-vitamin B12. De Novo total biosynthesis of vitamin B12 in the bacteria should

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include DMBI biosynthesis through riboflavin pathway. Propionibacterium freudenreichii can

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produce vitamin B12 through anaerobic biosynthesis pathway. As vitamin B12 production by P.

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freudenreichii is the growth-associated phenomena, the effect of different carbon sources (rice

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bran oil, argan oil), nutrients (DMBI) and amino acids (L-Serin, L-Tryptophan, L-cysteine, L-

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Methionine) on the growth of Propionibacterium freudenreichii PTCC1674 (pfre) were

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investigated. Through the statistical analysis of vitamin B12 production, rice bran oil (RBO) was

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selected as the sole carbon source. By applying Plackett-Burman method, significant parameters

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of vitamin B12 production were extracted and optimized based on Box-Behnken design of

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experiments. RBO, DMBI and CaCl2.2H2O concentrations and temperature were the four main

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effective parameters of vitamin B12 production. Via implemented response to surface

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methodology (RSM), the response was optimized to 2.94 mg/L, while 14% increase of vitamin

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B12 (cyanocobalamin) production was obtained at RBO concentration of 8.648% V/V,

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temperature of 38.3 (°C), DMBI concentration of 55.758 (mg/L) and elemental solution

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concentration of 2 (mg/L). It was concluded that pfre can grow on rice bran oil as a new carbon

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source while the changing of culture media composition alters the growth profile. Box-Behnken

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design effectively optimized parameters achieved from Plackett-Burman screening method.

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Graphical abstract

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Key Words: Vitamin B12 biosynthesis; Rice bran oil; RSM Optimization; Propionibacterium

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freudenreichii

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1. Introduction

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Vitamin B12 is one of the most important water-soluble vitamins in the group of vital nutrients

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(Martens et al., 2002). Vitamin B12 in some beings cannot be synthesized via their metabolisms,

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therefore, it has to be supplied by fortified food. Cyanocobalamin (Cyano-Cbl), synthetic and the

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most stable form of produced vitamin B12, is the outcome of fermentation processes which have

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been classified into two distinct categories.1 (Fang et al., 2017): 1) fully aerobic pathway, 2)

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semi-anaerobic pathway. In the first one, the Pseudomonas strains substantially plays role

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(Acevedo-Rocha et al., 2019) and Propionibacterium strains are identified as the bio-worker in

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latter (Wang et al., 2015; Survase et al., 2006). Propionibacteriums including freudenreichii and shermanii, which have been generally

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recognized as the safe microorganisms, were selected in this study and they can produce

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intracellular cobalamins via anaerobic pathway (Piwowarek et al., 2018b). Propionibacterium

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species are gram-positive bacteria which have been classified as aerotolerant bacteria (Thierry et

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al., 2011). Apart from propionic and acetic acids as the main products of these strains’

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fermentation (Suwannakham et al., 2006; Gonzalez-Garcia et al., 2017), many efforts paid on

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Vitamin B12, 5-aminolevulenic acid (Sonhom et al., 2012), trehalose (Cardoso et al., 2004), and

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exo-polysaccharides production (Dobruchowska et al., 2008) by employing Propionibacterium

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species. Recently, complete genome sequence of Propionibacterium freudenreichii DSM20271T

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has been revealed that contains valuable identical and practical information about secondary

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metabolite synthesis pathways (Koskinen et al., 2015). 1

Many studies have introduced Cyanocobalamin production as the vitamin B12 production. In this study, it has been intended as well.

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Biosynthesis of cobalamin has attracted so many efforts to elucidate the clear route

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of such large cobalt containing biomolecule synthesis. This pathway uses around 30 enzymatic

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reaction steps to carry out cobalamin synthesis in the intracellular forms through porphyrin and

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chlorophyll metabolism in the bacteria (Smith et al., 2018; Murooka et al., 2005). Cobalamin

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biosynthesis pathway could be highlighted from α-aminolivulenic acid (ALA) synthesis and end

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to adenosyl cobalamin (AdoCbl) production (Blanche et al., 1995). Meanwhile, aerobic and

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anaerobic pathways as the subsidiary of the mentioned route, deviate from precorrin-2 synthesis

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and turn back to the same pathway at adenopsyl cobyric acid bio-production step. Aerobic and

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anaerobic pathway distinguish from each other by cobalt insertion step (Falentin et al., 2010).

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Propionibacterium genera use the C-5 pathway to synthesize ALA. It has been shown that in C-5

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pathway in which, 3 enzymes were applied for ALA production, the NADPH-dependent

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glutamyl-tRNA reductase (GluTR) has catalyzed the limiting step in these triple reactions

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(Piwowarek et al. 2018b). Studies indicate how the deficiency of heme and the presence of

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glucose (as the ALA dehydratase inhibitor) would be favorable for ALA buildup and

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consequently can affect the Cyano-Cbl production (Kang et al., 2012).

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In fact, DMBI synthesis has been demonstrated as the important division of intracellular

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vitamin B12 production which has focused on lower ligand of cobalamin molecule synthesis.

