Received date: March 15, 2012; Accepted date: March 29, 2012; Published date: March 31, 2012
Citation: Ahmed H, Naouel O, Djilali B (2012) 1,3-Propanediol Production by Clostridium Butyricum in Various Fed-Batch Feeding Strategies. J Biotechnol Biomaterial 2:134. doi:10.4172/2155-952X.1000134
Copyright: © 2012 Ahmed H, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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The aim of this study is to propose different fed-batch strategies during the fermentation of glycerol into 1,3-Propanediol, which show the influence of the initial concentration in glycerol and the feed flow on the behaviour of Clostridium butyricum . The kinetic study of the conversion of glycerol into 1,3-Propanediol by Clostridium butyricum , showed that according to the initial concentration of glycerol and feed flow, this strain displays two different metabolic behaviours. This strain directs its metabolism towards its traditional metabolic pathways (production of 1,3-Propanediol, butyric and acetic acid), when the culture was started with a high initial concentration of glycerol and was fed with a weak rate. The high initial concentration of glycerol causes a deceleration of growth rate, which led to an increase in the growth phase duration. Feeding by a weak flow allowed maintaining the cells in full growth phase by providing the necessary substrate for its growth and its maintenance and, at the same time, to avoid the glycerol accumulation in the medium. A high feed flow involved a glycerol accumulation in the culture medium, which obliged the bacterium to direct its metabolism towards the production of acetic acid in significant quantities. The growth phase always showed the same duration, yielding similar final concentrations in biomass and metabolites, an identical metabolic behaviour was observed when the culture begin with a low initial concentration of glycerol, and whatever the flow of substrate applied. This behaviour may be explained by a growth inhibition caused by the accumulation of 1,3-Propanediol in concentrations of 20-25 g/L.
Glycerol; 1.3-Propanediol; Clostridium butyricum; Fermentation; Fed-batch
The 1,3-Propanediol of formula CH2OH-CH2-CH2OH is a neutral, stable, sticky, miscible liquid in the water and in the alcohols, the density as of 1.06 and boiling point with atmospheric pressure of 214°C . It is at present used in the chemical synthesis of polyesters and polyurethanes, agent of tobacco elaboration, the lubricant of type polyalcohol, solvent, and as food additive . The synthesis of 1,3-Propanediol can be realized with chemical way from the hydro carbonation of the oxide of ethylene or of hydration of acrolein in hydroxypropanol followed by a catalytic reduction . These expensive processes implement toxic products (acrolein) and generate intermediaries compound and secondary not useful whish make them little profitable. At present, the production of 1.3-Propanediol by biologic way represents a good alternative if the production cost allows it . Numerous bacterial strains realize the fermentation of glycerol in 1,3-Propanediol: Klebsiella pneumoniae, Escherichia coli, Citrobacter freundii, Lactobacillus brevis, Clostridium pasteurianum, Clostridium acetobutylicum and Clostridium butyricum [5-11]. The aim of this study is to propose different fed-batch strategies during the fermentation of glycerol into 1,3-Propanediol, which show the influence of the initial concentration in glycerol and the feed flow on the behaviour of Clostridium butyricum.
Bacterial strain and medium
Clostridium butyricum strain used in all our experiments was isolated by the team of professor Petitdemange (Biologic Chemistry Laboratory of the Faculty of Science of Nancy - France) able of growing on industrial glycerol and allows the appearance of only two coproducts (acetic and butyric acid). Glycerol used as carbon substratum and of energy is produced during the transformation of oil of colza in ester fuel.
Fermentation conditions and methods
We used medium RCM (Reinforced Clostridia Medium) in a concentration of 38 g/L accompanied with a degassing by the nitrogen (0.5 vvm) to reactivate Clostridium butyricum spores further to a thermal shock of 10 mn in 80-85°C. So it allows discount in culture and the maintenance of strain. The sterilization of the cultural mediums is 120°C for 20 mn. Fed batch cultures are led in a reactor APPLIKON of 7 L jar, incubated during 24 hours in 32°C, led by an agitation speed to 200 rpm to assure gas exchange and not to cut through bacteria. The regulation of the pH in 6.9 is assured by the addition of a solution concentrated by KOH 2 N. The experimental protocol of the various strategies of fermentation is presented in the table 1.
|Initial broth volume in the fermentor (L)||Feed rate glycerol
|Pulse feeding times
Table 1: Experimental protocol of the various strategies of fed batch fermentations.
The determination of the cellular concentration (biomass) is esteemed by measure of optical density (OD) by spectrophotometer in 660 nm .
