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2017-09-12 - Colloque/Présentation - poster - Anglais - 1 page(s)

Bayon-Vicente Guillaume , Zarbo Sarah, Gerbaux Pascal , Onderwater Robertus , Leroy Baptiste , Wattiez Ruddy , "Quantification of polyhydroxyalkanoates in the non-sulphur purple bacterium Rhodospirillum rubrum S1H" in Biopol 2017, MONS, Belgium, 2017

  • Codes CREF : Biochimie (DI3112), Microbiologie et protistologie [bacteriol.,virolog.,mycolog.] (DI3130)
  • Unités de recherche UMONS : Protéomie et Microbiologie (S828)
  • Instituts UMONS : Institut des Biosciences (Biosciences)
Texte intégral :

Abstract(s) :

(Anglais) Introduction Rhodospirillum rubrum S1H belongs to the group of Proteobacteria well-known for its huge metabolic diversity. Effectively, Rs. rubrum is able to grow chimio/photo-auto/heterotrophically depending on the culture condition (1-2). Previous studies conducted at the laboratory of Proteomic and Microbiology revealed the photoheterotrophic (e.g. anyxogenic phototrophy) production of polyhydroxyalkanoates (PHAs) in presence of volatile fatty acids (VFAs, e.g. acetate and butyrate)(3). Physiologically, PHAs represent a carbon and energy storage polymer produced when a nutrient is lacking in the medium (e.g., N, P, S) as it can be observed in other PHA producing bacteria. Considering their biodegradability and their physicochemical properties, these bioplastics could represent an interesting candidate to replace petroleum non-biodegradable plastics. Surprisingly, in our cultivation condition, no nutrient was limiting (4). Our hypothesis to explain this particular bioplastics production is that the use of reduced carbon source leads to a deregulation of the intracellular redox balance (e.g. in the NADH/NAD+, NADPH/NADP+ ratio). As the production of PHA implies the consumption of a molecule of NADPH (5), this metabolic pathway could be seen as an electron sink used by the cell to regulate the intracellular redox balance. Moreover, the particularity of Rs. rubrum lies in its ability to produce several homo or heteropolymers of PHAs with different physicochemical properties depending on the provided carbon source (6). Our laboratory has undertaken an extensive effort to study the link between environmental condition and carbon source with both the amount and the composition of the produced PHAs. Here we present first methodological results consisting firstly in the establishment of PHAs and cofactor quantification procedure in order to link these two parameters. Secondly, we present a first overview of the valerate assimilation by Rs. rubrum in order to use this source of carbon to produce P(HB-co-HV), a high added-value copolymer. Results and discussion 1. Quantification of PHAs PHAs monomers, obtained through methanolysis, have been studied via GC-MS. This methanolysis leads to the production of the related methyl-ester species. In the frame of this project only monomers corresponding to the P(HB-co-HV) have been studied, namely the 3-hydroxybutyrate and the 3-hydroxyvalerate. After some optimisation of the temperature gradient or the scan time, standard of the methyl-ester species related to those two monomers have been analysed through GC-MS in order to determine their retention time as well as the limit of detection and the dynamic range of the method for these compounds (fig. 1). It reveals that methyl-(R)-3-hydroxybutyrate (RT = 7.39 min) and methyl-(R)-3-hydroxyvalerate (RT = 10.32 min) are both detectable at 1 ng of standard injected on column and are reliably quantifiable between 2 and 10 ng of standard injected on column (fig. 1). The injection of compounds obtained by the methanolysis of only 0.5 mL of pelleted biomass and the comparison of MS spectrum with NIST library confirmed the presence of the compound of interest and efficiency of the method for quantification of PHAs in biomass samples. Figure 1 : detection of standard of methyl ester species of interest injected at 10 ng on column by GC-MS (A) and their dynamic range (B). Detection of (R)-methyl-3-hydroxybutyrate issued from the methanolysis of biomass resulting from 0.5 mL of bacteria cultivated in acetate 62.40 mM (OD680nm ≈ 1.0). 2. Valerate metabolism in Rs. rubrum In order to efficiently produce the P(HB-co-HV) copolymer by using valerate as main carbon source, the growth on this medium has to be characterised and metabolic pathway(s) necessary to assimilate the valerate have to be identified. First experiment showed the essential role of CO3-- on the growth of Rs. rubrum (fig. 2) as we have already observed in the case of butyrate (manuscript in preparation). It can be hypothesized that this results comes from the presence of CO3-- consuming steps in the assimilation pathway, such as the ethylmalonyl-CoA (EMC) pathway. In parallel, proteins have been extracted from biomass obtained in control condition (succinate as carbon source) or valerate condition in order to perform mass spectrometric analysis. This one revealed a higher relative abundance of some proteins involved in the EMC pathway such as the crotonyl-CoA reductase (p-value = 0.00012; ratio val/suc = 6.35), the methylmalonyl-CoA mutase (p-value = 0.05; ratio = 1.65) or the isovaleryl-CoA dehydrogenase (p-value = 0.011; ratio = 2.53). Interestingly, numerous proteins implicated in the PHA metabolism showed a significant change in their relative abundance. Two types of phasin, constitutive proteins of PHA granules, were found with a higher relative abundance (p-value = 0.00038; ratio = 48.24 / p-value = 0.02786; ratio = 2.05) whereas the polyhydroxyalkanoate depolymerase was found with a lower relative abundance (p-value = 0.00414; ratio = 0.58). It is also interesting to notice that a large number of proteins associated to stress or electron transport chain (17 proteins) present a higher relative abundance. This observation is consistent with the hypothesis presented earlier. Figure 2 : Culture of Rs. rubrum cultivated in 25 mM valerate and 50 mM of CO3--, black arrow represents the sampling time for MS/MS analysis (A). Influence of CO3-- on Rs. rubrum in presence of valerate, black arrows indicate the pulse of 10 mM CO3-- (B). Conclusion The quantification of PHA in complement with cofactor quantification will allow us to link the PHA production with the redox state of the carbon source and the intracellular redox state. The connection between them is of first interest in order to fully understand the PHA production leading to the production of cheap microbial biodegradable plastics using VFAs derived from, for example, wastewater treatments. First results concerning the assimilation of valerate and the observation of proteins implied in PHA metabolism are promising results for the production of the high added value compound P(HB-co-HV).