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(Iida and Kajiwara, 2007). Biologically active form of Vitamin B12 must be constructed by

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DMBI moiety instead of adenine as the lower ligand of cobalamin molecules (Deptula et al.,

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2015). DMBI synthesis as the part of riboflavin metabolism pathway was the elusive section of

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cobalamin synthesis pathway (Lawrence and Roth, 1995). Since endogenous production of the

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lower ligand in the cobalamin molecule was not observed through the growth of bacteria in the

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absence of cobalt ions (Hörig and Renz, 1977), there are difficulties to express DMBI production

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pathway independently. Although studies, which are using radioactively-labelled nutrients, had

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reflected signs of endogenous synthesis of DMBI in the bacterium like Propionibacterium; there

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was not a bright genome annotation and metabolic mechanism for the DMBI synthesis till recent

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years. By applying 14C-riboflavin as the nutrient in the culture medium, it has been proved that

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riboflavin acts as the DMBI precursor (Renz, 1970; Keck et al., 1998). Through interpretation of

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data, were prepared via cellular studies, a complex of 9-step reactions has been recommended as

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an oxygen-dependent pathway for FMN (flavin mononucleotide) conversion to DMBI in the

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Propionibacterium (Hörig and Renz, 1980). Since 2007, a group of enzymes for endogenous

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DMBI production were introduced (Gray and Escalante-Semerena, 2007). BluB enzyme is the

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key protein which converts FMNH2 (reduced flavin Mononucleotide) to the DMBI. This

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conversion needs molecular oxygen for oxidative fragmentation of heterocyclic compounds (Yu

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et al., 2012). The kinetic of this reaction is not Michaelis-Menten type and it shows substrate-

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inhibited kinetic at high concentrations of FMN. (Taga et al., 2007). Genomic data reveals that

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Propionibacterium freudenreichii comprises the BluB/CoT2 fusion gene and heterologous

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expression of such gene provides its responsibility for the DMBI synthesis as well as DMBI

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conversion to α-ribazol phosphate (Deptula et al., 2015). Recently, it has been observed that

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riboflavin addition (DMBI precursor) to the fermentation medium can improve cobalamin

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synthesis up to 4 folds (Chamlagain et al., 2016). It is considerable to know that in so many

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attempts of Cyano-Cbl production, DMBI contribution was illustrated as the semi-obligated

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component in the broth of Propionibacterium freudenreichii cultivation (Piao et al., 2004).

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Regulation mechanism of gene expression, which is responsible to control BluB/CoT2 protein

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action, should be studied well to understand main role of using exogenous DMBI. Nowadays,

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bioinformatics data besides experimental efforts, effectively explain metabolic pathways of

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microbial strains. Via employing information mentioned in the trustable databases such as

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KEGG (Kanehisa et al., 2007), BRENDA (Schomburg et al., 2004) and UniProt, it is possible to

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focus on the key elements of fermentation production more precisely. Useful visualized

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information about the Cobalamin biosynthetic pathway and whole metabolic pathway of

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Propionibacterium DSM20271 have been illustrated in Fig. 1. Many useful information about

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the BluB/CoT2 fusion gene and its expressed protein are accessible in these databases especially

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in BRENDA and UniProt.

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Fig. 1

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Design of experiments (DoE) has gradually become a part of experimental studies to

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reduce the number of experimental attempts as well as decreasing the cost and labor time of

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studies in vitamin production researches (Hajfarajollah et al, 2015; Piwowarek et al. 2018a).

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Such kind of planning in the experimental researches has been developed from the agricultural

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and biological studies (Mäkelä, 2017). DoE can provide more confidence during the conduction

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of the research. Response to Surface Methodology (RSM) is a multi-variable statistical routine

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which is using the DoE approaches to optimize the goal of the study with reasonable precision

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(Witek-Krowiak et al., 2014). Combination of RSM and screening step can also make more trust

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in ultimate results of an investigation. Plackett-Burman is a screening method which takes

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advantage of DoE to determine the significant factors (Plackett and Burman, 1946). Introducing

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kinds of simple and complex sugars (Quesada-Chanto et al., 1994), applying substituted carbon

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sources (Lebloas et al., 1994; Gardner and Champagne, 2005), using stimulators and adding

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single or groups of amino acids (Marwaha et al., 1983) are samples of Vitamin B12 fermentation

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optimization attempts. Many factors can affect the fermentation process (e.g Growth rate,

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metabolite concentration, process efficiency). Therefor the best DoE can considerably reduce the

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cost and number of experiments while simultaneously shows the interactive behavior of the

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variable on the response (Mohammed et al., 2014). Any method of optimization as well as the

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DoE model has its own accuracy and resolution which should be considered during method

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selection.

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Over the past decade, because of environmental issues, energy saving and the economic

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aspects, studies have been encouraged to choose nutrients for the microbial fermentation from

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renewable or recyclable resources. Introducing new carbon sources from the waste part of the

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industrial and agricultural attempts are the common challenge of this field. In the region like

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north of Iran, rice planting is the common practice and sometimes rice bran is introduced as the

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waste of the rice plant. Rice bran oil is a new carbon source identified for fermentation. Rice

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bran oil supplies free fatty acid presence in the broth (Ghosh, 2007). The organic compounds

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from C14 to C18 fatty acids (linoleic, oleic and palmitic acids) are the main carbon sources in the

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rice bran oil. Recently RBO has been considered in the field of biofuel (El Khatib et al., 2018)

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and chemical additive production (Salinas-Solano et al., 2018). In best of our knowledge, no

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report has found to use it as a carbon source of fermentation to yield valuable metabolites such as

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cobalamins.

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In this study, initially we have examined the possibility of growth of the bacteria

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(Propionibacterium freudenreichii) on the fermentation medium containing new carbon sources

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(RBO and argan oil) as well as the effect of DMBI and amino acid presence in the medium on

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the growth profile. Afterwards we have substantially focused on statistical optimization of

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Cyano-Cbl production using Propionibacterium freudenreichii. In this step RBO was the sole

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carbon source and statistical screening and RSM attempts have been conducted.

