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Improvement of the poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) production by dual feeding with levulinic acid and sodium propionate in Cupriavidus necator
New Biotechnology Volume 00, Number 00 July 2015
RESEARCH PAPER
Research Paper
Improvement of the poly(3-hydroxybutyrateco-3-hydroxyvalerate) (PHBV) production by dual feeding with levulinic acid and sodium propionate in Cupriavidus necator Nathalie Berezina1 and Bopha Yada Q1 Materia Nova R&D Centre, rue des Foudriers 1, 7822 Ghislenghien, Belgium
In the context of increasing volatility of oil prices, replacement of petroleum based plastics by bioplastics is a topic of increasing interest. Poly(hydroxyalkanoate)s (PHAs) are among the most promising families in this field. Controlling composition of the polymer on the monomeric level remains a pivotal issue. This control is even more difficult to achieve when the polymer is not synthesized by chemists, but produced by nature, in this case, bacteria. In this study mechanism and role of two 3-hydroxyvalerate (3HV) inducing substrates on the production of PHBV with high, 80%, 3-HV content were evaluated. It was found that levulinic acid contributes to biomass and bio-polymer content enhancement, whereas sodium propionate mainly contributes to 3-HV enhancement. Optimized proportions of feeding substrates at 1 g/L and 2.5 g/L, respectively for levulinic acid and sodium propionate allowed a 100% productivity enhancement, at 3.9 mg/L/hour, for the production of PHBV with 80% 3-HV.
Introduction Q3 Necessity of more sustainable and responsible behaviour rose these last years all around the world. Different aspects were concerned so far: energy, fuel, plastics, waste management, recycling, etc. [1]. Plastics are ubiquitous in today’s society, unfortunately the mainly used are from fossil origin and non-biodegradable. Substitution of traditional petroleum-based plastic materials, by bio-plastics, is not an easy task, new materials have to reach economic and technical constraints, or present specific characteristics to meet not yet addressed issues [2,3]. Poly(hydroxyalkanoate) (PHA) family represents a very promising group of bio-polymers [4]. This family presents a wide range of polymers with very diverse characteristics, depending on the length of the alkanoate substitute and of the co-polymers composition [5–7]. The most studied part of the PHA family concerns the short-chain length PHA (PHASCL), which main representatives are poly(3-hydroxybutyrates) (PHB) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) [8]. The production of PHB
Corresponding author: Berezina, N. ([email protected]), Yada, B. ([email protected]) 1
Current address: Ynsect, 1 rue Pierre Fontaine, 91058 Evry, France.
http://dx.doi.org/10.1016/j.nbt.2015.06.002 1871-6784/ß 2015 Published by Elsevier B.V.
can be achieved by many strains and at relevant yields and production rates [9–11]. These polymers can be used either for technical [12], medical [13] or bioremediation [14,15] purposes. However, certain applications require less brittle, more elastic polymer with lower glass transition temperature (Tg). This can be achieved by modulation of PHB composition and the incorporation of the 3-hydroxyvalerate (3-HV) monomeric units [16,17]. Some, rather rare, strains, such as Haloferax mediterranei [18,19] or Halomans campisalis [20], are able to produce PHBV on unrelated carbon sources. However, the main PHBV producers, such as Cupriavidus genus (previously known as Alcaligenes, among others) were shown to produce the biopolymers with noticeable 3-HV content when cultured on the odd-carbon numbered inducing substrates [21,22]. Recently, we have shown [23] that in the case of Cupriavidus necator DSM 545 the combination of levulinic acid with sodium propionate gave the best results for the enhancement of 3-HV content in PHBV. In the present study the interactions between those compounds, as well as their optimal concentrations and proportions were studied in order to enhance the PHBV production with high 3-HV content. www.elsevier.com/locate/nbt
Please cite this article in press as: Berezina, N., and Yada, B., Improvement of the poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) production by dual feeding with levulinic acid and sodium propionate in Cupriavidus necator, New Biotechnol. (2015), http://dx.doi.org/10.1016/j.nbt.2015.06.002
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Materials and methods General All chemicals were from Sigma Aldrich, C. necator DSM 545 was from the DSMZ collection. Sartorius Certomat IS incubators were used for the fermentations.