Weight dry (g/L) = C.DO
C. Coefficient of conversion (0.548)
The organic acids (acetic and butyric acid) are determined by HPLC (spectrophotometer to UV) and the glycerol’s with the 1.3-Propanediol are measured by HPLC (differential Refractometer). The characteristics of the analysis by HPLC are: Eluant: H2SO4 in 0.04 N, debit: 0.7 mL/mn, temperature: 65°C, column: polypore H 25 cm 7 mm, injector: 10 μL, lasted analysis 15 mn/sample.
Gram colouring is made regularly to control at the same moment any risk of contagion and presence or not of spores. The colouring is based on the differences of composition of the bacteria wall. Indeed, if bacteria are positive Gram, they seem purple and rose if they are negative Gram .
Kinetic explanation of fed batch fermentations
The made analysis allow to follow evolution during the time of the concentrations of the present constituents in the medium of culture: biomass X=f(t), residual glycerol S=f(t) and metabolites: P=f(t).
Volumetric rate r’’’x (g/L.h ) and specific rate of growth μ (h-1)
Volumetric rate r’’’s (g/L.h ) and specific rate of glycerol consumption QS (g/g.h)
Volumetric rate r’’’p (g/L.h ) and specific rate of metabolite production πp (g/g.h)
Volume in the reactor in L (V) Yields
The yields on conversion of the glycerol in biomass and products represent the slopes of the following equations:
The modelling consists in the annexation of kinetic laws in balances materials followed by the resolution of obtained differential equations [14,15]. The modelling is made on Matlab software by the method of differential equations resolution of Runge-Kutta.
Results of the batch fermentation
A batch culture begun with an initial concentration in glycerol of 70 g/L is made to determine profiles and kinetic parameters of the fermentation to choose flow and pulse of feeding in fed batch cultures. According to results mentioned on the figure 1, it appears that the growth of Clostridium butyricum on the glycerol is characterized by an absence of the phase of latency which indicates that the inoculated cells were in full exponential phase and the perfect adaptation of the strain in the culture medium (glycerol). This growth begins with an initial concentration of biomass of 0.1 g/L and arrives after 20 hours of fermentation at a cellular concentration of 2.7 g/L. This cellular multiplication leads to the production of 14.62 g/L of 1.3-Propanediol, 4.05 g/L of butyric acid and 1.85 g/L of acetic acid at the end of fermentation.
Clostridium butyricum multiplication on glycerol is characterized by a maximal specific rate of growth (μmax) of 1.82 h-1 obtained after 20 hours from fermentation. The classic pathway of the strain growth leads to a strong production of 1.3-Propanediol with a specific rate (π1,3PD max) of 9.84g/g.h and a weak production of secondary metabolites: butyric and acetic acid respectively with maximal specific rates (πBut and πAcet) of 4.27 and 0.32 g/g.h (Table 2)
|QGlyc max(g/g.h)||π1,3PD max (g/g.h)||πBut max(g/g.h)||ΠAcét max(g/g.h)|
Table 2: Specific rate of growth, consumption of glycerol and production of 1,3-propanediol, butyric and acetic acid during batch fermentation.
Results of fed batch fermentations
Cultures with 50 g/L of glycerol and with flow of 2.5, 3.5, 4.5, 5 and 10 g/h: The interest of this work is to study the influence of the feeding flow on the behaviour of Clostridium butyricum in fed batch cultures begun with an initial concentration of glycerol of 50 g/L and an initial volume in the fermentor of 1.5 L. The pulse was fixed at 0.03 L/h and the flow varies from 2.5 to 10 g/h by variation of the concentration of glycerol in the substratum of feeding. Study was led by comparison of the evolution of concentrations in biomass, glycerol and metabolites. Obtained results show a variation of the behaviour of Clostridium butyricum according to the adopted flow. This strain directs its metabolism towards the way of production of 1.3-Propanediol and of butyric acid or towards the pathway of production of the acetic acid according to the concentration of glycerol accumulated in the reactor.