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2. Material and Method

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2.1. Microorganism

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Propionibacterium freudenreichii subsp. freudenreichii PTCC1674 (pfre) was supplied from

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Persian Type Culture Collection (PTCC) in the live culture form. The solid culture composition

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based on PTCC recommendation was: casein peptone of 10 g, yeast extract of 5 g, sodium lactate

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of 10 g and agar of 10 g in 1000 ml deionized water. All the components were properly mixed

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and autoclaved; then, subcultures were prepared from PTCC’s bacteria to obtain stock-culture of

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bacteria for all the attempts of this study.

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2.2. Chemicals

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Glucose, peptone casein, yeast extract and bacteriological agar were supplied from Merck

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(Darmstadt, Germany), sodium lactate 60%, Cyanocobalamin (Vitamin B12), betaine

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hydrochloride from Sigma-Aldrich (Germany), L-glutamic acid, CoCl2.6H2O purchased from

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Sam Chun (Seoul, S. Korea), MgCl2.6H2O,MnCl2.4H2O, K3PO4, KH2PO4,NaH2PO4.2H2O,

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NaCl, Ca Pantonate, ZnCl2,FeSO4.7H2O, CaCl2.2H2O, CaCO3, Di-ammonium phosphate, 5,6

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dimethyl benzimidazole, L-methionine, L-cysteine, L-tryptophan, L-seine are supplied from

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Merck (Darmstadt, Germany). Rice bran oil and argan Oil have been supplied from commercial

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local market.

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2.3 Methods

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2.3.1 Inoculum preparation

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According to the adaptation of bacteria with the growth media and to cross over to log phase,

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pfre cells have been transferred from stocked culture (agar media) to the pre-culture medium.

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The composition of inoculum media was: glucose, 20 g/L; peptone, 5 g/L; yeast extract, 10 g/L;

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KH2PO4, 2 g/L; di-ammonium phosphate, 5 g/L; and pH of medium was adjusted to 7 using

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NaOH solution (3M). Temperature set at 30°C and incubator-shaker agitated at 150 rpm under

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anaerobic conditions.

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2.3.2. Growth analysis

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The nutrient concentrations which were fixed during all attempts of microbial growth analysis,

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was: Peptone, 5 g/L; yeast extract, 10 g/L; Na-lactate, 2g/L; KH2PO4, 2 g/L; di-amonium

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phosphate, 5 g/L; MgSO4.7H2O, 1 g/L; CoCl2.6H2O, 5 mg/L; MnCl2.4H2O, 2 mg/L; NaCl, 5

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mg/L; FeSO4.7H2O, 10 mg/L; CaCO3, 5 mg/L. The other components of the medium alter

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according to the factors defined in the study. After medium preparation and autoclaving,

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microbial colonies at log phase of growth, were introduced into the 150 ml of the cultivation

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broth. The pH of fermentation medium was adjusted to 7 using 3M NaOH solution. Optical

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density (OD, λ=600) was recorded by means of NanoDrop-One of Thermo SCIENTIFIC

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Company. Fermentation procedure includes 2 parts. First 3 days undergoes anaerobic condition

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and it has been followed by another 3 days under aerobic condition. A 5 % V/V from inoculum

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culture transferred to the fermentation medium under sterile protocol conditions (unwanted

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microbial pollution is the main threat during this process). All the flasks placed in the anaerobic

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jar which the nitrogen gas was purged in and all the set-up put in the incubator-shaker at 30°C

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and 150 rpm. After the first step, all the flasks were taken out from the anaerobic jar and the

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process was followed under aerobic condition for the next 3 days. Samples were taken every 12

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hours for optical density measurement. In the last day of aerobic stage, during analysis of DMBI

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and amino acids effect on the growth profile, samples were taken every 24 hours

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2.3.3. Experimental design and statistical analysis for Cyano-Cbl production

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2.3.3.1. Plackett-Burman analysis

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For the determination of the main effective variables on the response; Plackett-Burman (PB)

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method needs at least N trials for N-1 variables which N=4k (k is an integer, greater than 1).

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These multifactorial experimental design accurately programmed by MiniTab.17 software that

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was used in this study. The subject of “dummy factor” or inert factor is considerable in PB

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design. The main role of dummy factors is to estimate random and experimental error (Chauhan

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et al. 2007). Having the dummy factor is not obligatory; but, it can provide some redundancy of

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the statistical procedure. Participating at least 3 dummy factors is recommended by some

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investigators and some others proposed one third of the whole variables are preferred to be

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dummy factors (Jain et al. 2010). Such recommendations would be desire to observe if the

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Plackett-Burman supposed to apply solely. Since we have followed the Plackett-Burman method

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by the response to surface methodology, it has been only used to associate the effect of

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parameters on the response comparatively. Effect of each variable by the Plackett-Burman

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method evaluate by Eq. 1:

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E( ) = 2 (∑

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Which E( ) is the calculated effect of variable ith, and the ∑

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of trials which include the upper level of the variable ith, and ∑

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results of trials which include lower level of the variable ith. N is the number of trials.

(

)

−∑

(

) )⁄

(1) (

)

(

is the summation of results ))

is the summation of

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12 runs of PB design for 11 independent variables assessments were performed. Responses

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extracted and all the comparative analysis was conducted. ANOVA analysis was carried out by

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the computer software of MniTab 17.1.

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During the first step of statistical analysis, most of the constituents of the culture media were

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assessed via the Plackett-Burman screening method and the details will be discussed in the next

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section.

