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Analysis of Co-producing Riboflavin in ABE

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Published: 8th Jun 2018 in Biology

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A dynamic metabolic analysis of co-producing riboflavin in ABE (acetone-butanol-ethanol) fermentation by Clostridium acetobutylicum ATCC 824

  • Xinhe Zhao, Mayssa Kasbi, Jingkui Chen, Sabine Peres, Mario Jolicoeur*


Riboflavin, which is also called vitamin B2, is synthetized intracellularly from the enzymatic reaction involving the precursors flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), and plays a significant role in cells metabolism, which is naturally synthetized and widely found in plants and animals, but not in higher animals. It must acquire it from their diet, with a 2 mg dose of vitamin B2 per day that is necessary for human being (Cai & Bennett, 2011; Zamboni, 2003). Currently, the global market of riboflavin (that is continuously increasing) is estimated at 3,000 tons per year, with a commercial production by chemical and biochemical synthesis and fermentation methods; fermentation processes only accounting for up to 2,500 tons per year(Lim, Choi, & Park, 2001). Since the 1940s, various bacteria have been studied to produce riboflavin (Arzberger, 1943), with clostridia being the first commercial scale production. With the potential of genetic and metabolic engineering, microorganism species that are more efficient have been developed for riboflavin production, using yeasts such as Candida flareri, Candida guilliermondii, Candida famata and Mycobacerium pheli. and fungi such as Aspergillus terreus, Eremothecium ashbyii and Ashbya gossypii (Lim et al., 2001). Generally, bacteria and yeast express low yield levels, but the yield in riboflavin can be increased after improving the culture management, such as for Clostridium acetobutylicum which reaches 100mg/L by a bipyrimidine induction (Hickey, 1945), and Candida flareri which reaches 600mg/L in an optimized medium (Lim et al., 2001). Through industrial demonstration, after optimization work for instance, fungi species (Ashbya gossypii) expressed high yields compared to yeasts and bacteria while reaching ~5g/L in 8 days of culture (Lim et al., 2001). Meanwhile, various medium and culture methods were tried to improve riboflavin production (Legg & Beesch, 1945; Pollard, Rodgers, & Meade, 1948)(Legg & Beesch, 1945). Of interest, Roche Vitamins AG developed a riboflavin plant with yearly 3000t production in Germany, utilizing a metabolically re-engineered Bacillus subtilis strain (Alosta, 2007). It should be understood that, in order to adequately evaluate the commercial interest of the innovation described therein, further research (e.g. medium and culture management optimization) is required to reach the cell’s maximum production capacity.

Riboflavin is a by-product of ABE (acetone-butanol-ethanol) fermentation by Clostridium acetobutylicum, but at undetectable to very low concentration levels in the medium naturally. The production level reported here is seen as the minimal level under the novel culture protocol discovered. As reported, during ABE fermentation by clostridia, riboflavin is produced in too low concentration to be detected in the medium, so riboflavin industrial scale production during ABE fermentation has never been mentioned in previous reports. However, in our innovation, we found the addition of sodium acetate, a chemical component, significantly improves riboflavin production from undetectable levels to 200mg/L (Fig 1 to 3). We have indeed visually detected an anomaly in the culture broth with orange matter that then turned out being riboflavin. Riboflavin as byproduct that adds to biobutanol production, which may confer a higher economics competitiveness. With respect to the strain of Clostrdium acetobutylicum, as the typical strain for ABE fermentation, its ability to produce butanol at a commercial competitive yield is currently unsuspected by the community. Therefore, obtaining riboflavin as a byproduct by Clostrdium acetobutylicum in a (re)new concept, will bring to the industrial community a new cell platform to produce riboflavin as well as butanol.

