High-Cell-Density cultivation of pseudomonas putida IPT 046 and medium-chain-length polyhydroxyalkanoate production from sugarcane carbohydrates

We studied high-density cultures of Pseudomonas putida IPT 046 for the production of medium-chain-length polyhydroxyalkanoates (PHA_MCL) using an equimolar mixture of glucose and fructose as carbon sources. Kinetics studies of P. putida growth resulted in a maximum specific growth rate of 0.65h⁻¹. Limitation and inhibition owing to NH₄ ⁺ ions were observed, respectively, at 400 and 3500 mg of NH₄ ⁺/L. The minimum concentration of dissolved oxygen in the broth must be 15% of saturation. Fed-batch strategies for high-cell-density cultivation were proposed. Pulse feed followed by constant feed produced a cell concentration of 32 g/L in 18 h of fermentation and low PHA_MCL content. Constant feed produced a cell concentration of 35 g/L, obtained in 27 h of fermentation, with up to 15% PHA_MCL. Exponential feed produced a cell concentration of 30 g/L in 20 h of fermentation and low PHA_MCL content. Using the last strategy, 21% PHA_MCL was produced during a period of 34 h of fed-batch operation, with a final cell concentration of 40 g/L and NH₄ ⁺ limitation. Using phosphate limitation, 50 g/L cell concentration, 63% PHA_MCL and a productivity of 0.8 g/(L·h) were obtained in 42 h of fed-batch operation. The PHA_MCL yield factors from consumed carbohydrate for N-limited and P-limited experiments were, respectively, 0.15 and 0.19 g/g.

CER:

CO₂ evolution rate (mol/[L·h])

k _La :

volumetric coefficient for oxyen transfer (h⁻¹)

K _O2 :

constant for Monod equation

OUR:

oxygen uptake rate (mol/[L·h])

P _Xr :

active biomass productivity (g/[L·h])

qCO₂ :

specific CO₂ evolution rate (mol/[g·h])

qO₂ :

specific oxygen uptake rate (mol/[g·h])

r _SX :

substrate consumption rate for growth (g/L·h])

s :

substrate (g)

S _I :

inhitial concentration of substrate (g/L)

T :

time (h)

V :

volume (L)

V _O :

initial volume (L)

Vesp :

specific feed rate (g/[g·h])

xr :

active biomass (g)

xr _o :

initial amount of active biomass (g)

Xr :

concentration of active biomass (g/L)

X _O :

initial concentration of biomass (g/L)

Y _Xr/F :

conversion yield of fructose to biomass (g/g)

Y _Xr/G :

conversion yield of glucose to biomass (g/g)

Y _Xr/G+F :

conversion yield of carbohydrate to biomass (g/g)

Y _Xr/O :

conversion yield of oxygen to biomass (g/g)

Y _Xr/S :

conversion yield of substrate to biomass (g/g)

μ:

specific growth rate (h⁻¹)

μ _F :

specific consumption rate of fructose (h⁻¹)

μ _G :

specific consumption rate of glucose (h⁻¹)

μ _N :

specific consumption rate of ammonium ions (h⁻¹)

μ _Xr1 :

specific growth rate on glucose (h⁻¹)

μ _Xr2 :

specific growth rate on fructose (h⁻¹)

θ:

freed rate (L/h)

CRIT:

critical

MAX:

maximum

Authors and Affiliations

1. Biotechnology Group, Division of Chemistry, Institute for Technological Research of São Paulo State-IPT, Av. Professor Almeida Prado, 532, CEP 05508-901, São Paulo, S.P., Brazil

   Simone Cardoso Diniz, Marilda Keico Taciro, José Gregório Cabrera Gomez & José Geraldo da Cruz Pradella

Corresponding author

Correspondence to José Geraldo da Cruz Pradella.

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