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S3: Catabolic network

r0

128.10-6 THF + 14.10-6Methyl-THF + 293.10-6Methenyl-THF + 200.10-6 Formyl-THF + 555.10-6 Sulfur + 0.010 Ammonia + 0.052 ATP + 4699.10-6NADH + 0.012 NADPH + 3239.10-6 Acetyl-CoA + 361.10-6 Erythrose-P + 93.10-6 Fructose-P + 423.10-6 Glucose-P + 1727.10-6 -Ketoglutarate + 2358.10-6 Oxaloacetate + 597.10-6 Pentoses-P + 769.10-6 PEP + 1676.10-6 Glycerate-P + 2488.10-6 Pyruvate + 440.10-6 Succinyl-CoA + 107.10-6 O2



1683.10-6 CO2 + 718.10-6 Acetate + 440.10-6 Succinate +19.10-6 Glycerol-P + 3679.10-6 CoA-SH + 634.10-6 Methylene-THF + 1 g of Biomass

r1

1 ATP + 1 CoA-SH + 1 Butyric acid



1 Butyryl-CoA

r2

1 Butyryl-CoA



1 FADH + 1 Crotonyl-CoA

r3

1 Crotonyl-CoA



1 S-3Hydroxybutyryl-CoA

r4

1 S-3Hydroxybutyryl-CoA



1 NADH + 1 AcetoacetylCoA

r5

1 AcetoacetylCoA + 1 NADPH



1 R-3 Hydroxybutyryl -CoA

r6

1 R-3 Hydroxybutyryl -CoA



1 CoA-SH + 1 HydroxyButyrate monomer

r7

1 CoA-SH + 1 AcetoacetylCoA



2 Acetyl CoA

r8

1 Acetyl CoA + 1 Oxaloacetate



1 CoA-SH + 1 Isocitrate

r9

1 Isocitrate



1 NADH+ 1 -ketoglutarate + 1 CO2

r10

1 Isocitrate



1 NADPH+ 1 -ketoglutarate + 1 CO2

r11

1 -ketoglutarate + 1 CoA-SH



1 NADH+ 1 CO2 + 1 Succinyl-CoA

r12

1 Succinyl-CoA



1 ATP + 1 Succinate + 1 CoA-SH

r13

1 Succinate



1 Fumarate + 1 FADH

r14

1 Fumarate



1 Malate

r15

1 Malate



1 NADH+ 1 Oxaloacetate

r16

1 ATP + 1 Oxaloacetate



1 PEP + 1 CO2

r17

1 Malate



1 NADPH+ 1 Pyruvate + 1 CO2

r18

1 Pyruvate + 1 CoA-SH



1 NADH+ 1 Acetyl CoA + 1 CO2

r19

1 PEP



1 ATP + 1 Pyruvate

r20

1 PEP



1 Glycerate-P

r21

1 ATP + 1 Glycerate-P



1 Glycerate-1,3 diP

r22

1 NADH+ 1 Glycerate-1,3 diP



1 Triose-P

r23

2 Triose-P



1 Fructose di-P

r24

1 Fructose di-P



1 Fructose-P

r25

1 Fructose-P



1 Glucose-P

r26

1 Glucose-P



1 NADPH+ 1 Gluconate 6-P

r27

1 Gluconate 6-P



1 2-Keto-3-deoxygluconate 6-P

r28

1 2-Keto-3-deoxygluconate 6-P



1 Pyruvate + 1 Triose-P

r29

1 Isocitrate



1 Succinate + 1 Glyoxylate

r30

1 Acetyl CoA + 1 Glyoxylate



1 CoA-SH + 1 Malate

r31

1 Ribose-P



1 Pentose-P

r32

1 Xylulose-P



1 Pentose-P

r33

1 Triose-P + 1 SedoHeptulose-P



1 Ribose-P + 1 Xylulose-P

r34

1 Fructose-P + 1 Triose-P



1 Erythrose-P + 1 Xylulose-P

r35

1 Fructose-P +1 Erythrose-P



1 Triose-P + 1 SedoHeptulose-P

r36

1 ATP + 1 Acetate + 1 CoA-SH



1 Acetyl CoA

r37

1 Glycerol-P



1 NADH+ 1 Triose-P

r38

1 THF + 1 NADPH+ 1 CO2



1 Formyl-THF

r39

1 Methylene-THF + 1 NADPH



1 Methyl-THF

r40

1 Methylene-THF



1 Methenyl-THF + 1 NADH

r41

1 Methylene-THF



1 THF + 1 CO2



r42

1 NADH+ 0.5 Oxygen



6 Proton

r43

0.5 Oxygen + 1 FADH



3 Proton

r44

3 Proton



1 ATP

r45

1 ATP






Respiratory Chain according to Ishaque and Aleem, 1970 and ATP spilling reaction

S4: Metabolites list

i

name

0

Xr (Biomass)