Fermentations
Research Paper
Mineral medium: 6.7 g of Na2HPO42H2O, 1.5 g of KH2PO4, 1 g of (NH4)2SO4, 0.2 g of MgSO47H2O, 0.01 g of CaCl22H2O, 0.06 g of Fe(NH4)2(SO4)2 and 1 mL of trace element solution for 1 L. Trace element solution: 0.3 g of H3BO3, 0.2 g of CoCl2, 0.1 g of ZnSO4 7H2O, 0.03 g of MnCl24H2O, 0.02 g of NaMoO42H2O, 0.02 g of NiCl26H2O and 0.01 g of CuSO45H2O for 1 L. The pH value was adjusted at 7. 100 mL inoculates were grown for 24 hours on mineral medium in 300 mL shaking flasks, at 308C, 150 rpm. Further inoculations were performed with 5% of 24 hours starter culture and the only C source was a mix of levulinic acid and sodium propionate at different combinations with the concentrations ranging from 1 to 5 g/L. The screening experiments were performed in 300 mL shaking flasks filled with 100 mL medium and the scale up was made in the 3 L shaking flasks filled with 1 L medium.
Analysis The 3-hydroxyvalerate content was determined from 1H [24] and 13 C [25] NMR spectra, performed on a Bruker 500 MHz instrument, and is given in mol %. The PHA content in cells was determined by the thermogravimetric analysis (TGA) [26] performed with a Q500 TA instrument. The concentrations of levulinic acid and sodium propionate were evaluated by HPLC, performed with an Alliance Waters instrument, equipped with a Photodiode Array (PDA) detector (Waters 2996) operating at 220 nm, using a Metacarb 67H column from Varian, and deionized water containing 1% H2SO4 as mobile phase. All experiments were performed at least twice. All values presented here are averaged, and the error bars represent standard deviation.
Results and discussion Screening of different proportions of levulinic acid and sodium propionate To evaluate the most suitable proportions of the two, previously shown to be the most promising [23], 3-HV inducing substrates, the experiments with different initial concentrations ranging from 1 to 5 g/L of each, were performed (Fig. 1). The time-course of biomass (Fig. 1a) shows that a rather long period, 24 hours, is needed in any case for the acclimation of cells to those substrates. Also, as soon as the concentration of sodium propionate reaches 5 g/L, the biomass enhancement is no longer observed. The experiments with low amount of sodium propionate (1 g/L) concomitant with levulinic acid concentration below 5 g/L, have reached the maximum cell dry mass (CDM), after 48 hours of experiment. The highest biomass was observed with the highest concentration of levulinic acid (5 g/L) when the sodium propionate concentration was kept at a minimum (1 g/L). These observations are consistent with the analysis of PHA content in cells (Fig. 1b), no PHA were produced at 24 hours of fermentation. The amount of PHA was more relevant at higher 2
FIGURE 1
Time courses for CDM (a) and PHA content in cells (b), during the screening experiments. The different initial concentrations of levulinic acid and sodium propionate are indicated in g/L, following this order (1:2.5 corresponds to the experiment with the initial concentrations of levulinic acid and sodium propionate of 1 and 2.5 g/L, respectively).
concentration of levulinic acid. It decreased between 48 and 72 hours when levulinic acid was fed at 1 g/L and increased when it was at 5 g/L. At 2.5 g/L of initial levulinic acid, the evolution of PHA content was dependant on the concentration of sodium propionate: it slightly decreased (merely remained stable) at 1 g/ L and increased at 2.5 g/L of the latter substrate. For a better understanding of the phenomena occurring during these fermentations we have also monitored the consumption of substrates (Fig. 2). With an initial sodium propionate concentration of 5 g/L no consumption of either sodium propionate or levulinic acid was observed (Fig. 2c,f,i), consistent with the above observations (Fig. 1a). Also, at small concentration of sodium propionate, 1 g/L, it has been almost linearly consumed in 48 hours. Levulinic acid at small concentration, 1 g/L, was concomitantly consumed (Fig. 2a), its consumption being delayed at higher concentrations, 2.5 g/L (Fig. 2d) and 5 g/L (Fig. 2g). This can explain the limited to 48 hours growth of cells at small, 1 g/L, sodium propionate concentrations concomitant with small to moderate, 1 to 2.5 g/L, levulinic acid concentrations; as well as the decrease of PHA content in cells between 48 and 72 hours of cells’ cultivation in those experiments, since, after the exhaustion of sodium propionate and levulinic acid, the PHA become the only source of carbon for cells. Initial sodium propionate at 2.5 g/L requires 72 hours for being totally consumed, and levulinic acid concentration is hardly modified during the whole fermentation process in these experiments (Fig. 2b,e,h). These observations
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FIGURE 2
Time courses of the concentrations of levulinic acid and sodium propionate at different initial concentrations (introduced amounts) during the screening experiments.