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2.3.3.2. RSM analysis and optimization

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Response surface methodology is applied to benefit from mathematical and statistical tools for

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approaching the optimum outcome of the response. RSM using 2 defined level of variables by

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the operator and one center point level for each one. Full factorial design, Central composite

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design, Box-Behnken design, Doehlert design are the most common statistical designs

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accompanied to generate the complex of runs which lead to the best fitting of the predicted

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model on the experimental data (Karimifard et al. 2018). The purpose which all DoE scenarios

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follow, is the material and computing cost reducing as well as extracting the most accurate model

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of the response. More level of the variables cause to more truthful predicting and eventually

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proper optimization, but the number of runs dramatically increases. Full factorial design with 3

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levels for the variables requires 3 trials which N is the number of variables. It means, for the

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study with 4 factors it needs 81 trials while we have done by 27 runs through Box-Behnken

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design. We have used the full quadratic model to solve the Box-Behnken design. Stepwise

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analysis was applied and also Box-Behnken transformation (natural log) was implemented

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during the calculations. Logarithmic transformation introduced better curve fitting while center

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point is defined in triplicate. Optimization was executed by the MiniTab 17.1 optimizer

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application. Assessment of factors interaction is one the most important advantages of the RSM.

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Of course, RSM model can only consider binary interactions and upper level interactions are

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omitted. In spite of many advantages of RSM, this method cannot reflect the reliable extrapolate

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out of defined levels. Therefore, making groups of variables and running the RSM, separately,

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over these groups of variables to avoid the higher number of trials, is not a suitable optimization

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approach.

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The significant factors which were obtained by Plackett-Burman analysis, were introduced to

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RSM optimization through Box-Behnken design. Also, the constant concentration of the culture

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media during all experiments were assigned as; peptone casein, 15 g/L; yeast extract, 10 g/L; L-

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glutamic acid, 0.2 % W/V; betaine hydrochloride, 1 g/L; (NH4)2HPO4, 10 g/L; FeSO4.7H2O, 20

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mg/L; ZnCl2, 2 mg/L; MgCl2.6H2O, 20 mg/L. RBO, DMBI, CaCl2.2H2O, CoCl2.6H2O

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concentrations as well as the temperature of the fermentation, vary by DoE of the RSM analysis.

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As explained before, fermentation should follow both steps of anaerobic and aerobic conditions.

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After 3 days of anaerobic cultivation to grow the pfre appropriately, and synthesis of corrinoid

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part of Cobalamin molecule, aeration is necessary to produce endogenous DMBI. Eventually, the

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active form of Cobalamin synthesis is completed. The pH adjustments and the lab apparatus

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were the same as mentioned in growth analysis section.

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2.3.4. Extraction and quantification of Cyano-Cbl

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As the cobalamins are produced in intracellular form during the pfre fermentation, proper and

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efficient method for cobalamins extraction has important role. The general protocol mentioned in

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literature is autoclave extraction and cyanidization of cobalamin molecule (Kumar et al., 2010).

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Adenosyl, hydroxyl, and methylcobalamin are the natural form of cobalamins which are unstable

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in extracellular media and all of the mentioned compounds are light sensitive. To stabilize the

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active form of Vitamin B12, the natural cobalamins should be converted to the cyanide form.

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Cyanide ions were displaced instead of the upper ligand of cobalamin molecules. Based on

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Hugenschmidt et al. (2010) method with slight alteration, for extraction of cobalamins, a 40 ml

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of the broth was centrifuged under refrigeration at 4°C, for 15 min at 8000g acceleration. The

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supernatant was discarded and pellets were washed and dispersed in 0.2 M phosphate buffer

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(pH=6), centrifugation and washing of cells were duplicated. Now, the pellets were dispersed in

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0.1 % KCN in phosphate buffer (pH=6) solution. The vial was vigorously shaken via vortex lab

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equipment. According to the literature (Van Wyk and Britz, 2010), the percentage of KCN

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solution varies between 0.1 up to 0.5 % ; also, the pH of KCN solution changes from 4.5 to 11.

267

The lower limit of the mentioned range can extract and convert properly but some others were

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used a higher excess amount of KCN at a wide range of pH, in order to make sure for

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Cyanidization. Of course, in the upper limits of the KCN percentage and pH, di-cyanocobalamin

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formed more than Cyano-Cbl which is not the target of our study. Dispersed cells in the KCN-

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Phosphate buffer were placed in the autoclave at 121°C for 15 min; then, centrifuged for 20 min

272

at 4°C under 8000g gravitational force. Supernatant of later step passed through the 0.45 µm

273

syringe filter and now it is ready to be analyzed by HPLC. Van Wyk and Britz (2010) method

274

was applied for HPLC analysis with some modifications. Smartline KNAUER 2300 (Germany)

275

HPLC system equipped with a reversed phased Eurosphere C18 column (250 mm×4.6 mm,

276

5µm) at 361 nm set up for UV detector used to analyze the produced Cyano-Cbl. The flow rate

277

was 0.75 ml/min at ambient temperature. In the prior studies, UV detection wavelength varies

278

from 254 to 580 nm while the Cyano-Cbl UV scanning shows 3 picks in 278, 361, 550 nm which

279

the 361 nm is the most dominant pick between them (Kumar et al. 2010). Therefore, we applied

280

361 nm for the detection of Cyano-Cbl. The mobile phase was employed in the linear gradient

281

format of 15:85 - 50:50(V/V) methanol-water for duration of 25 min, limit of detection by HPLC

282

method was mentioned 0.005 µg/ml which is well quantified limit for fermentation production of

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Cyano-Cbl.

