Protocol for propionic acid production in a bioreactor
Culture –
Propionibacterium acidipropionici ATCC 4875 Make stock culture in test tube and please store stock cultures in a refrigerator. Transfer to a fresh slant is done around 2-3 days before preparation of inoculum.
Synthetic Medium –
Synthetic medium is to be used for the preparation of inoculum. 100 mL (20 g/L lactose) medium with the following composition is to be used: 1 g yeast extract, 0.5 g trypticase, 0.025 g K2PHO4, 0.005 g MnSO4, 2 g lactose. pH is to be adjusted to 6.5 with 3 N NaOH/HCl. For sterilization, lactose and rest of the medium components (basal medium) are to be autoclaved separately at 121º C and 15 psig for 20 min. Mix the two solutions aseptically under laminar flow hood.
Preparation of Inoculum –
20 mL of the synthetic medium is to be taken in a 100 mL serum bottle, Cap it by a rubber stopper and seal by aluminium cap by crimper. Before inoculation, N2 is to be sparged through the serum bottle for about 15 min. An exit valve with sterile filter has to be provided at the flask outlet to prevent build up of excessive pressure inside it. Inoculation is to be done using a two days old slant growth scrapped in 1 mL sterile distilled water. A uniform suspension is to be made in the water before aseptically transferring the inoculum to the serum bottle with the help of a sterile syringe. Growth is to be carried out at 30º C in a shaker (Adolf Kuhner AG, Basel, Switzerland) maintained at 150 rpm. Use sterile syringes to withdraw samples. Transfer the inoculum at around 40 h using 6% (v/v) inoculum.
Shake Flask Studies –
For shake flask cultivation studies, use 500-mL custom made flasks (carefully modified by glass blowing) so that it can be closed by rubber stopper and sealed by aluminium caps using crimper. Use synthetic medium as described above. Sparge nitrogen gas through the flask for 15 min to create anaerobic conditions. Make provision for an exit valve through sterile filter to prevent build up of excessive pressure during sparging of nitrogen. Inoculate the flask with 6% inoculum. Initiate the growth by agitating at 30º C and 150 rpm. Withdraw samples at regular intervals using sterile syringes. Maintain pH between 6 and 7 during growth by intermittant addition of sterile 3 N NaOH.
Bioreactor cultivation –
Initiate fermentation in a bioreactor at an initial concentration of lactose (using) 50 g/L (7-l Bioengineering (Bioengineering, Switzerland) glass reactor) working volume 5 liter. Autoclave the fermentor with distilled water in it, in situ. After autoclaving, when the temperature drops down to 60º C, the water is to be removed through the sampling port by exerting N2 pressure. Continue flushing the headspace of the reactor with nitrogen gas to maintain anaerobiosis throughout the fermentation. The agitator is to be kept at 150 rpm. Control the temperature at 30±1 ºC and maintain the pH at 6.5±0.1 by automatic addition of sterile 6 N NaOH. A pH value of 6.5 has been reported to be suitable for propionic acid accumulation (Hsu and Yang 1991). Grow the inoculum in a custom-made 500-ml serum flasks (headspace flushed with nitrogen gas). Aseptically inoculate the bioreactor containing 5 l of medium with 250 ml of 1-day-old log-phase culture. Collect 10 mL liquid samples at regular intervals Measure the optical density and centrifuge the remaining volume of the sample at 10,000 g for 20 min. Store the supernatant at –20 ºC for HPLC analysis to establish the product concentrations.
Assay Methods
Biomass Estimation –
Measure the cell concentration in fermentation broth by measuring the optical density at 660 nm (OD660) of the suspension in a 1.5 mL polystyrene cuvette in a (Beckman Model 24) spectrophotometer. A standard curve is to be plotted between cell dry weight and absorbance which is to be used to determine cell weights in the unknown samples based on their absorbance values. Dilute the samples with distilled water if the OD exceeds 0.4 values. It had been established that one unit of optical density was equivalent of 0.42 g/L of cells.
Substrate/Product Estimations Using HPLC –
Use High performance liquid chromatography (HPLC) to assay the samples from fermentation experiments for lactose, propionic , acetic and pyruvic acid concentrations in the fermentation broth. The column used was organic acid analysis column (Model HPX-87H, Bio-Rad, USA). Maintain the temperature of column at 580 C. Use mobile phase as 0.01 N (0.005 M) H2SO4 at a flow rate of 0.6 mL/min. Refractive index (RI) detector (Differential Refractometer (model R401), Waters, USA) is to be used for analysis. Centrifuge all the fermentation samples at 12,000 rpm for 20 min and make it cell free. Filter each sample using a 0.45 µm disposable filter (Millipore) before injection.
Protocol for operation of model simulator
Bioreactor –
The Bioreactor portion illustrates the different components which are adequately described in the theory link. It also describes the purging of the sterile air (please see the air filter in the air inlet) from the sparger and rotation of the impeller. The impeller is bottom driven. This kind of arrangement is possible with BioEngineering AG Switzerland Bioreactor. This kind of agitation gives more space on the lid so that there is no overcrowding of different inlet ports & sensors and it is particularly useful when flame is used to connect acid /alkali pipes and when transfer of inoculum is done to the reactor. The constant temperature water flows in the jacket of the reactor (marked green) to maintain the temperature of the reactor.
Select parameters –
Beneath Select parameters label there are spaces for Simulation Start time, Time increment step and Simulation end time wherein the User have the option to enter different relevant numbers to start the simulations using different time intervals till the end of the simulation. The maximum limits are also indicated User has to ensure that the values do not exceed the maximum values.
Beneath above table there is another table which allows the Users to input the values of different model parameters (the minimum and maximum range is indicated in the brackets and the users has to ensure the input with in the range).
Underneath above table there is yet another table which allows the Users to input the initial conditions for the model simulation at time t=0.
Run Simulation –
After entering the above entries the User can enter the RUN Simulation button and the graph and table for X, S, P vs time is printer on the screen.
More graphs are available for Batch Microbial Cultivation wherein the user also sees the rate specific rates and yields of the biomass substrate and product vs time. This data along with the kinetic profile can be used to understand the culture behavior.
Out put Graph –
This section describes the kinetic profile of different fermentations in graphs.
Save Results –
By pressing this button the User goes to the Excel Sheet where all the values of kinetic profile is tabulated which can used for the detailed analysis of the system and /or making more desired trends in the Excel Sheet.