suggest that the consumption of substrates follows strict order, sodium propionate being digested prior to levulinic acid. Finally, 3-HV content in the obtained PHBV showed a very interesting dependence on the initial substrate concentrations: when the concentration of levulinic acid progressed (sodium propionate concentration being the same) – the 3-HV content decreased, while when the sodium propionate concentration increased (the levulinic acid concentration being the same) – the 3-HV content increased (Fig. 3). The screening experiments thus showed that the enhancements of biomass and of PHA content in cells were mostly due to the levulinic acid, whereas the enhancement of 3-HV content was mostly due to the sodium propionate, moreover the 5 g/L concentration of sodium propionate appeared to be inhibiting for the cells. The possibility of the inhibition of growth of PHA producing strains by the 3-HV inducing substrates was already reported [27]. Therefore, our results are rather consisting with those previously reported [28] when C. necator was cultured on a mixture of 3 fatty acids, that is, acetic, butyric and propionic. Also the inhibition by valerate was reported on a Pseudomonas hydrogenovora strain [29] but actually at lower concentration, that is, 2 g/L.
Kinetic considerations Screening experiments have shown that the two studied substrates mainly contribute to two different aspects of PHA production: levulinic acid mainly contributes to biomass and PHA content
FIGURE 3
The 3-HV content in PHBV obtained during the screening experiments with different initial concentrations of levulinic acid and sodium propionate.
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enhancements, whereas sodium propionate mainly contributes to 3-HV enhancement. We have also noticed (Fig. 2) that the kinetics of consumption of these two substrates were very different, sodium propionate being consumed primarily to levulinic acid. It becomes therefore very tempting to try to figure out a kinetic model which can describe the whole process. The considered reactions took place in cells, representing a pool of enzymes, being thus very difficult to model. However, the previously observed fact that sodium propionate is transformed before the beginning of the levulinic acid transformation, combined with the observation that, when the concentration of sodium propionate becomes inhibitory (5 g/L) and no transformation of levulinic acid is observed, suggest that both, sodium propionate and levulinic acid, have the same receptor at the cell wall and that the sodium propionate has to bind first to it, being the preferential substrate. Considering that, we can make several simplifying hypothesis, that is, considering that the binding to the cell wall is the limiting kinetic step and thus making all the modelling considering the whole cell as en enzyme working with two substrates, sodium propionate and levulinic acid, and delivering two products biomass (and PHA content in cells) and 3-HV content in PHBV.
A
B
P
A
In this case the whole process can be considered as a classical enzymatic two-substrate reaction. This situation was abundantly studied [30–32] and different mechanisms are generally classified into two categories: Ping-Pong and sequential mechanisms, the latter one being either ordered or random (Fig. 4). In the Ping-Pong mechanism one or more products must be released before all substrates can react. In sequential mechanisms, all substrates must combine with the enzyme before the reaction can take place. However, in ordered sequential mechanism, substrates react with the enzyme and the products are released in a specific order, whereas in random sequential mechanism the order of substrate combination and product release is not mandatory. In previously described situation the random approach (Fig. 4c) is clearly not the one observed. To discriminate between the two others, one have to determine whether we are facing the ternary EAB complex (ordered-sequential mechanism) (Fig. 4b) or primary release of the first product (P) with the subsequent modification of E to E0 before the consumption of the second substrate (Ping-Pong mechanism) (Fig. 4a). The difficulty here relies on the fact that biomass and PHA production on the one hand and 3-HV content on the other, can hardly be considered as independent products P and Q, showing thus severe limitation of the high simplification
Q Ping-Pong bi-bi mechanism
E
(EA
A
B
E'P)
E'
(E'B
P
B
E
EQ)
Q
ordered sequential bi-bi mechanism E
EA
(EAB
C A
P
B
E
EQ
EPQ)
Q random-sequnetial bi-bi mechanism
E
EAB
B
A
E
EPQ
Q
P
FIGURE 4
Different models of two-substrate enzymatic kinetic mechanisms. E and E0 stand for enzymes, A and B for substrates, P and Q for products. 4
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the production of 3HB and 3HV monomers, however more specific work is needed to confirm this hypothesis.