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However, the synthesis of riboflavin involves guanosine triphosphate and the purine pathway, which is subject to various energy and redox regulatory mechanisms. When combining solvents metabolism in clostridia, although which is clear in both acids and solvets generation mechanism currently (Zhao, Condruz, Chen, & Jolicoeur, 2016), it should be a completely new project. Depending on our previous work and research capacity having the expertise to unravel strategies to overcome these regulatory mechanisms that may limit the productivity in this biosystem. Besides metabolic analysis, present work exhibited a global dynamic model mainly including carbon flow from pentose (xylose), which verified acetate activation on riboflavin accumulation in ABE system. In fact, various modeling works have been done in clostridia culture system during previous reports (Li et al., 2011; Mayank, Ranjan, & Moholkar, 2013; Shinto et al., 2007; Votruba, Volesky, & Yerushalmi, 1985). Impressed, Shinto et al. simulated both glucose and xylose carbon flux forward ABE production by WinBEST-KIT software. However, all completed work not only ignored riboflavin metabolism, but without reports related a global intracellular metabolic simulation. In fact, a rigorous intracellular simulation provided further scope to verify a model’s precision, and what’s we are doing in present work.

2. Materials and methods

2.1 Cell culture and sampling

Present culture applied typical strain of Clostridium acetobutyicum ATCC 824, which stored in liquid nitrogen with 20% glycerol protection. Batch culture was carried out in 3.6 L bioreactor (Bio , Montreal, Quebec) with 2 L medium, and was inoculated 5% (v/v) seed prepared in 500 ml serum bottle. Seed medium applied RCM medium and culture medium applied CGM medium, medium preparation  referred previous work (Zhao et al., 2016). 60mmol and 0 mmol sodium acetate (NaAc) was supplied initially in culture medium. Filtered 100% nitrogen was sparged from the bottom of bioreactor for keeping anaerobic condition. Culture was carried out in 93 h maximum under 37 „ƒ and 100 rpm. Taking samples flexible between about 12 h and treated immediately and stored in -80 „ƒ for intracellular and extracellular analysis. Sampling and metabolites extraction methods referred to previous work described (Ghorbaniaghdam, Chen, Henry, & Jolicoeur, 2014; Zhao et al., 2016).

2.2 Metabolites analysis

Riboflavin was identified by LC/MS/MS (Agilent, Quebec, Canada) with . Riboflavin standard (Sigma, Quebec, Canada ) retention time.

Meanwhile, acetone, butanol and ethanol were analyzed by GC-FID, Perkin-Elmer Clarus 480 GC with Elite-Wax ETR column. Acetate and butyrate were carried out in a HPLC with a PDA detector (Waters, Canada). All intracellular metabolites (including energy, redox, sugar phosphate and organic acids), and extracellular amino acids and extracellular xylose were analyzed by LC/MS/MS system. All methods and equipment were described in previous work (Ghorbaniaghdam et al., 2014; Zhao et al., 2016).

2.3 Kinetic modeling

2.3.1 Model hypothesis and description    

Based on previous works on dynamic modeling (Cloutier, Perrier, & Jolicoeur, 2007; Ghorbaniaghdam, Henry, & Jolicoeur, 2013; Leduc, Tikhomiroff, Cloutier, Perrier, & Jolicoeur, 2006) , a metabolic kinetic model in ABE fermentation system was carried out. Multi Michiaelis-Menten was applied as biological mathematical expression to describe each reaction velocity (Equation ZZZ) (Leduc et al., 2006). Present model mainly exhibited carbon flow which related to major metabolic pathway, such as PPP pathway, glycolysis and acids, solvents formation. Figure XXX and Table YYY displayed the network and stoichiometry of reactions used in present model. Because of only carbon metabolism framework considered, nitrogen source and regulators (e.g. energy and redox) were ignored in this model. A total of 21 intracellular reactions and 77 parameters total were considered in this model. Meanwhile, biomass specific growth rate was assumed obtaining precursors from R5P, X5P and Ru5P (contribute to (deoxy)ribonucleic acid, and nucleotides); PYR (to cell walls polysaccharide formation (Schaffer & Messner, 2005)); F6P (to phospholipids formation). Therefore, cells contribution terms were deducted when describing above metabolites (Table YYY). One gram dry weight cells (gWD) was considered as simulated unit in this model, and flux speed was described mmol per gDW per hour. All extracellular molecule was hypothesized entry cells and release out accessibility, ignored all resistance when molecule passing cells membrane.

