Steps in Batch 1,3-PD Fermentation and Model Development
Microorganism for 1,3-PD production
Clostridium diolis DSM 15410 (previously known as Clostridium butyricum DSM 5431)is used for 1,3-PD production. The microorganism has to be maintained on Reinforced Clostridium Medium (RCM) and stored at 40C. Subculturing is required every 20 days.
Flasks Modification for maintaining anaerobic conditions
Ordinary Erlenmeyer flasks (500 ml) have to be modified for carrying out anaerobic cultivation outside the anaerobic tent. The mouth size (30 mm) is reduced to 10 mm and flasks are provided with a rim to facilitate sealing with rubber and aluminium caps by means of a crimper. Sparge nitrogen gas in the flasks to purge out the dissolved oxygen of broth and maintain anaerobic conditions.
Inoculum Growth medium and culture conditions
Develop the inoculum in 120 ml serum bottles which contain 50 ml sterile medium having composition (per litre): pure glycerol 20 g; K2HPO4 3.4 g; KH2PO4 1.3 g; (NH4)2SO4 2 g; MgSO4.7H2O 0.2 g; CaCl2.2H2O 0.02 g; FeSO4.7H2O 5 mg; Yeast extract 2 g; Trace element solution (TES) 2 ml. The trace element solution (per liter) consisted of: 70 mg ZnCl2; 0.1 g MnCl2.4H2O; 60 mg H3BO3; 0.2 g CoCl2.2H2O; 20 mg CuCl2.2H2O; 25 mg NiCl2.6H2O; 0.9 ml HCl (37%). Sparge the bottles with nitrogen and incubate inoculated bottles at 330C and 150 rpm in an orbital incubator shaker.
Transfer aseptically exponentially growing culture from the serum bottle (24 h) to sterile nitrogen gassed medium in a specially modified 500 ml Erlenmeyer flask containing 100 ml medium. Incubate the inoculated flasks in an orbital shaker at 330C and agitate at 150 rpm for 12 h after which use the actively growing culture from the flask as inoculum for the bioreactor.
Batch 1,3-PD fermentation in bioreactor using statistically optimized medium
Batch cultivation of C. diolis is carried out in a 3.7 L bioreactor (Bioengineering AG, Switzerland) (working volume 1.5 L) using statistically optimized nutrient medium recipe. The medium consists (per litre): pure glycerol 54.45 g; K2HPO4 3.21 g; KH2PO4 2.75 g; (NH4)2SO4 2 g; MgSO4.7H2O 0.2 g; CaCl2.2H2O 0.02 g; FeSO4.7H2O 5 mg; Yeast extract 2 g; Trace element solution (TES) 2 ml. The trace element solution (per liter) consisted of: 70 mg ZnCl2; 0.1 g MnCl2.4H2O; 60 mg H3BO3; 0.2 g CoCl2.2H2O; 20 mg CuCl2.2H2O; 25 mg NiCl2.6H2O; 0.9 ml HCl (37%). Sterilize bioreactor and tubing by autoclaving at 1210C and 15 psi for 40 minutes. Use 10% (v/v) actively growing culture from the shake flask as inoculum for the bioreactor. Control medium pH in bioreactor at 7.0 by automatic addition of 2N NaOH/HCl through acid/base peristaltic pumps. Agitate the medium in the bioreactor using a flat blade turbine impeller rotating at 150 rpm and maintain temperature at 330C by circulating chilled water using FP 50 chiller (Julabo, Germany). Sparge nitrogen gas at 0.5 vvm in the bioreactor to provide anaerobic conditions during the fermentation. Withdraw samples at regular intervals of 3 h from the bioreactor and analyze for biomass, residual glycerol and 1,3-PD.
Development of batch mathematical model and optimization of model parameters
Using the batch kinetics and inhibition (both substrate and product) data propose the mathematical model for growth and 1,3-PD production by C. diolis. To identify the approximate values of the model parameters, use the original experimental kinetic data and plug these values of model parameters in the model equations to perform simulations. Minimize the error between the model simulations and experimental observations of different process variables by minimising the Sum of Squares of Weighed Residues (SSWR) by non-linear regression using the original algorithm of Rosenbrock assisted by the computer programs and methodology as described by Volesky and Votruba.
1,3-PD MODEL EQUATIONS:
The model consists of a system of differential material balance equations which describe the batch dynamics of growth & 1,3-PD production. These equations are summarized below:
µ = µmax (S/S + KS) [1- (S / Sm)a] [1- (P/Pm)b] (1)
qs = - {(1/Ymax) µ + m} (2)
qp = K1µ + K2 (3)
where μ- specific growth rate, qs - specific substrate consumption rate, qp- specific product formation rate, Sm- critical substrate concentration, m- maintenance energy constant, Pm- critical product concentration.
Eq. (1) describes the limitation of specific growth rate by substrate. The specific growth rate also features inhibitions by high substrate and product concentrations. These inhibitions (S/P) are described by Luong’s inhibition expression (terms 2 and 3) and contribute in the model equation for specific growth rate.
Eq. (2) describes the specific substrate consumption rate of C. diolis. It features contributions for growth and product associated substrate consumption and also its use for maintenance functions of the cell.
1,3-PD production is shown to occur during both growth and stationary phases of cultivation in Eq. (3) which therefore describes specific product formation rate containing both growth and non-growth associated product constants.
Initial values of process variables: Biomass: 0.24 g/L, glycerol: 52.68 g/L , 1,3-PD: 0.05 g/L
Values of model parameters of 1,3-PD fermentation: µmax - 0.65 h-1 , Ks - 12.8 g/L, 1/Ymax - 13.96 g/g, m- 0.23 g/g/h, K1- 7.3 g/g, K2- 0.15 g/g/h, Sm - 98.3 g/L , Pm - 65.2 g/L , a- 1.12, b- 1.0.