A 1,2
Scale-up of the optimized conditions
0,8 0,6 0,4 0,2 0,0 0
20
40
60
80
100
time (h) CDM (g/L)
concentration (g/L)
B
3 2,5 2 1,5 1 0,5 0 0
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time (h) levulinic acid
The conditions chosen for the scale-up experiment were the ones allowing the highest 3-HV content, that is, 1 g/L initial levulinic acid and 2.5 g/L initial sodium propionate (Fig. 5). As previously observed, the consumption of sodium propionate at 2.5 g/L initial concentration is rather slow during the first 48 hours and deeply accelerates after this period (Fig. 5b), being consistent with the start of the exponential cells’ growth, beginning after 50 hours of fermentation (Fig. 5a). Also the time-course of the consumption of the substrates seems to confirm the Ping-Pong kinetic hypothesis, as levulinic acid concentration remains almost unchanged until 72 hours of the fermentation process and starts to decline only at 74 hours, when 84% of sodium propionate were consumed (Fig. 5b). Finally, using these conditions 0.3 g/L of PHBV with 3-HV content of 80% were obtained. Moreover, the volumetric productivity reached 3.9 mg/L/hour, exceeding by 100% the previously reported results [23].
Conclusion sodium propionate
FIGURE 5
Time courses for CDM (a), and substrates concentration in the medium (b), during the scale-up experiment.
hypothesis. However, the kinetics of consumption of the 2 considered substrates (Fig. 2) suggest that sodium propionate is consumed before the beginning of the decline of levulinic acid concentration in the reaction medium, therefore implying that the ternary EAB complex is not a correct description of what is happening during this transformation. In this case the Ping-Pong mechanism hypothesis seems to be the one to be privileged. Those assumptions are also consistent with the previous work reported by Jaremko and Yu [33], they have shown that the metabolism of levulinic acid by C. necator is split into acetylCoA and propionyl-CoA pathways, which confirms the preference for the consumption of propionate. Another interesting point is the influence of levulinic acid on the monomeric composition of the resulting PHA. Indeed, it was previously reported [34–36] that this substrate also induces the 4HV co-monomer and the final polymer is a terpolyester P(3HB-co3HV-co-4HV), the latter remaining the minor one. In our experiences we did not observed any significant trace of 4HV, this can be due to the total absence of even-numbered carbon substrates in our work, thus mainstreaming the metabolism of levulinic acid to
A precise study of the influence of initial concentration of levulinic acid and sodium propionate on the production of PHBV by C. necator with high 3-HV mol fraction was achieved. Even if this dual feeding was originally designed for the 3-HV content enhancement, the two substrates were found to play different roles, levulinic acid being mainly dedicated to cells growth and PHA enhancement, whereas sodium propionate – to the actual 3-HV enhancement. Also, for the first time a Ping-Pong-like kinetics for the consumption of levulinic acid and sodium propionate were suggested, thus allowing more specific understanding of inherent processes regulating biomass, PHA and 3-HV contents production and improvement. It was thus found that the most efficient combination for the production of PHBV with high 3-HV content is 1 g/L initial levulinic acid and 2.5 g/L initial sodium propionate concentrations. This allows the volumetric productivity of 3.9 mg/L/hour which is 100% greater than the previous results [23]. This method remains economical, as it uses only 3.5 g/L of carbon substrates for the production of PHBV with high, 80%, 3-HV content comparing to previously reported studies, with 20 g/L of inducing substrates [25,37]. Finally, to go forward and circumvent the inhibition effect observed here, an application of continuous strategy may be worthwhile, indeed, this technique has been previously described as valuable for such type of improvements [15,38,39].
