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Model-based intensification of a fed-batch microbial process for the maximization of polyhydroxybutyrate (PHB) production rate

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Abstract

An integrated metabolic–polymerization–macroscopic model, describing the microbial production of polyhydroxybutyrate (PHB) in Azohydromonas lata bacteria, was developed and validated using a comprehensive series of experimental measurements. The model accounted for biomass growth, biopolymer accumulation, carbon and nitrogen sources utilization, oxygen mass transfer and uptake rates and average molecular weights of the accumulated PHB, produced under batch and fed-batch cultivation conditions. Model predictions were in excellent agreement with experimental measurements. The validated model was subsequently utilized to calculate optimal operating conditions and feeding policies for maximizing PHB productivity for desired PHB molecular properties. More specifically, two optimal fed-batch strategies were calculated and experimentally tested: (1) a nitrogen-limited fed-batch policy and (2) a nitrogen sufficient one. The calculated optimal operating policies resulted in a maximum PHB content (94% g/g) in the cultivated bacteria and a biopolymer productivity of 4.2 g/(l h), respectively. Moreover, it was demonstrated that different PHB grades with weight average molecular weights of up to 1513 kg/mol could be produced via the optimal selection of bioprocess operating conditions.

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Abbreviations

AcAcCoA:

Acetoacetyl coenzyme A

AcCoA:

Acetyl coenzyme A

AL:

Azohydromonas lata cultivation medium

AL*:

A. lata feeding medium for the discrete single-step policy

AL**:

A. lata feeding medium for the continuous policy

ATP:

Adenosine triphosphate

CS:

Continuous feeding policy

CoA-SH:

Coenzyme A

C/N:

Carbon to nitrogen mass ratio

DNS:

Dinitrosalicylic acid

DSM:

German collection of microorganisms and cell cultures

FPT:

Fixed pivot technique

FTIR:

Fourier transform infrared spectroscopy

GC:

Gas chromatography

GPC:

Gel permeation chromatography

MWD:

Molecular weight distribution

NADH:

Nicotinamide adenine dinucleotide

phaA :

β-Ketothiolase

phaB :

NADH-depended acetoacetyl reductase

phaC :

PHA synthase

phaZ :

PHA depolymerase

PHAs:

Polyhydroxyalkanoates

PHB:

Polyhydroxybutyrate

PID:

Proportional–integral–derivative controller

RI:

Refractive index

SS:

Single-step feeding policy

UV/Vis:

Ultraviolet–visible spectroscopy

3-HB:

3-Hydroxybutyric acid, 3-hydroxybutyrate

3-HBCoA:

3-Hydroxybutyric coenzyme A

δ(t − t i ):

Kronecker delta function

DCW:

Dry cell weight (g/l)

[D n ]:

“Dead” polymer chains concentration (mol/l)

D.O.:

Dissolved oxygen concentration (% g/g)

[E-OH]:

Depolymerase concentration (mol/l)

[E-SH]:

Synthase dimer concentration (mol/l)

[H2O]:

Water concentration (g/l)

\(J_{{{\text{M}}^{\#} }} (t )\) :

Monomer production rate (mol/h)

k d :

Polymer degradation kinetic constant (1/h)

k H :

Henry’s law constant for oxygen (mol/(l atm))

k i :

Monomer initiation kinetic constant (1/h)

k L α :

Volumetric oxygen transfer coefficient (l/h)

k m :

Specific growth saturation constant (g/g)

k m1 :

Intermediate monomer initiation constant (1/h)

k m2 :

Intermediate polymer propagation constant (l/(mol h))

k p :

Polymer propagation kinetic constant (1/h)

k t , k ′ t :

Chain transfer to water kinetic constants (1/h)

k 1–5 :

Macroscopic model kinetic constants

[M]:

Monomer concentration (mol/l)

M n :

Number average molecular weight (g/mol)

[MS#]:

Monomer-synthase complex (E-SH-M#) concentration (mol/l)

M w :

Weight average molecular weight (g/mol)

MWM :

Monomer unit (3-HB) molecular weight

\({\text{MW}}_{{{\text{O}}_{ 2} }}\) :