In an initial concentration in glycerol of 50 g/L and with a weak flow (2.5 g/h), we noticed a weak cellular density at the end of fermentation (1.36 g/L). This growth limited by Clostridium butyricum has a repercussion on the quantity of 1.3-Propanediol and butyric acid produced (respectively of 13 g/L and 3.56 g/L). An accumulation of the glycerol is noticed at the end of fermentation with a concentration of 110 g/L. This high concentration of glycerol in the fermentor became toxic for the strain which directed its metabolism to the pathway of acetic acid production with a concentration of 5.67 g/L. For cultures fed with 3.5, 4.5 and 5 g/h, we noticed a very strong cellular density in the reactor at the end of fermentation (2.5, 2.51 and 2.11 g/L). This cellular growth is accompanied by a strong production of 1.3-Propanediol respectively with concentrations of 26.7, 35.36 and 25.6 g/L. The concentration of the acetic acid produced during these fermentations is very weak with regard to the first fed batch culture (Figure 2, 3 and Table 3).
|Parameters Flow||Final biomass (g/L)||Final 1.3-Propanediol (g/L)||Final butyric a. (g/L)||Final acetic a. (g/L)||Final glycerol (g/L)|
Table 3: Concentrations of biomass, glycerol, 1,3-propanediol, butyric and acetic acid in g/L obtained at the end of fed-batch cultures.
Concerning culture fed with a pulse of 10 g/h, we noticed a strong accumulation of the glycerol in the culture medium, which arrives to 230 g/L. This concentration of toxic substratum for the strain influenced cellular growth (1.9 g/L). The concentration of the acetic acid produced during this fermentation is to the order of 6.33 g/L. The table 4 indicates kinetic parameters obtained during Clostridium butyricum fed batch fermentation. Cultures fed with 3.5 and 5 g/h allow having a very high μmax of the order of 0.53 and 0.4 h-1. The specific rate of production of the acetic acid is especially raised during the last fermentation with an πAc max of 0.64 g/g.h.
|Parameters Flow||µmax(h-1)||QGlyc max(g/g.h)||π1.3PD max (g/g.h)||πBut max(g/g.h)||πAc max(g/g.h)|
Table 4: Kinetics parameters of fed-batch fermentations.
Cultures with 100 g/L of glycerol and with streams of feed of 2.5 and 10 g/hour: To study the incidence of the initial concentration in substratum on profiles and kinetics of conversion of the glycerol in 1,3-Propanediol by Clostridium butyricum, two fed batch cultures begun by 100 g/L of glycerol and fed by 2.5 and 10 g/hour were made. The analysis of the profiles of the evolution of the biomass concentration and metabolites production during these two fed batch cultures (Figure 4, 5 and Table 5) shows us:
|Parameters Flow||Final biomass (g/L)||Final glycerol (g/L)||Final 1.3-propanediol (g/L)||Final butyric a. (g/L)||Final acetic a. (g/L)|
Table 5: Concentrations of biomass, glycerol, 1,3-propanediol, butyric and acetic acid in g/L obtained at the end of fed batch culture.
During the first fermentation fed by 2.5 g/h of glycerol and initially the begun by 100 g/L of substratum, biomass obtained at the end of fermentation is of 1.4 g/L with an exponential phase which spreads out during all the duration of fermentation. This cellular growth is accompanied by a strong consumption of glycerol (residual glycerol is of 31.1 g/L) and a strong production of 1,3-Propanediol and butyric acid respectively of 31.1 g/L and 11.55 g/L. The concentration of the acetic acid produced during the fermentation is very weak of the order of 4.1 g/L.
For the second culture fed by 10 g/h of glycerol and initially the begun by 100 g/L of substratum, an absence of the phase of latency is observed which indicates the viability of cells during the fermentation. Obtained cellular density is of the order of 1.99 g/L during the first hours of the fermentation. At the time of the feed in glycerol, cellular lyse is observed involving a decrease of the biomass and accumulation of glycerol in the fermentation medium (288.2 g/L). The answer of the strain to this physiological stress is manifested by a weak production of 1,3-Propanediol and butyric acid respectively of 3.13 and 1.49 g/L and a strong accumulation of the acetic acid of the order of 13.68 g/L.
Clostridium butyricum strain is characterized with three different metabolic pathways :
- The pathway of synthesis of the 1,3-Propanediol, which does not produce energy but uses the reducing elements.
- The pathway glycerol-butyric acid which leads by two molecules of consummate glycerol to the creation of three ATP molecules, two NADH+H+ molecules and two molecules of hydrogen.
- The pathway glycerol-acetic acid, which leads by molecule of consummate glycerol to the forming of two ATP molecules, two NADH+H+ molecule and a molecule of hydrogen.
Fran this analysis, it springs that the way glycerol-acetic acid is indispensable for the bacterium because it supplies necessary energy for the growth and the maintenance of the cell.