Acknowledgements The authors are thankful to the Walloon region and FEDER Q4 European funds for the financial support through the SINOPLISS 565515-346987 program.
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Research Paper
Improvement of the poly(3-hydroxybutyrateco-3-hydroxyvalerate) (PHBV) production by dual feeding with levulinic acid and sodium propionate in Cupriavidus necator Nathalie Berezina1 and Bopha Yada Q1 Materia Nova R&D Centre, rue des Foudriers 1, 7822 Ghislenghien, Belgium
In the context of increasing volatility of oil prices, replacement of petroleum based plastics by bioplastics is a topic of increasing interest. Poly(hydroxyalkanoate)s (PHAs) are among the most promising families in this field. Controlling composition of the polymer on the monomeric level remains a pivotal issue. This control is even more difficult to achieve when the polymer is not synthesized by chemists, but produced by nature, in this case, bacteria. In this study mechanism and role of two 3-hydroxyvalerate (3HV) inducing substrates on the production of PHBV with high, 80%, 3-HV content were evaluated. It was found that levulinic acid contributes to biomass and bio-polymer content enhancement, whereas sodium propionate mainly contributes to 3-HV enhancement. Optimized proportions of feeding substrates at 1 g/L and 2.5 g/L, respectively for levulinic acid and sodium propionate allowed a 100% productivity enhancement, at 3.9 mg/L/hour, for the production of PHBV with 80% 3-HV.
Introduction Q3 Necessity of more sustainable and responsible behaviour rose these last years all around the world. Different aspects were concerned so far: energy, fuel, plastics, waste management, recycling, etc. [1]. Plastics are ubiquitous in today’s society, unfortunately the mainly used are from fossil origin and non-biodegradable. Substitution of traditional petroleum-based plastic materials, by bio-plastics, is not an easy task, new materials have to reach economic and technical constraints, or present specific characteristics to meet not yet addressed issues [2,3]. Poly(hydroxyalkanoate) (PHA) family represents a very promising group of bio-polymers [4]. This family presents a wide range of polymers with very diverse characteristics, depending on the length of the alkanoate substitute and of the co-polymers composition [5–7]. The most studied part of the PHA family concerns the short-chain length PHA (PHASCL), which main representatives are poly(3-hydroxybutyrates) (PHB) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) [8]. The production of PHB
Corresponding author: Berezina, N. ([email protected]), Yada, B. ([email protected]) 1
Current address: Ynsect, 1 rue Pierre Fontaine, 91058 Evry, France.
http://dx.doi.org/10.1016/j.nbt.2015.06.002 1871-6784/ß 2015 Published by Elsevier B.V.
can be achieved by many strains and at relevant yields and production rates [9–11]. These polymers can be used either for technical [12], medical [13] or bioremediation [14,15] purposes. However, certain applications require less brittle, more elastic polymer with lower glass transition temperature (Tg). This can be achieved by modulation of PHB composition and the incorporation of the 3-hydroxyvalerate (3-HV) monomeric units [16,17]. Some, rather rare, strains, such as Haloferax mediterranei [18,19] or Halomans campisalis [20], are able to produce PHBV on unrelated carbon sources. However, the main PHBV producers, such as Cupriavidus genus (previously known as Alcaligenes, among others) were shown to produce the biopolymers with noticeable 3-HV content when cultured on the odd-carbon numbered inducing substrates [21,22]. Recently, we have shown [23] that in the case of Cupriavidus necator DSM 545 the combination of levulinic acid with sodium propionate gave the best results for the enhancement of 3-HV content in PHBV. In the present study the interactions between those compounds, as well as their optimal concentrations and proportions were studied in order to enhance the PHBV production with high 3-HV content. www.elsevier.com/locate/nbt
Please cite this article in press as: Berezina, N., and Yada, B., Improvement of the poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) production by dual feeding with levulinic acid and sodium propionate in Cupriavidus necator, New Biotechnol. (2015), http://dx.doi.org/10.1016/j.nbt.2015.06.002
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Materials and methods General All chemicals were from Sigma Aldrich, C. necator DSM 545 was from the DSMZ collection. Sartorius Certomat IS incubators were used for the fermentations.