Oxygen molecular weight (g/mol)

[M#]:

Monomer coenzyme A (M-SCoA) concentration (mol/l)

[N]:

Ammonium sulfate concentration (g/l)

N s :

Number of samples

O.D.:

Optical density @ 600 nm

[O2]:

Oxygen concentration (g/l)

PDI:

Polydispersity index

[P]:

PHB concentration (g/l)

[P n ]:

“Live” polymer chains (P n -ES) concentration (mol/l)

\([{\text{P}}_{n}^{*} ]\) :

“Intermediate” polymer chains (P n -ES-M#) concentration (mol/l)

Q :

Volumetric flow rate (l/h)

R :

Ideal gas constant ((l atm)/(mol K))

\(R_{{{\text{D}}_{n} }}\) :

Net formation rate of “dead” polymer chains (mol/(l h))

\(R_{{{\text{M}}^{\#} }}\) :

Net consumption rate of monomer (mol/(l h))

\(R_{{{\text{MS}}^{\#} }}\) :

Net formation rate of synthase-monomer complex (mol/(l h))

R n :

Ammonium sulfate consumption rate (g/(l h))

\(R_{{{\text{O}}_{ 2} , {\text{gl}}}}\) :

Oxygen mass transfer rate (g/(l h))

\(R_{{{\text{O}}_{ 2} }}\) :

Oxygen mass cells uptake rate (g/(l h))

R p :

PHB production rate (g/(l h))

\(R_{{{\text{P}}_{n} }}\) :

Net formation rate of “live” polymer chains (mol/(l h))

\(R_{{{\text{P}}_{n}^{ *} }}\) :

Net formation rate of “intermediate” polymer chains (mol/(l h))

R s :

Sucrose consumption rate (g/(l h))

R X :

Residual biomass growth rate (g/(l h))

[S]:

Sucrose concentration (g/l)

T :

Absolute temperature (K)

V :

Volume (l)

w P :

Intracellular PHB content (g/g)

[Χ]:

Residual biomass concentration (g/l)

Y NX :

Ammonium sulfate consumption coefficient for residual biomass synthesis/maintenance (g/g)

\(\dot{Y}_{\text{NX}}\) :

Ammonium sulfate consumption rate yield coefficient for residual biomass synthesis/maintenance (g/(g h))

Y P/S :

Accumulated polymer per consumed sucrose yield (g/g)

Y SP :

Sucrose consumption coefficient for PHB accumulation (g/g)

Y SX :

Sucrose consumption coefficient for residual biomass synthesis/maintenance (g/g)

Y X/S :

Total produced biomass per consumed sucrose yield (g/g)

\(\dot{Y}_{\text{SX}}\) :

Sucrose consumption rate yield coefficient for residual biomass synthesis/maintenance (g/(g h))

z :

Oxygen compressibility factor

μ s :

Specific growth rate (l/h)

eq:

Equilibrium

g:

Gas phase

in:

Feeding stream

l:

Liquid phase

max:

Maximum value

o:

Initial value

out:

Sample or outflow stream

P:

PHB

T:

Total

X:

Residual biomass

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Authors and Affiliations

  1. Chemical Process and Energy Resources Institute (CPERI), Centre for Research and Technology Hellas (CERTH), 57001, Thermi, Thessaloniki, Greece

    Giannis Penloglou, Athina Vasileiadou & Costas Kiparissides

  2. Department of Chemical Engineering, Aristotle University of Thessaloniki (AUTH), 54124, Thessaloniki, Greece

    Athina Vasileiadou, Christos Chatzidoukas & Costas Kiparissides

Authors
  1. Giannis Penloglou
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  2. Athina Vasileiadou
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  3. Christos Chatzidoukas
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  4. Costas Kiparissides
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Correspondence to Costas Kiparissides.

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Penloglou, G., Vasileiadou, A., Chatzidoukas, C. et al. Model-based intensification of a fed-batch microbial process for the maximization of polyhydroxybutyrate (PHB) production rate. Bioprocess Biosyst Eng 40, 1247–1260 (2017). https://doi.org/10.1007/s00449-017-1784-0

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