284 285 286

3. Results and Discussion

287

3.1 Growth analysis

288

Attention-grabbing surveillance meanwhile is a similar growth profile when the same group of

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nutrients was used. Using RBO and Argan oil as carbon sources clearly have the same profile of

290

growth. Higher concentration of RBO, 8% V/V shows higher cell density (OD600=2.762) in

291

compare with less concentration of RBO, 4% V/V. At identical concentration of RBO and Argan

292

oil, RBO makes higher cell density at the end of the anaerobic stage of pfre growth (Fig. 2a).

293

Since RBO and Argan oil are containing free fatty acids, having the same profile of growth is

294

predictable; however, the composition of fatty acids in RBO seems to be easily utilized under

295

anaerobic stage of the growth. It confirms that the type of carbon source firmly affected on the

296

growth of pfre. The interesting finding is approximately the same cell density (OD600=0.9±0.05)

297

was obtained at the end of the aerobic stage of fermentation. Having equal endpoint at the late

298

stage of aeration regardless of kind and concentration of carbon sources; it needs to be further

299

investigation to assess impact of aeration on cell density variation.

300

Utilization of DMBI in the course of fermentation process and its effect on growth of pfre

301

also was studied. Fig. 2b shows DMBI addition from the beginning of anaerobic fermentation

302

can completely differs the route of fermentation in the anaerobic and aerobic stages of the

303

process. For 30 g/L of glucose as the sole carbon source shows great impact on aerobic

304

fermentation (OD600=0.372). The growth profile is completely reversed when the DMBI is

305

added. In the absence of DMBI during the aeration, cell density decreased to the minimum level

306

and bounce back to its asymptote, while in the presence of DMBI, cell density starts from the

307

considerable lower cell density and goes upward up to its maximum and comes down. Both

308

experiment, with and without DMBI, were containing the same amount of simple sugar as the

309

carbon source and approximately have the same cell density. It is considerable although these

310

experiments have crossed the different route in their growth, they terminate the process at the

311

same final cell concentration. It shows DMBI can affect the metabolic regulation of pfre in the

312

aerobic and anaerobic part of fermentation. The other finding is the same type of growth profile

313

when the 15 g/L of glucose as the carbon source is replaced with 4% V/V of Rice Bran Oil in the

314

DMBI containing medium. This experiment shows completely increasing trend of the growth, up

315

to its upper level. The final cell density is considerably higher than the DMBI containing system

316

without RBO as the supplementary carbon source. Therefore using combined carbon source

317

(simple glucose 15 g/L and complex RBO 4% V/V shows greater cell density which is beneficial

318

for the production of Cyano-Cbl although its growth profile influenced by DMBI presence in the

319

culture media.

320

The other term which was studied, is the effect of amino acids adding in the fermentation media.

321

It was figured out that kinds of amino acids in the broth can affect the growth of the cobalamin

322

producing bacteria. In the same media composition with modification of amino acids content,

323

variation of cell density is illustrated. The most important finding via focusing on the growth

324

profile under amino acids enriched medium, is the monotonic profile of growth through aerated

325

process (see Fig. 2c). In the Figs. 2a and 2b clearly the extremum point is visible while in Fig. 2c

326

monotonic profile reflects the effect of amino acids on the growth and metabolic regulation in

327

the pfre. Growth-associated cobalamin synthesis has been proved in Propionibacterium genus

328

(Wang et al. 2014). Different carbon sources, as well as the effect of the combination of nutrients

329

were studied on the growth of pfre. Cell density as a sign of growth is the first step of cobalamin

330

synthesis. Hitherto result of experiments describe the complicated effect of the fermentation

331

medium composition. Therefore obtaining trustable optimization of Cyano-Cbl growth-

332

associated production, required to examine via statistical optimization methods. In the next part,

333

the screening and optimization of Cyano-Cbl production were conducted. Fig. 2

334 335

3.2 Statistical analysis

336

3.2.1 Plackett-Burman method

337

Items which were assessed by Plackett-Burman method for screening and their levels are

338

summarized in Table 1. Since there was no report of using Rice Bran Oil as a sole carbon source

339

in fermentation for cobalamin production; we needed to demonstrate Propionibacterium

340

freudenreichii subsp. freudenreichii PTCC1674 (identical to DSM20271T) generating

341

cobalamins and Vitamin B12 (Cyano-Cbl) as an intracellular secondary metabolite. Therefore,

342

Plackett-Burman routine was selected to investigate and prove the production of such

343

metabolites and extract the parameters mainly affected on production via pfre. Selection of

344

factors which should be examined, was the critical part of this work. RBO was selected as sole

345

substituted carbon source, Peptone and Yeast Extract concentration are the general factors

346

engaged to the growth of bacteria. Marwaha et al. (1983) showed, between 11 amino acids as

347

well as among choline chloride and betaine as stimulators of cobalamin production by pfre, L-

348

Glutamic acid and betaine have substantial role. DMBI and cobalt ion from CoCl .6H O, are the

349

main parts of adenosyl- and methyl-cobalamin molecules produced in intracellular form by this

350

genera. Also, they have been introduced as the influencer on pathways which lead to cobalamin

351

synthesis (Wang et al. 2015). Di-ammonium phosphate has been proved as the most effective

352

ammonium salt. It is required to investigate the effect of other trace elements as obligatory

353

cofactors for the growth cycle and cobalamins production. In addition, temperature could not be

354

exempted because of its effect on the growth of bacteria.