Fermentations
Research Paper
Mineral medium: 6.7 g of Na2HPO42H2O, 1.5 g of KH2PO4, 1 g of (NH4)2SO4, 0.2 g of MgSO47H2O, 0.01 g of CaCl22H2O, 0.06 g of Fe(NH4)2(SO4)2 and 1 mL of trace element solution for 1 L. Trace element solution: 0.3 g of H3BO3, 0.2 g of CoCl2, 0.1 g of ZnSO4 7H2O, 0.03 g of MnCl24H2O, 0.02 g of NaMoO42H2O, 0.02 g of NiCl26H2O and 0.01 g of CuSO45H2O for 1 L. The pH value was adjusted at 7. 100 mL inoculates were grown for 24 hours on mineral medium in 300 mL shaking flasks, at 308C, 150 rpm. Further inoculations were performed with 5% of 24 hours starter culture and the only C source was a mix of levulinic acid and sodium propionate at different combinations with the concentrations ranging from 1 to 5 g/L. The screening experiments were performed in 300 mL shaking flasks filled with 100 mL medium and the scale up was made in the 3 L shaking flasks filled with 1 L medium.
Analysis The 3-hydroxyvalerate content was determined from 1H [24] and 13 C [25] NMR spectra, performed on a Bruker 500 MHz instrument, and is given in mol %. The PHA content in cells was determined by the thermogravimetric analysis (TGA) [26] performed with a Q500 TA instrument. The concentrations of levulinic acid and sodium propionate were evaluated by HPLC, performed with an Alliance Waters instrument, equipped with a Photodiode Array (PDA) detector (Waters 2996) operating at 220 nm, using a Metacarb 67H column from Varian, and deionized water containing 1% H2SO4 as mobile phase. All experiments were performed at least twice. All values presented here are averaged, and the error bars represent standard deviation.
Results and discussion Screening of different proportions of levulinic acid and sodium propionate To evaluate the most suitable proportions of the two, previously shown to be the most promising [23], 3-HV inducing substrates, the experiments with different initial concentrations ranging from 1 to 5 g/L of each, were performed (Fig. 1). The time-course of biomass (Fig. 1a) shows that a rather long period, 24 hours, is needed in any case for the acclimation of cells to those substrates. Also, as soon as the concentration of sodium propionate reaches 5 g/L, the biomass enhancement is no longer observed. The experiments with low amount of sodium propionate (1 g/L) concomitant with levulinic acid concentration below 5 g/L, have reached the maximum cell dry mass (CDM), after 48 hours of experiment. The highest biomass was observed with the highest concentration of levulinic acid (5 g/L) when the sodium propionate concentration was kept at a minimum (1 g/L). These observations are consistent with the analysis of PHA content in cells (Fig. 1b), no PHA were produced at 24 hours of fermentation. The amount of PHA was more relevant at higher 2
FIGURE 1
Time courses for CDM (a) and PHA content in cells (b), during the screening experiments. The different initial concentrations of levulinic acid and sodium propionate are indicated in g/L, following this order (1:2.5 corresponds to the experiment with the initial concentrations of levulinic acid and sodium propionate of 1 and 2.5 g/L, respectively).
concentration of levulinic acid. It decreased between 48 and 72 hours when levulinic acid was fed at 1 g/L and increased when it was at 5 g/L. At 2.5 g/L of initial levulinic acid, the evolution of PHA content was dependant on the concentration of sodium propionate: it slightly decreased (merely remained stable) at 1 g/ L and increased at 2.5 g/L of the latter substrate. For a better understanding of the phenomena occurring during these fermentations we have also monitored the consumption of substrates (Fig. 2). With an initial sodium propionate concentration of 5 g/L no consumption of either sodium propionate or levulinic acid was observed (Fig. 2c,f,i), consistent with the above observations (Fig. 1a). Also, at small concentration of sodium propionate, 1 g/L, it has been almost linearly consumed in 48 hours. Levulinic acid at small concentration, 1 g/L, was concomitantly consumed (Fig. 2a), its consumption being delayed at higher concentrations, 2.5 g/L (Fig. 2d) and 5 g/L (Fig. 2g). This can explain the limited to 48 hours growth of cells at small, 1 g/L, sodium propionate concentrations concomitant with small to moderate, 1 to 2.5 g/L, levulinic acid concentrations; as well as the decrease of PHA content in cells between 48 and 72 hours of cells’ cultivation in those experiments, since, after the exhaustion of sodium propionate and levulinic acid, the PHA become the only source of carbon for cells. Initial sodium propionate at 2.5 g/L requires 72 hours for being totally consumed, and levulinic acid concentration is hardly modified during the whole fermentation process in these experiments (Fig. 2b,e,h). These observations
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FIGURE 2
Time courses of the concentrations of levulinic acid and sodium propionate at different initial concentrations (introduced amounts) during the screening experiments.
