The report is about the bench-scale fermentation experiment which was conducted as a part of the cell factories module. The objective of the study was to set up a 5l capacity batch vessel and to grow Escherichia coli in submerged culture to monitor growth, dissolved Oxygen, pH and glucose utilization.
Escherichia coli has been used in lab scale studies for a long time as it is one of the most studied and well known microorganism. The growth characteristics for this bacterium are very simple, making it an important species in biotechnology and microbiology. The most common lab strains used are K-12 and B. Escherichia coli is a facultative anaerobe and also the doubling time for Escherichia coli is very less making it one of the favourite microorganism for bench scale study (2).
For a bench scale fermentation study, the most commonly used fermenter is the 5l fermenter controlled by a small programmable logic controller which helps in controlling the various parameters used in the fermentation. It has a temperature, pH and dissolved oxygen control. The mixing in the reactor is usually achieved by the use of impellers connected to a top drive motor. The agitation rate can be controlled by PLC controller. The inlet and exit gases are sterilized through air filters of appropriate size. All the main measurements like pH, temperature and dissolved oxygen can be controlled manually or with the PLC controller which in turn is connected to the computer.
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The microorganism used for the study was Escherichia coli and was propagated at 37°C.The medium used for the fermentation consisted of yeast extract(5g/l),peptone(40g), salts and glucose(10g/l).The fermentor used was Electrolab Ltd- F erMac 310/60 Fermenter. Initial set points were 37°C, pH- 7, Speed-600rpm and DO2 100%
DO2 electrode and pH electrode was calibrated
Medium and glucose was prepared
Medium was added to the vessel and was kept for sterilisation at 121°C at 15 psi
Glucose flask was connected and fermenter was run in glucose.
Sample of 20 ml was taken
Innoculum was added aseptically and air was turned on
Samples were taken hourly
Growth was measured by monitoring the OD at 650 nm
Glucose depletion was measured using glucose meter
pH and DO2 was monitored continuously from computer
RQ and other parameters were monitored continuously from Mass spectrophotometer
At the end of fermentation 10 ml sample was taken and dry weight was measured.
RESULTS AND DISCUSSION
TABLE 1: OD and Cell count
Dilution to sample
OD at 650nm
Dilution factor for plating
ln cell count
Fig 1: OD vs. Time
Fig 2: Cell count vs. Time
Fig 3: ln Cell count vs. Time
Fig 4: ln OD vs. Time
Table 2: Glucose depletion with time.
Fig 5: Glucose Depletion vs. Time
Fig 6: pH vs. Time
Fig 7:DO2 VS Time
Specific Growth Rate
From the plot of Figure 4 we can get the slope which will be equal to specific growth rate
Slope of the graph is 0.776582 h-1
It can also be found from the Figure 3
Slope of the graph is 1.08 h-1
It's derived from the formulae, x = x0eµt
The value shows that the growth is happening at good rate inside the fermenter.
Mean Doubling time
td = ln 2/µ
so td = ln 2/0.776582
= 53.55 minutes
Always on Time
Marked to Standard
td = ln 2/1.08
= 38.5 minutes
The doubling time for E.coli under optimum conditions is 20 minutes and this result gives us a finding that the difference may be due to the nutrient used and also due to the fermenter characteristics.
Final cell yield
Amount of sample taken = 10 ml
Dry cell weight= 5.0463 - 5.0077 =0.0386g
Initial concentration of Glucose= 10g/l
Final concentration of Glucose= 0.3267g/l
Amount of glucose used up= 10- 0.3267= 9.6733 g/l
Dry cell weight in 1L= 3.86g
Final Cell yield = 3.86/9.6733= 0.399 g dry cell weight per g of glucose consumed
The Experiment was done over a span of seven hours in a bioreactor with samples taken out hourly and checked for growth by measuring the optical density at 650 nm and the number of cells by first serially diluting the sample and then plating them to find the number of viable cells. Figure 1 clearly shows the growth kinetics of E.coli with four distinctive phases. The lag phase, where there is no increase in cells followed by an exponential phase where cell numbers increase exponentially with time and then a stationary phase at which the population reached its maximum size. Finally there is the death phase where a decline in number of cells is seen. In this experiment it was seen that the lag phase and stationary phase was not very distinctive and this may be attributed to the samples taken and the error in serial dilution done (1).
On Figure 6, it was seen that the pH decreased from a value of 7 at start to 6.4 in between and then finally stabilized at around 6.7.This can be attributed to the formation of Acetate in case of E.coli. This is due to the mechanism of overflow metabolism where the initial concentration of glucose is high and as a result the organism uses is it for the aerobic acetate formation and the system changes from pure respiratory to mixed respiratory and fermentative resulting in reduction in pH and at the later stage there is the ammonia production and hence the pH increased (2).
On evaluation of Dissolved oxygen vs. Time it was seen that the value decreased during the initial stages and kept on fluctuating and at the end of the fermentation got steady. It can be attributed to the growth of the microorganism in the frementer. The microorganism uses the oxygen in the medium effectively at first and after sometime and as the DO2 became limiting the formation of secondary metabolites started and also the over flow metabolism caused the fluctuating use of the dissolved oxygen. The change can also be attributed to the fact that a batch reactor is used.
On evaluation of glucose depletion vs. Time, it was seen that there was constant change in glucose level in the medium showing that the organism used the glucose effectively and one value was contradictory which might have been due to sampling mistake.
The major problem of overflow metabolism which causes the cells to produce byproducts and use up energy inefficiently could be avoided by different mechanism of glucose induction (2). The feed induction should be done through exponential feed, which would decrease the high influx of glucose at the start and thereby preventing the overflow metabolism and increasing the cell yields.
The operator errors that happened in the experiment had a major effect on the results that were drawn and if this could be avoided the overall effectiveness can be increased. Use of flow cytometry can greatly increase the quality of the results as it can get the viable cells count and also accurate results better than the measurement of optical density and cell count by plating.
Good and Bad points of the Fermenter used
Sterilization is easy
Aseptic conditions can be maintained
Shear problem is negligible
Low risk of contamination
Easy to control
High agitator loads cause stability problems
Constant monitoring needed
Incomplete control of metabolism
Probes have to calibrated frequently
The study was done on the growth of E.coli in a fermenter. The growth characteristics were studied effectively and the specific growth rate, doubling time and final cell yield was found out. The doubling time was found to be above the optimum doubling time for E.coli which might have happened due to the conditions that prevailed inside the fermenter and also the medium used. It was observed that acid formation occurred by monitoring the pH and it was attributed to the overflow metabolism that occurred due to the high influx of glucose at the start. The problems with using a batch reactor was analysed and also modifications were suggested.
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