355 356

Table 1 Through screening, four parameters were identified which had the highest effect on the

357

Vitamin B12 (Cyano-Cbl) production. RBO concentration, CaCl2.2H2O, DMBI, and temperature

358

were significant effective parameters on the cobalamin synthesis, respectively (Fig. 3a). Of

359

course, the effect of other parameters is illustrated in Fig. 3b. The high slope in Fig. 3b indicates

360

the high effect on the objective of PB screening; while, negative or positive slopes shown direct

361

or inverse effect on cobalamin synthesis. RBO concentration is the direct and most effective item

362

in this study. As the cobalamin production follows growth-associated perception; therefore, high

363

concentration of carbon source would have an influence on our goal. We are optimistic, high cell

364

density at the end of the production process assists us to obtain high concentration of Cyano-Cbl.

365

DMBI is the second effective parameter which is directly affecting on the cobalamin synthesis.

366

Earlier, presence of DMBI in the fermentation broth showed making changes especially after

367

aeration in cell density profile. Fortunately, it differs the downward manner of cell density

368

profile to the upward as well as it is the main lower ligand of intracellular produced cobalamins.

369

It was predictable that DMBI as a part the cobalamin molecules should be one of the direct

370

effective parameters. The last direct effective parameter on the objective of present work is the

371

temperature. Temperature generally can enhance the bacteria growth rate in the operative range.

372

Therefore, more accumulated bacteria at the end of the process can be in the benefit of the

373

defined objective for the screening. The last assessed effective item is CaCl2.2H2O

374

concentration. Trace elements have shown as part of the effective parameters on cobalamin

375

synthesis. Ca pantothenate showed before as an effective component on the cobalamin synthesis

376

(Kośmider et al. 2012) and our study confirmed its inverse effect on our objective. The most

377

considerable parameter which is brought up by the Plackett-Burman screening study is the effect

378

of CoCl2.6H2O as a reverse affecting factor. Although, it has a considerable effect on the

379

cobalamin synthesis, it has been ranked on the fifth position and close to CaCl2.2H2O as the

380

effective trace element supplier (see Fig. 3).

381

Fig. 3

382

The inverse effect of CoCl2.6H2O is an interesting finding; because, the cobalt ion is the main

383

upper ligand which is constructing the complex structure of intracellular cobalamins. It is

384

expected to have a direct effect on the achieved Cyano-Cbl at the end of the process; but, it is

385

completely opposing the objective. Extensive research showed heavy metals hinder the growth

386

of bacteria in the range of their concentration (Seidametova et al. 2004; Bainotti et al. 2000).

387

Pfre also has illustrated that cannot tolerate the presence of high concentration of Cobalt ions. It

388

should be added to the fermentation medium because it is part of the cobalamin molecule and

389

also decreasing the growth rate of pfre. Although, CoCl2.6H2O presence could increase cell death

390

rate, its contribution in this process is obligatory. Since the Plackett-Burman method is a kind of

391

resolution III statistical method for screening the effective parameters; it can only reflect the

392

estimates of parameters which have significant effect on our objective. Placket-Burman cannot

393

separate the combination of main and interaction effects and it may cause error. So it is

394

preferred, the Plackett-Burman method follows a more precise technique which analyzes the

395

interaction of parameters. Hence this research applied RSM after obtaining the main effective

396

parameters through PB design. Since CoCl2.6H2O and CaCl2.2H2O have the same trend on the

397

produced Cyano-Cbl concentration, through the RSM optimization; we have introduced

398

“elemental solution” factor which is containing equal molarity of CaCl2.2H2O and CoCl2.6H2O.

399

Elemental solution factor was used instead of CaCl2.2H2O concentration in the RSM

400

optimization.

401 402

3.2.2 RSM method

403

After screening, the Response Surface Methodology was adapted to optimize four parameters

404

extracted through Plackett-Burman screening method. We have followed the Box –Behnken

405

(BBD) design instead of Central Composite Design (CCD). In CCD out of the defined range of

406

parameters by the “α” factor, is tested and it would be helpful when we need to check further

407

than the achieved limits. So having more number of experiments is the characteristic of CCD in

408

compare with BBD. Via BBD, studying of the response or the research goal is accomplished

409

with less experiments. The level of the parameters in this part of present study were chosen quite

410

wide, according to the nature of the selected parameters (Table 2). The design table of the

411

experiments and the responses (Cyano-Cbl produced concentration) is illustrated in Table 3.

412

With consideration of ANOVA results (Table 4), linear and squared parameters of the model are

413

the considerable and among six possible parameter interactions, half of them are effective in the

414

model, based on approved confidence level. To the model summary residual squared (R2),

415

adjusted residual squared (R2-adj) and predicted residual squared (R2-pred) were 97.43, 95.82

416

and 90.98%, respectively; which conform to DoE rules, with acceptable lack of fit. The

417

important perception is the effect of the elemental solution on the couple of effective interactions

418

in the model. By the Plackett-Burman, it has been obtained that the effect of CaCl2.2H2O and

419

CoCl2.6H2O is against of the other effective parameters and now RSM study reflects that these

420

elements, apart from their direct effects can interact with the other parameters to alter the final

421

concentration of produced Cyano-Cbl. The other perception is the role of DMBI in the making of

422

interactions. The lower ligand of Cyano-Cbl molecule which is favorable to add to the

423

fermentation culture also interacts with temperature and elemental solution. Refer to the

424

interaction plot Fig. 4, obviously DMBI-Elemental Solution and the RBO-Elemental Solution are

425

the synergistic interaction to increase the resulted Cyano-Cbl while the Temperature-DMBI

426

interaction causes the complicated effect on the response of this study. It has been shown that the

427

profile of Mean of Response-Temperature has the high positive slope at a lower concentration of

428

DMBI while the slope turns to negative amounts by DMBI concentration increasing. Since

429

Plackett-Burman study proved the temperature as the direct effective parameter on the response,