With respect to modeling process, one auto-generating system was carried out implemented by Matlab software. The program called for a “.txt” file (included stoichiometry of the reactions Table YYY) to generate kinetic equations and mass balance files automatically, then simulate metabolites concentration curve compared with experimental data finally. During the programming, reactions inhibition (Equation ZZZ) and activity (Equation ZZZ) were considered. The Matlab tool “ode23s” was used for ordinary differential calculation. Meanwhile,

: reaction rate of each intracellular biochemical reaction; : constant of the maximum reaction rate. : substrate of each substrate; n: number of substrates and co-factors involved in each reaction. μ is describe from μmax and accounting for all substrates and co-factors involved.

: biomass specific growth rate; maximum biomass specific growth rate; : substrate of inhibition term; : inhibition parameter.

Table 1 Stoichiometric reactions in the model




XYL => X5P


X5P = R5P


X5P => 0.5 GTP


GTP => 0.6 RIBO


2 X5P + R5P => 2 F6P + GA3P


F6P => 2 GA3P


GA3P => G13DP


G13DP => PEP






PYR => ACoA + CO2




ACoA => EtOH


2 ACoA => AACoA














BCoA => BuOH


X5P + R5P + F6P + PYR => X

=>: unidirectional reation;

=: reversible reaction

2.3.2 Model calibration and parameters sensitivity analysis

A “Semi-fixed” methods was developed during parameters calibration. The principle of this method is part of metabolites simulation were fixed to experimental data by general formula (y=ax+b), which auto generating parameters (a, b) to keep fixing. Based on fixed simulation results by general formula, then adjusting remained parameters by kinetics. Meanwhile, Matlab Optimization Toolbox of “lsqnonlin” was applied for optimizing parameters depending on minimized objective function to identify advanced parameters. Equation ZZZ displayed the least square optimization function for generating objective function. Meanwhile, the minimization of object function was also used for parameter sensitivity analysis. Changing each parameter value a range of -100% to 200% while fix all other values, obtained a global parameter sensitivity view.

3 Results and Discussion

3.1 Na-Acetate Induces Riboflavin Production in ABE Fermentation

3.1.1 Riboflavin production

Serendipity, a yellow broth what (?) obtained during ABE fermentation with 60 mM NaAc supplemented by xylose as carbon source. After recrystallization process, a yellow acicular crystal emerged under microscope (Figure ZZZ). Molecular weight was identified 376 by LC-MS-MS (Figure ZZZ), which suggested riboflavin overproduced by Clostridium acetobutylicum ATCC824. The final result revealed that the new yellow product was riboflavin and reached 0.2 g L-1. Present studying provided a novel method co-producing riboflavin in ABE fermentation system, which reached maximal 0.20 g L-1 (0.53 mM) riboflavin with 60mM sodium acetate (NaAc) supplemented, but with only 0.035 g L-1 (0.09 mM) obtained in control. Although the riboflavin quantity was lower than Ashbya gossypii, the mainly species applied both in research and industry with more than 5g L-1 (Lim et al., 2001), co-produced by biobutanol production still be very interesting for riboflavin industry. In fact, Cai & Bennet obtained 0.07g L-1 by a genetic stain of C. acetobutylicum ATCC 824(pJpGN) in a buffering culture (Cai & Bennett, 2011), and Hickey also obtained 0.1 g L-1 through adding 2, 2-bipyrimidine in the medium (Hickey, 1945). Therefore, comparing previous reports, present research provided an inexpensive and effective method to co-produce riboflavin by clostridia species.

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Fig.1. 56 hour bioreactor fermentation broth after centrifugation. The orange sediment is the riboflavin, white sediment is biomass.