1

Butyric acid

2

Butyryl-CoA

3

Crotonyl-CoA

4

S-3Hydroxybutyryl-CoA

5

AcetoacetylCoA

6

R-3 Hydroxybutyryl -CoA

7

HydroxyButyrate monomer

8

Acetyl CoA

9

Oxaloacetate

10

Isocitrate

11

-ketoglutarate

12

Succinyl-CoA

13

Succinate

14

Fumarate

15

Malate

16

PEP

17

Pyruvate

18

Glycerate-P

19

Glycerate-1,3 diP

20

Triose-P

21

Fructose di-P

22

Fructose-P

i

name

23

Glucose-P

24

Gluconate 6-P

25

2-Keto-3-deoxygluconate 6-P

26

Glyoxylate

27

Ribose-P

28

Pentose-P

29

Xylulose-P

30

SedoHeptulose-P

31

Erythrose-P

32

Acetate

33

Glycerol-P

34

Formyl-THF

35

Methyl-THF

36

Methylene-THF

37

Methenyl-THF

38

Oxygen

39

CO2

40

CoA-SH

41

FADH

42

NADH

43

NADPH

44

Proton

45

ATP

S5: Feeding strategy and indicators used to readjust the flow to the biological requirement

In order to avoid inhibitory effect of volatile fatty acids, the cultivation was carried out in Fed-Batch condition at limiting carbon concentration (null residual concentration or lower than 0.25 g.L-1) monitored by an exponential or linear profile adjusted to the biological demand.



Figure 1: System considered for mass balance calculations



1. Calculation of exponential feed performed according to mass balance:

,

assuming a null residual concentration of substrate ( and ) :

with  :

during the exponential growth phase, , yielding ,

with a constant growth rate, and ,

assuming a constant biomass on substrate yield:

- during PHB production associated with growth (phosphorus feeding), exponential feeding was applicable considering an apparent growth rate and a global carbon yield :

- during PHB production without phosphorus feeding, we were not able to apply an exponential carbon feeding rate, the carbon flow was manually readjusted as successive constant or linear flows with the help of the indicators listed below.



2. Indicators used to readjust the feeding to biological requirement:

The following measurements and online calculations enabled culture monitoring and feeding readjustments:



  1. pO2: an increase in the O2 partial pressure value (pO2) without action on airflow or stirring speed indicated a limitation (carbon or of another element) or an inhibition of the strain resulting of acid accumulation in the broth

  2. N/C: nitrogen fed on carbon fed ratio was calculated with added mass (estimated instantly by weighing). This ratio indicated the partitioning of nitrogen (exclusively fed by pH regulation) between biomass production and neutralization of residual acids in the media:

    1. During biomass production stage, the N/C must be about 0.25 mole.Cmole-1 (according to biomass composition : C1H1.77O0.44N0.25, 4% ash, Aragao, 1996). The accumulation of butyric acid cannot be detected since the N/C corresponding to its neutralization was also 0.25 mole.Cmole-1. However with others acids (e. g., acetic acid or propionic acid), the N/C increased if nitrogen was used for neutralization (see Figure 2 below).

    2. During PHB or PHV production, N/C should asymptotically approach the limit value of 0.04 mole.Cmole-1 (corresponding to a PHB or PHV accumulation of 80% of the DCW) regardless of the substrate. An increase in this ratio revealed an accumulation of the acid in the fermentation broth.



Figure 2: Expected evolution of nitrogen fed on carbon fed ratio (N/C) during a Fed-Batch culture depending on nitrogen distribution

  1. RQ: the respiratory quotient calculated from dioxygen and carbon dioxyde measurement in outlet gazes indicated the partitioning of butyric acid between biomass (RQ between 0.61 and 0.63 according to the metabolic descriptor), PHB (RQ between 0.45 and 0.67) and CO2 if the substrate was totally decarboxylated because of the toxicity of the substrate (RQ of 0.8)

  2. Acid concentration: residual butyric acid concentration in the fermentation broth can be quantified in 10 minutes by CPG as described in the Material and Methods section. The substrate flux adapted to biological requirements can be subsequently recalculated, if necessary.

S6: Schematic flux distribution during growth at maximal growth rate (A), during PHB production without any growth (B), during PHB production associated with a low growth rate (C) - of 0.05h-1 (D)



References

1 - Aragao, G.M.F., 1996. Production de poly-beta-hydroxyalcanoates par Alcaligenes eutrophus : caractérisation cinétique et contribution à l'optimisation de la mise en oeuvre des cultures Génie Biochimique et Alimentaire. Institut National des Sciences Appliquées de Toulouse, Thèse n° d'ordre : 403, Toulouse.

2 - Ingraham, J.L., Maaloe, O., Neidhardt, F.C., 1983. Growth of the bacterial cell. Sinauer Associates, Inc, Sunderland, Massachusetts.

3 - Ishaque, M., Aleem, M.I.H., 1970. Energy coupling in Hydrogenomonas eutropha. Biochimica et Biophysica Acta (BBA) - Bioenergetics, 223, 388.





* Corresponding author: Estelle GROUSSEAU, Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés – INSA, UMR INSA/CNRS 5504 - UMR INSA/INRA 792, 135 Avenue de Rangueil, 31077 TOULOUSE CEDEX 4, France. Tel. +33 (0)5 61 55 94 70. Email: estelle.grousseau@insa-toulouse.fr



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