430

probably high temperature is troublesome on the DMBI consuming by pfre which cause to

431

reduce the produced Cyano-Cbl. Eq. 1 expresses the RSM predicted model by the following

432

regression:

433 434 435

Ln(R) = -2.691 + 0.2034 A + 0.0687 B + 0.0492 C - 0.2355 D - 0.01008 + 0.00678 + 0.00738 A×D - 0.000989 B×C+ 0.000750 C×D Fig. 4

436

Table 2

437

Table 3

438

Table 4

- 0.000115 (Eq. 1)

439

The model optimizer of Minitab.17 software predicted 2.726 mg/L of Cyano-Cbl as the highest

440

response through medium condition including the RBO Concentration of 8.648 % V/V,

441

temperature of 38.285 (°C), DMBI Concentration of 55.758 mg/L and Elemental Solution

442

Concentration of 2 mg/L. The recommended experiment was implemented and the achieved

443

concentration was 2.937 mg/L of Cyano-Cbl which is reflecting 14% increase in the resulted

444

concentration of product, based on the highest achieved concentration during implemented RSM

445

experiments. Figs. 5a, 5b and 5c show the contour plots of fitted model. Fig. 5

446 447

Conclusion

448

It has been shown that the rice bran oil can be used as the sole carbon source of vitamin B12

449

microbial production. Growth profile of pfer by versatile constituents of culture medium was

450

investigated and the effect of carbon sources, amino acids and lower ligand of cobalamin

451

participation in medium was studied. It is shown that ingredients can completely alter the growth

452

profile. Via Plackett-Burman method, screening of the effective parameters was implemented

453

and RBO, DMBI and CaCl .2H O concentration beside the temperature were extracted as the

454

main effective parameters of Cyano-Cbl production. RSM analysis of four effective variables

455

with some modification (equimolar CaCl2.2H2O -CoCl2.6H2O solution instead of CaCl2.2H2O

456

solely) was carried out and finally 14% increase in response (produced Cyano-Cbl) was

457

obtained. In-silico studies would be very helpful to receive better output from Propionibacterium

458

freudenreichii subsp. freudenreichii and we shall publish our best attempts in this field in the

459

near future.

460 461

Acknowledgement

462

Present work as a part of Ph.D. thesis of Rouhollah Hedayati, was supported by the Babol

463

Nushirvani University of Technology research grant no: BNUT/925150010/98. Many thanks to

464

Biotechnology Research Lab at Babol Noshirvani University of Technology (BNUT), Babol,

465

Iran for all cooperation.

466 467 468 469

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470 471 472

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637 638 639 640

Table 1. Parameters of culture media and their limitations studied by Plackett-Burman method on vitamin B12 (cyanocobaamin) production Parameter RBO Peptone Yeast Extract L-Glutamic Acid Dimethyl Benzimidazol Betain Hydrochloride (NH4)2 HPO4 Fe SO4 CoCl2 CaCl2 Temperature

641 642

Lower bound 4% V/V 5 g/L 10 g/L 0.05 %(w/v) 15 mg/L 0.2 (w/v) 2 g/L 2 mg/L 2 mg/L 2 mg/L 30 °C

Upper bound 8% V/V 15 g/L 25 g/L 0.2 %(w/v) 75 mg/L 1 (w/v) 10 g/L 20 mg/L 10 mg/L 10 mg/L 40 °C

643 644 645

Table 2. RSM parameters extracted from Plackett-Burman study to run vitamin B12 (cyanocobalamin) optimization, elemental solution of equimolar concentration of CoCl2 and CaCl2

646

Parameter RBO Temperature DMBI Elemental Solution 647 648

Coded Letter A B C D

Lower Limit (-1) 4 % V/V 30 °C 20 mg/L 2 mg/L

Upper Limit (+1) 10 % V/V 40 °C 75 mg/L 10 mg/L

649 650

Table 3. Box-Behnken design for RSM analysis and the related response, “Response” represents Vitamin B12 (cyanocobalamin) concentration Run NO. A 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27

651 652

-1 1 0 0 0 -1 0 1 0 0 0 0 1 0 -1 0 -1 1 0 0 0 1 0 0 1 -1 -1

B

C

D

-1 -1 0 0 0 1 0 1 0 -1 1 1 0 0 0 -1 0 0 1 -1 -1 0 1 0 0 0 0

0 0 -1 -1 1 0 0 0 1 1 -1 1 0 0 0 -1 0 0 0 0 0 -1 0 0 1 1 -1

0 0 -1 1 -1 0 0 0 1 0 0 0 -1 0 1 0 -1 1 1 1 -1 0 -1 0 0 0 0

Response (mg/L vitamin B12) 2.204 1.540 0.898 1.140 0.933 0.825 2.423 1.995 2.340 1.808 2.275 1.610 1.105 1.495 1.850 1.550 1.813 1.183 1.805 1.862 2.574 1.502 1.387 1.582 0.774 1.450 0.784

653 654

Table 4. ANOVA investigation of the resulted responses. Stepwise selection of terms has been chosen and α=0.15 to enter as well as to remove

655

Analysis of Variance for Transformed Response Source DF Adj. SS Adj. MS 0.31805 10 3.18045 Model 4 2.83349 0.70837 Linear RBO Concentration 1 1.22589 1.22589 Temp 1 0.14146 0.14146 DMBI Concentration 1 0.60676 0.60676 Elemental Solution 1 0.85939 0.85939 3 0.21441 0.07147 Square 1 0.04941 0.04941 RBO Concentration2 DMBI Concentration2 1 0.04567 0.04567 2 Elemental solution 1 0.07057 0.07057 3 0.13255 0.04418 2-Way Interaction RBO Concentration× Elemental Solution 1 0.03139 0.03139 Temp× DMBI Concentration 1 0.07394 0.07394 DMBI Concentration× Elemental Solution 1 0.02722 0.02722 16 0.08389 0.00524 Error Lack of Fit 14 0.08115 0.00580 Pure Error 2 0.00275 0.00147 26 3.26435 Total 656 657 658 659 660 661 662 663 664