3.1.2 ABE fermentation in co-producing riboflavin environment

Present accidentally found abundant riboflavin accumulation was attributed to NaAc supplemented. Generally, people believe a buffering function by NaAc addition in ABE fermentation (Hüsemann & Papoutsakis, 1990), but which still be suspected when people found acetic acid supplemented also promoted solvents production (Holt, Stephens, & Morris, 1984). Then, Chen & Blaschek investigated enzymes including coenzyme A transferase, acetate kinase and butyrate kinase, all their activity were highly expressed in sodium acetate addition experiment with higher solvents production (Chen & Blaschek, 1999). Therefore, acetate supplemented plays complex roles in ABE fermentatio”n. In present culture, compared to control group, supplemented 60mM NaAc effectively buffered cells culture (Figure ZZZ), and accompanied biomass growth was stimulated, maximal optical density (OD600) reached 19.2 and 17.7 in Batch A and B versus 5.7 in control group (Figure ZZZ). Meanwhile, solvents were obviously promoted in NaAc supplemented culture, for example, butanol and acetone reached 145.29 mM and 23.14 at 45 h by Batch A, and reached 101.02 mM and 12.51 mM at 48 h by Batch B, but control culture just obtained 36.29 mM and 14.79 maximum respectively.

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In two representative batches culture from five, Batch A’s riboflavin yield kept higher level than batch B (Figure ZZZ), although both of them started in the same initial value. But the riboflavin specific yield reached similar level when both of two batches reached maximal value. Batch A reached 0.20 g L-1 with 3.5 g DW L-1 biomass which corresponding specific yield of 0.0571 g gDW-1, while Batch B obtained 0.0576 g gDW-1 (0.167 g L-1 riboflavin with 2.9 g DW L-1 biomass). Therefore, the difference between groups attributed to the biomass error but not riboflavin generating capacity. The biomass growth in ABE culture was easily affected by pH conversion. The metabolic conversion mechanism from acidogenesis to solventogenesis has been proved which significantly related to cells growth and solvents production【】. A phenomena called “acid crash” would occur when generated excess acids, cells growth would be inhibited and even be blocked by low pH, and this phenomena related to %%%%%%%%%% (Maddox et al., 2000; Wang et al., 2011). In present culture Batch B, pH sharply decreased from 6.4 (at 7 h) to 4.7 (at 19 h) (-³pH=1.7), but at the same period Batch A just decreased 1.1(-³pH) with 6.2 to 5.1. Then, cells in Batch B spent longer time (21 h) to recover back from pH 4.6 (at 23 h) to 5.2 (at 44 h), but Batch A just used 12 h (from pH 5.1 to pH 5.6) (Figure ZZZ). Lower pH in Batch B not only caused biomass induced, but also depressed butanol and ethanol stimulation. As main product, butanol and ethanol reached 145.28 mM and 23.14 mM at 57 h in Batch A, while Batch B reached 101.02 mM and 12.51 mM at 48 h, respectively (Figure ZZZ).

  • Compare control group, production of riboflavin didn’t affect ABE production, on the contrary, which accompanied high level solvents stimulation.

3.2 Intracellular metabolic analysis

3.2.1 GTP plays key role in riboflavin production

Riboflavin generation pathway mainly concentrated to GTP metabolism which has been reported in many related species. Lim et. al., summarized three metabolic models from three representative organisms including Candida famata, Bacillus subtillis and Ashbya gossypii, which belongs to yeast, bacteria and fungi respectively (Lim et al., 2001). In C. famate, GTP generated from Ru5P directly, while in B. subtilis it has to experience IMP, XMP and GMP, then reached GTP. However, in A. gossypii, GTP generation experienced more complex metabolic pathway, which including glyoxylate reactions in peroxisome and citrate circle in mitochandrium from fatty acid decomposing. But the same reactions exhibited after GTP. By DBP synthase catalysis from R5P, DBP reacted with ARP, a metabolite from GTP, generate DMRL and then reach to riboflavin(Lim et al., 2001). Therefore, GTP was the entrance of riboflavin biosynthesis in organism metabolism (Alosta, 2007). Present study investigated GTP concentration influence on riboflavin accumulation. In NaAc supplemented Batch A and B, GTP generated a peak from initial to about 40 h, which reached 0.19 μmol gDWˆ’1 (at 25 h) and 0.15 μmol gDWˆ’1 (at 19h), respectively. But control culture reached 0.74 μmol gDWˆ’1 at 24 h, and deceased to 0.28 μmol gDWˆ’1 at 70h (Figure ZZZ). Therefore, GTP was accumulated in control culture compared to NaAc supplemented, or in another word, acetate was benefit for release GTP to generating downstream metabolites, i.e. riboflavin.