suggest that the consumption of substrates follows strict order, sodium propionate being digested prior to levulinic acid. Finally, 3-HV content in the obtained PHBV showed a very interesting dependence on the initial substrate concentrations: when the concentration of levulinic acid progressed (sodium propionate concentration being the same) – the 3-HV content decreased, while when the sodium propionate concentration increased (the levulinic acid concentration being the same) – the 3-HV content increased (Fig. 3). The screening experiments thus showed that the enhancements of biomass and of PHA content in cells were mostly due to the levulinic acid, whereas the enhancement of 3-HV content was mostly due to the sodium propionate, moreover the 5 g/L concentration of sodium propionate appeared to be inhibiting for the cells. The possibility of the inhibition of growth of PHA producing strains by the 3-HV inducing substrates was already reported [27]. Therefore, our results are rather consisting with those previously reported [28] when C. necator was cultured on a mixture of 3 fatty acids, that is, acetic, butyric and propionic. Also the inhibition by valerate was reported on a Pseudomonas hydrogenovora strain [29] but actually at lower concentration, that is, 2 g/L.
Kinetic considerations Screening experiments have shown that the two studied substrates mainly contribute to two different aspects of PHA production: levulinic acid mainly contributes to biomass and PHA content
FIGURE 3
The 3-HV content in PHBV obtained during the screening experiments with different initial concentrations of levulinic acid and sodium propionate.
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enhancements, whereas sodium propionate mainly contributes to 3-HV enhancement. We have also noticed (Fig. 2) that the kinetics of consumption of these two substrates were very different, sodium propionate being consumed primarily to levulinic acid. It becomes therefore very tempting to try to figure out a kinetic model which can describe the whole process. The considered reactions took place in cells, representing a pool of enzymes, being thus very difficult to model. However, the previously observed fact that sodium propionate is transformed before the beginning of the levulinic acid transformation, combined with the observation that, when the concentration of sodium propionate becomes inhibitory (5 g/L) and no transformation of levulinic acid is observed, suggest that both, sodium propionate and levulinic acid, have the same receptor at the cell wall and that the sodium propionate has to bind first to it, being the preferential substrate. Considering that, we can make several simplifying hypothesis, that is, considering that the binding to the cell wall is the limiting kinetic step and thus making all the modelling considering the whole cell as en enzyme working with two substrates, sodium propionate and levulinic acid, and delivering two products biomass (and PHA content in cells) and 3-HV content in PHBV.
A
B
P
A
In this case the whole process can be considered as a classical enzymatic two-substrate reaction. This situation was abundantly studied [30–32] and different mechanisms are generally classified into two categories: Ping-Pong and sequential mechanisms, the latter one being either ordered or random (Fig. 4). In the Ping-Pong mechanism one or more products must be released before all substrates can react. In sequential mechanisms, all substrates must combine with the enzyme before the reaction can take place. However, in ordered sequential mechanism, substrates react with the enzyme and the products are released in a specific order, whereas in random sequential mechanism the order of substrate combination and product release is not mandatory. In previously described situation the random approach (Fig. 4c) is clearly not the one observed. To discriminate between the two others, one have to determine whether we are facing the ternary EAB complex (ordered-sequential mechanism) (Fig. 4b) or primary release of the first product (P) with the subsequent modification of E to E0 before the consumption of the second substrate (Ping-Pong mechanism) (Fig. 4a). The difficulty here relies on the fact that biomass and PHA production on the one hand and 3-HV content on the other, can hardly be considered as independent products P and Q, showing thus severe limitation of the high simplification
Q Ping-Pong bi-bi mechanism
E
(EA
A
B
E'P)
E'
(E'B
P
B
E
EQ)
Q
ordered sequential bi-bi mechanism E
EA
(EAB
C A
P
B
E
EQ
EPQ)
Q random-sequnetial bi-bi mechanism
E
EAB
B
A
E
EPQ
Q
P
FIGURE 4
Different models of two-substrate enzymatic kinetic mechanisms. E and E0 stand for enzymes, A and B for substrates, P and Q for products. 4
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the production of 3HB and 3HV monomers, however more specific work is needed to confirm this hypothesis.
