Production of polyhydroxyalkanoates by purple phototrophic bacteria using

Production of polyhydroxyalkanoates by purple phototrophic bacteria using wastewater treatment products Guillaume Bayon-Vicente

Plastic production in the world Plastic. Europe - Plastics, the facts 2018

Polyhydroxyalkanoates, future worldwide plastics ? Pros Bio-based Biodegradable Agriculture Packaging and everyday’s life Broad range of application Dentistry Drug delivery

How are they produced now ?

Rhodospirillum rubrum, future PHA producer model ? Photosynthesis repressed by presence of oxygen High metabolic versatility (photoheterotrophy, photoautotrophy, chemoheterotrophy) Assimilation of a wide range of carbon sources Volatil fatty acids (VFAs) Acetic Propionic acid (C 2) (C 3) Butyric Valeric acid (C 4) (C 5)

Rhodospirillum rubrum, future PHA producer model ? Poly(hydroxybutyrate-co-hydroxyvalerate)

Our bioprocess advantages Final price Bacterial selection Bioprocess Substrate O 2 VFAs Use of LED Waste Light regime Cheap Knowledge based process optimisation Easily available

Knowledge based process optimisation Ø Accumulation inside granules Ø Granules formed by phasins Ø Stock of energy, electron and carbon Ø Another nutrient (N, S, P) is limiting

Working hypotheses Reduced carbon sources Photoreduction of NAD+ Redox state NAD(P)H NAD(P)+ H+ H+ Biomass Carbon source ATP synthase NAD+ ADPATP + Pi Electron sink Pha. A Pha. C Pha. B NADH dehydrogenase NADH

1. Use of reduced carbon source

The assimilation of valeric acid Compounds Formule Redox state Malate C 4 H 6 O 5 +2 Pyruvate C 4 H 6 O 4 +1 Acetic acid C 2 H 4 O 2 0 CH 1. 7 O 0. 4 N 0. 2 S 0. 003 P 0. 01 -0, 45 Propionic acid C 3 H 6 O 2 -1 Butyric acid C 4 H 8 O 2 -2 Valeric acid C 5 H 10 O 2 -3 Biomass Production of the high added value compound poly(hydroxybutyrate-co-hydroxyvalerate) (P(HB-co-HV)

The assimilation of valeric acid Excess of HCO 3 - is mandatory for Rs. rubrum growth is presence of valerate

The assimilation of valeric acid Redox homeostasis Pathway Peak Name Q 2 RS 31 Q 2 RU 73 Q 2 RS 30 Q 2 RS 29 Q 2 RS 28 Q 2 RTI 5 Q 2 RW 01 Q 2 RW 00 Q 2 RXS 5 Q 2 RWY 1 Q 2 RUH 6 Q 2 RT 29 Q 2 RWF 3 Q 2 RYH 0 Group Ferredoxin β-lactamase-like Electron transfer flavoprotein-ubiquinone oxidoreductase Electron transfer flavoprotein beta-subunit Superoxide dismutase Stress protein Electron-transferring-flavoprotein dehydrogenase Cytochrome bd ubiquinol oxidase. subunit I Alkyl hydroperoxide reductase/ Thiol specific antioxidant Ferredoxin Glutaredoxine Glutathion peroxidase Ref. Seq Locus Tag Rru_A 2264 Rru_A 1522 Rru_A 2265 Rru_A 2266 Rru_A 2267 Rru_A 1760 Rru_A 0893 Rru_A 0894 Rru_A 0265 Rru_A 0560 Rru_A 1418 Rru_A 1916 Rru_A 0020 Rru_A 0332 p-value 0. 00527 0. 03968 2. 9782 E-05 0. 00027 0. 02503 0. 00053 0. 00717 0. 03873 0. 01435 0. 00082 0. 0024 0. 00657 0. 02147 0. 05 Fold Change val/suc # identified peptides 4. 65 5 3. 81 4 3. 13 4 3. 02 5 2. 39 3 2. 09 5 1. 91 5 1. 87 3 1. 62 3 1. 60 5 1. 47 5 1. 45 5 1. 29 5 Pathway Peak Name Group Ref. Seq Locus Tag p-value Fold Change val/suc # identified peptides PHA production Q 2 RQI 1 Q 2 RP 67 Phasin Rru_A 2817 Rru_A 3283 0. 00038 0. 02786 48. 24 2. 06 5 5 Q 2 RNZ 5 Polyhydroxyalkanoate depolymerase Rru_A 3356 0. 00414 0. 58 3 Calvin cycle Peak Name Q 2 RRP 5 Group Ribulose bisphosphate carboxylase Ref. Seq Locus Tag cbb. M - Rru_A 2400 p-value 0, 0103 Fold Change val/suc 1, 708385498 # identified peptides 89

The assimilation of valeric acid Excess of HCO 3 - Progressive adding of HCO 33 m. M HCO 3 - Redox « feast and famine » process improve PHA production

Associating high production with high added value

2. Impact of light regime

Light intensity influence on acetate cultivated bacteria Light intensity increase from 50 to 150 µmol photons Proteomic analysis Dramatic impact on WT acetic acid cultivated bacteria growth

Light intensity influence on acetate cultivated bacteria Acetate Comp post Acetate Comp pre Acetate NC post Acetate NC pre Succinate post Succinate pre

Light intensity influence on acetate cultivated bacteria • Proteins linked to photosynthesis or pigment synthesis : 31 • Proteins linked to redox homeostasis : 7 • Polyhydroxyalkanoate synthesis repressor

Light intensity influence on acetate cultivated bacteria • • • NADH: flavodoxin oxidoreductase/NADH oxidase Flavodoxin Zinc containing alcohol dehydrogenase superfamily Thioredoxin Ferredoxin • Glycogen operon protein Glutathion peorixdase • Sugar fermentation stimulation NAD(P)H dehydrogenase

Intracellular storage analysis - Polyhydroxyalkanoates Light intensity increase from 50 to 150 µmol photons Light regime switch has an impact on PHA production

Intracellular storage analysis - Glycogen Succinate Acetate Comp Acetate Non. Comp Light intensity increase from 50 to 150 µmol photons

3. Conclusion and future prospects

Conclusion Intracellular redox environment Redox state NAD(P)H NAD(P)+ Biomass Environment Carbon source PHA production

Further Prospects Fermentation phenomenon PHA production Volatile fatty acids Pigment production

Further Prospects Mix of volatile fatty acids Acetic acid Propionate Butyrate Acetic acid is assimilated first

Acknowledgement The Prot. Mic team