Interconversion between guanosine and adenosine has been proved, for example, GTP energized the process of AMP synthesis from amino acids, meanwhile, ATP provides energy for GTP and GDP formation (Michal, 1999). Meanwhile, all of them play various role to provide energy in carbon and nitrogen synthesis and degradation. Therefore, as precursor of riboflavin, GTP was significantly affected by adenosine phosphate groups. Figure ZZZ exhibited GTP and three adenosine phosphates (AMP, ADP and ATP) tendency following with culture process. Similar trend shown in AMP and ADP comparing to GTP concentration evolution. In control, AMP and ADP reached maximal 1.55 μmol gDWˆ’1 (63 h) and 0.75 μmol gDWˆ’1 (24 h), but in both NaAc cultures AMP always kept in a low stable level (0.02-0.21 μmol gDWˆ’1), and ADP were both lower than control (maximal 0.36 and 0.29 μmol gDWˆ’1 in Batch A and B respectively). Interestingly, ATP dramatically accumulated in NaAc supplemented cultures, both of them reached peak at 25 h with 0.75 and 0.36 μmol gDWˆ’1 , which higher than control expressed level with 0.19 μmol gDWˆ’1 at 24 h. Therefore, a preliminary speculation, GTP according with AMP and ADP transformation but contrary to ATP when comparing NaAc affecting riboflavin accumulation. Figure ZZZ further revealed that GTP/ATP in control expressed extraordinary higher level (ratio=7) than other group ratios and NaAc culture, which indicated in NaAc being benefit for ATP accumulation but promoted GTP, AMP and ADP degradation in different degree.

Figure ZZZ. Energy transformation in Batch A, B and control culture in xylose by C. acetobutylicum ATCC 824. Square is Batch A, circle is Batch B, triangle is control;

3.2.2 Amino acids metabolism

Riboflavin production was affected by amino acids has been reported in many microorganisms. For instance, Giri & Krishnaswamy investigated a yeast stain Saccharomyces Cerevisise cultivated by various amino acids. Their results shown methionine, glycine and arginine employed as the most efficient organic nitrogen source in sequence, which promoted riboflavin yield to 40.5, 34.8 and 32.8 μg mL-1 (control reached 26.3 μg mL-1) (Giri & Krishnaswamy, 1953). Meanwhile, except asparagine, methionine and glycine effectively promoted riboflavin accumulation by Laciobacillus casei, which was carried out by Burkholder (Burkholder, 1943). Therefore, investigating amino acids metabolism on riboflavin production in present research plays a significant role.

As previous discussion, GTP is a significant flux joint and plays key role in riboflavin generation. However, GTP not only was employed as energy provider and riboflavin precursor, but served as significant intermediate in purine metabolism. Therefore, part species of amino acids would be related to GTP and riboflavin production when they participated into purine metabolism. For instance, glycine has been proved generating 5C and 7N atoms and glutamine was responsible for 3N and 9N generating in purine ring atoms. (Michal, 1999). Meanwhile, glycine was easily catalyzed to serine by glycine hydroxymethyltransferase and to threonine by L-threonine aldolase. Therefore, investigating amino acids assimilation for riboflavin metabolism is significantly. In present study, 15 amino acids were tracked during two NaAc addition batch and one control culture, Figure ZZZ plotted various amino acids consumption tendency. In summary, 11 amino acids were consumed obviously, 3 ones (proline, histadine and asparatic acid) kept in a stable level, and interestingly, one amino acid (glutamine) accumulated.

Figure ZZZ. Fifteen amino acids degradation in Batch A, B and control culture in xylose by C. acetobutylicum ATCC 824. Black square is Batch A, red circle is Batch B, blue triangle is control.

As mentioned above, a robust glycine consumption contributed to a high riboflavin production which according with shown in Figure ZZZ (j). In Batch A, the highest riboflavin culture experiment, glycine concentration decreased faster than others, which consumed 76.3% in the first 19 h when others still kept at initial level. The similar tendency exhibited in threonine, another riboflavin sensitive organic nitrogen source, but the limitation concentration kept at 0.2 mM which different with the most of other amino acids degradation (Figure ZZZ (b)). Meanwhile, as the main nitrogen source in the medium, asparagine was utilized at first 40 h but recovered immediately after this time point. But, the more probability of abundant asparagine flow direction is going to cells growth, but its influence for riboflavin generation was still non-ignorable. In fact, Cells growth mainly concentrated on the period of asparagine consumption, which revealed the nitrogen demand for cells multiplying. However, asparagine accumulated after cells’ exponential phase (40h) which indicated a reversed stimulation by asparagine after cells accessed stable phase (Figure ZZZ (n)).