A 1,2
Scale-up of the optimized conditions
0,8 0,6 0,4 0,2 0,0 0
20
40
60
80
100
time (h) CDM (g/L)
concentration (g/L)
B
3 2,5 2 1,5 1 0,5 0 0
20
40
60
80
100
time (h) levulinic acid
The conditions chosen for the scale-up experiment were the ones allowing the highest 3-HV content, that is, 1 g/L initial levulinic acid and 2.5 g/L initial sodium propionate (Fig. 5). As previously observed, the consumption of sodium propionate at 2.5 g/L initial concentration is rather slow during the first 48 hours and deeply accelerates after this period (Fig. 5b), being consistent with the start of the exponential cells’ growth, beginning after 50 hours of fermentation (Fig. 5a). Also the time-course of the consumption of the substrates seems to confirm the Ping-Pong kinetic hypothesis, as levulinic acid concentration remains almost unchanged until 72 hours of the fermentation process and starts to decline only at 74 hours, when 84% of sodium propionate were consumed (Fig. 5b). Finally, using these conditions 0.3 g/L of PHBV with 3-HV content of 80% were obtained. Moreover, the volumetric productivity reached 3.9 mg/L/hour, exceeding by 100% the previously reported results [23].
Conclusion sodium propionate
FIGURE 5
Time courses for CDM (a), and substrates concentration in the medium (b), during the scale-up experiment.
hypothesis. However, the kinetics of consumption of the 2 considered substrates (Fig. 2) suggest that sodium propionate is consumed before the beginning of the decline of levulinic acid concentration in the reaction medium, therefore implying that the ternary EAB complex is not a correct description of what is happening during this transformation. In this case the Ping-Pong mechanism hypothesis seems to be the one to be privileged. Those assumptions are also consistent with the previous work reported by Jaremko and Yu [33], they have shown that the metabolism of levulinic acid by C. necator is split into acetylCoA and propionyl-CoA pathways, which confirms the preference for the consumption of propionate. Another interesting point is the influence of levulinic acid on the monomeric composition of the resulting PHA. Indeed, it was previously reported [34–36] that this substrate also induces the 4HV co-monomer and the final polymer is a terpolyester P(3HB-co3HV-co-4HV), the latter remaining the minor one. In our experiences we did not observed any significant trace of 4HV, this can be due to the total absence of even-numbered carbon substrates in our work, thus mainstreaming the metabolism of levulinic acid to
A precise study of the influence of initial concentration of levulinic acid and sodium propionate on the production of PHBV by C. necator with high 3-HV mol fraction was achieved. Even if this dual feeding was originally designed for the 3-HV content enhancement, the two substrates were found to play different roles, levulinic acid being mainly dedicated to cells growth and PHA enhancement, whereas sodium propionate – to the actual 3-HV enhancement. Also, for the first time a Ping-Pong-like kinetics for the consumption of levulinic acid and sodium propionate were suggested, thus allowing more specific understanding of inherent processes regulating biomass, PHA and 3-HV contents production and improvement. It was thus found that the most efficient combination for the production of PHBV with high 3-HV content is 1 g/L initial levulinic acid and 2.5 g/L initial sodium propionate concentrations. This allows the volumetric productivity of 3.9 mg/L/hour which is 100% greater than the previous results [23]. This method remains economical, as it uses only 3.5 g/L of carbon substrates for the production of PHBV with high, 80%, 3-HV content comparing to previously reported studies, with 20 g/L of inducing substrates [25,37]. Finally, to go forward and circumvent the inhibition effect observed here, an application of continuous strategy may be worthwhile, indeed, this technique has been previously described as valuable for such type of improvements [15,38,39].
Acknowledgements The authors are thankful to the Walloon region and FEDER Q4 European funds for the financial support through the SINOPLISS 565515-346987 program.
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