The analysis of the kinetic parameters of two fermentations shows that for the first culture the exponential phase of the cellular growth extends during all the duration of fermentation with a μmax which is weak but constant of the order of 0.064 h-1 against a μmax of the second fermentation of 0.1158 h-1 during the first hours of fermentation (during the batch phase). On the other hand the specific rate of production of 1,3-Propanediol and butyric acid are more brought up during the first culture respectively of 1.266 and 0.431 g/g.h. As answer of physiological stress led by the accumulation of glycerol in the reactor the specific rate of production of the acetic acid (πAc max) is sharply superior during the second fermentation of the order of 2.479 g/g.h against 0.695 g/g.h for the first fed-batch culture (Table 6)
|Parameters Flow||µmax (h-1)||QGlyc max
Table 6: Kinetics parameters of fed-batch fermentation.
Carbon balance: To verify the reliability of obtained results, we are going to establish the carbon balance during the fed-batch fermentation begun by 100 g/L of glycerol and fed by 2.5 g/h with substratum. This balance allows us to compare the quantity of carbon brought in the beginning of fermentation and quantity found in the end. We consider that the chemical formula of the biomass is of C5H7NO2 (chemical comparison averages bacteria). Furthermore, during the forming of a mole of acetic acid there is release of CO2 mole while during the forming of a mole of butyric acid there are two moles of CO2. We consider also that the main products of the cellular metabolism are the 1,3-Propanediol, butyric acid and acetic acid (Table 7 and Figure 6).
|Constituents||Quantity (g)||Carbon (g)||% of carbon|
|Constituents||Quantity (g)||Carbon (g)||% of carbon|
Table 7: Carbon balance.
The carbon balance is curled in a percentage of 96.7 % what brings us to conclude that uncertainty on measures is acceptable.
Modelling of fermentations: To quantify the effect of the feed flow in glycérol in the évolution of Clostridium butyricum and in the profiles of production of metabolic, we attempted to translate under shape of mathematical equations these various effects. This obtained model allows estimating performances fermentors of a culture by the knowledge of the initial concentration in glycerol, the feed flow and the concentration of glycerol in the substratum of food. To realize this modelling we estimated the specific rate of growth (μ), of consumption of substratum (Qglyc) and production of metabolic (πP) (Table 8 and Figure 7)
|Parameters||Theoretical values||Experimental values|
Table 8: Values of the kinetic parameters in the obtained model.
For the growth and the consumption of the glycerol:
For the metabolic production:
r’’’x : Volumetric rate of production of biomass (g/L.h)
r’’’Glyc : Volumetric rate of consumption of the glycerol (g/L.h)
r’’’p : Volumetric rate of production metabolic (g/L.h)
YX/Glyc : Yield biomass/glycerol (g/g)
YP/Glyc : Yield products/glycerol (g/g)
μ: Specific growth rate (h-1)
μmax: Maximal specific rate of growth (h-1)
S: Glycerol concentration (g/L)
Ks: Saturation constant (g/L)
Kp: Inhibition by the glycerol constant (g/L)
Ki: Inhibition by the 1,3-Propanediol constant (g/L)
To implement a strategy of successful fermentation allowing optimizing the production of 1,3-Propanediol, the kinetic characteristics of conversion of the glycerol at Clostridium butyricum were studied. The aim of this work was to make kinetic studies on the production of the 1,3-Propanediol from the glycerol by Clostridium butyricum and to study the influence of the parameters of environment (initial content in glycerol and feed flow) on the behavior of this strain. This method allowed defining the best conditions of growth and production of the 1,3-Propanediol. Different fed batch strategies were realized on glycerol as only source of carbon and energy; they began with weak and strong initial contents in glycerol and fed with weak and strong flow of glycerol, allowed us to retain different behaviors of Clostridium butyricum. According to obtained results, we can conclude that the pathway of production of the 1,3-Propanediol is associated to the one of some butyric acid. The accumulation of the glycerol in the fermentation medium provokes a disorientation of the Clostridium butyricum cellular metabolism towards the pathway of production of the acetic acid and contribution controlled by substratum in the fermentation medium allows the production of an important quantity of 1.3-Propanediol and butyric acid. This accumulation of the glycerol in the fermentation medium blocks and inhibits assimilation of the glycerol in the internal of the cell, which provokes a very strong decrease of kinetic parameters and yield on conversion of the glycerol in 1,3-Propanediol and butyric acid. The modeling showed a big resemblance between the experimental reality and the realized model.
I hold in thank particularly Pr. Michel Fick to have me to help to realize this work as well as all the team of the laboratory GPBA.