F-Value

P-Value

60.66 135.10 233.80 26.98 115.72 163.90 13.63 9.42 8.71 13.46 8.43 5.99 14.10 5.19

0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.007 0.009 0.002 0.001 0.026 0.002 0.037

4.22

0.0208

665 666

Fig. 1. General pathway for Propionibacterium freudenreichii DSM 20271

667 668 669

Colored part are the annotated genes from the experimentally analyzed genome of this bacteria. It contains helpful information about the parts of cobalamin synthesis.( extracted from KEGG database, based on Koskinen et al., 2015 recommended genome)

670 671 672 673 674

Cell growth RBO4%

OD600

RBO8% Argan4%

70

90

675

110 Time(hr)

130

150

676 677

2a

678

Cell growth

Glu30

OD600

Glu30,D Glu15,D,R

70

90

110

130

Time(hr)

679 680

2b

150

Serin

OD600

Tryptophan cystein methionine

70

681

90

110

130

150

Time(hr)

682

2c

683 684 685

Fig. 2. Cell growth of pfre at different condition of fermentation broth a) Effect carbon sources. b) Effect of the introducing the DMBI in the culture media (D represents DMBI and R for RBO) c) Effect of amino acid added to the media

686 687 688 689 690

Pareto Chart of the Effects Term

0.4860 Factor A B C D E F G H J K L

A K E L J B F

Name Rice Bran Oil Pep Yeast Extract L-Glutamic Acid DMBI Betaine (NH4)2 HPO4 Fe SO4 CoCl2 CaCl2 Temp

H G C D 0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

Effect

691

3a

692

Main Effects Plot for R Fitted Means Rice Bran Oil

Pep

Yeast Extract

L-Glutamic Acid

DMBI

Betaine

1.25

1.00

Mean of R

0.75

0.50 4

10

5

(NH4)2 HPO4

15

2

Fe SO4

10

0.05

CoCl2

0.20

15

CaCl2

70

0.2

1.0

Temp

1.25

1.00

0.75

0.50 2

10

2

20

2

10

2

10

30

40

693 694

3b

695 696 697

Fig. 3. Plackett-Burman comparative outcomes to elucidate the effective parameters on vitamin B12 (cyanocobalamin) production a) Pareto plot of effective parameters on the response b) Main effect plot of the responses, direct or inverse

698

699 700

In t e r a c t io n P l o t f o r R Fitted Means

RBO concentr * Temp (C) 2.4

1.6

Mean of R

0.8

RBO concentr * DMBI concent

Temp (C) * DMBI concent

RBO concentr * Elemental so

Temp (C) * Elemental so

DMBI concent 20 47.5 75

2.4

1.6

0.8

DMBI concent * Elemental so

2.4

1.6

0.8 5.0

7.5

RB O co n c e n t r

10.0

30

35

Te m p (C )

40

20

45

DMB I c o n c e n t

701 702 703 704 705 706 707 708 709 710 711

Fig. 4. Interaction plot of interactive behavior of RSM analysis

70

Elemental so 2 6 10

C o n t o u r P l o t o f R v s RB O c o n c e n t r a t i o n ( % V / V ) , E l e m e n t a l s o l u t i o n ( m g / L )

RBO c o n ce n t r a t i o n ( %V/ V)

10 1.00 1.25 1.50 1.75 2.00 2.25

9

8

R < – – – – – – >

1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.50

7

6

5

4

2

3

4

5

6

7

8

9

10

E l e m e n t a l s o l u t i o n ( mg / L )

712

5a

713

C o n t o u r P lo t o f R v s D M B I c o n c e n t r a t io n ( m g / L ) ; T e m p ( C )

D M BI c o n c e n t r a t i o n ( mg / L )

70

1.00 1.25 1.50 1.75 2.00 2.25

60

50

R < – – – – – – >

1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.50

40

30

20 30

32

34

36

38

40

T e mp ( C )

714

5b

715

C o n t o u r P lo t o f R v s E le m e n t a l s o lu t i ; D M B I c o n c e n t r a t i o n 10

El e me n t a l s o l u t i o n ( mg / L )

9

1.00 1.25 1.50 1.75 2.00 2.25

8 7

R < – – – – – – >

1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.50

6 5 4 3 2

716

20

30

40

50

60

70

D M B I c o n c e n t r a t i o n ( mg / L )

717

5c

718 719 720

Fig. 5. Contour plot shows the behavior of the response by the alteration of the pairs of the studied parameters. From “a” to “c” figures clearly has been shown how the effective parameters interaction affecting the response orientation

721 722 723

Footnotes: 1. Many studies have introduced Cyanocobalamin production as the vitamin B12 production. In this study, it has been intended as well.

• • • • •

Rice bran oil (RBO) used as new carbon source to microbial production of Vitamin B12. Propionibacterium freudenrichii subsp. freudenrichii PTCC1674 (pfre) produced Vitamin B12 using RBO. As the vitamin B12 production by pfre is the growth associated, the effect of different condition on the growth were investigated. Plackett-Burman method applied for screening of 12 factors to obtain effective parameters on the Vitamin B12 production. Response surface methodology through Box-Behnken design considerably optimized Vitamin B12 production.