Interestingly, glutamine accumulated from initial 0.1 mM to about 0.3 mM at 80 h, which indicated the direction of glutamine conversion Figure ZZZ (i). Glutamine synthetase played the key role on transforming glutamate to glutamine with one mole ATP degradation (Michal, 1999). However, glutamate was generated by many amino acids through α-KG (also called 2-oxo-glutarate) in citrate cycle, such as asparagine, phenylalanine and tyrosine. Therefore, as the first possibility, accumulated glutamine was transformed from other degraded amino acids in present research. However, the specific mechanism of glutamine accumulated in clostridia was still unclear.

Therefore, apart from purine metabolism related to amino acids uptake, as nitrogen source which plays more important role for cells growth and solvents production. Depending on different metabolic joints by various amino acids, five groups were distinguished which including group PYR, ACoA, OAA, α-KG and SCOA group (Figure ZZZ). In group PYR, ACoA and SCOA, included 9 amino acids were all consumed in different degrees. Interestingly, in group α-KG, histidine and proline were both kept in stable level while arginine consumed, but glutamine stimulated from initial. >>>>>>>>

Figure ZZZ. Metabolic pathway on amino acids.

3.2.3 Carbon and redox metabolic analysis

Main intracellular carbon metabolism by pentose in clostridia has been studied by Zhao et al, recently. They tracked main carbon flux joints under sodium iron environment and compared with control in anaerobic bottles (Zhao et al., 2016). However, in co-producing riboflavin system, Ru5P not only play a part in pentose phosphate pathway, but being employed GTP and further riboflavin production. Figure ZZZ exhibited a regressive phenomenon when cells accessed into solventogenesis in NaAc supplemented culture, Ru5P concentration recovered back to initial level. But in control group, it kept in a high level during all solvents producing period. In fact, all investigated intracellular carbon metabolites reached higher level in control culture than NaAc and kept all their superiority during solventogenesis. We attribute this phenomenon to a robust “highway” system when cells accessed into solventogenesis in NaAc supplemented environment, which dredged accumulated metabolites in acidogenesis for the solvents production.

Figure ZZZ. Intracellular carbon metabolites in Batch A, B and control culture in xylose by C. acetobutylicum ATCC 824. Square is Batch A, circle is Batch B, triangle is control.

One mole NADPH needed from Ru5P to GTP, and another one mole consumed by uracil reductase for GTP degradation. Therefore, investigating NADPH pool fluctuation was necessary for riboflavin production. Apart from riboflavin metabolism, NADP(H) redox pair also optionally participated ethanol/butanol dehydrogenase, and ferredoxin-NADP reductase (Gheshlaghi, Scharer, Moo-Young, & Chou, 2009). Interestingly, NADP(H) redox pair expressed similar trend, and kept stable ratio of NADPH/NADP in all culture period (Figure ZZZ). Differently, NAD(H) redox pair expressed inversely, when the NAD concentration in control reached highest level (7.00 μmol gDWˆ’1 versus 3.10 and 2.42 μmol gDWˆ’1 in Batch A,B), but which obtained the lowest level in NADH tracking (5.72 μmol gDWˆ’1 versus 19.13 and 29.32 μmol gDWˆ’1 in Batch A, B). Accordingly, the ratio of NADH/NAD was also lower than NaAc supplemented culture. In another word, NaAc addition promoted conversion from NAD to NADH, but which noneffective for NADP(H) pair (Figure ZZZ).

Figure ZZZ. Redox pairs tracking in Batch A, B and control culture in xylose by C. acetobutylicum ATCC 824. Square is Batch A, circle is Batch B, triangle is control.

3.3 Kinetic modeling

3.3.1 model calibration and parameters identification

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