This experiment has been conducted to accumulate data on the growth of Escherichia coli (E. coli) and to monitor how it grows under certain conditions. It has been demonstrated that the levels of glucose and dissolved oxygen were found to affect the rate of growth of E. coli proportionally with a lack of oxygen resulting in the lowering of the pH. In this experiment the growth of E. coli was studied at constant temperature (37 0C) at which it grows ideally. Experimental results for the growth of Escherichia coli showed good agreement with theory. Introduction
An Escherichia coli is a rod-shaped bacterium measuring about 2.5µm by 0.5µm.It is found in the guts of vertebrates. E. coli is a normal resident of the human colon and therefore grows ideally at 37 0C. In fact, the presence of most strains of E. coli keeps other more harmful bacteria away by starving them of food. They also help to make vitamin K which is an important vitamin for blood clotting. In some cases, E. coli infection can …show more content…
cause kidney failure and haemorrhaging (bleeding) in the gut. In the majority cases, infections of the pathogenic strain of E. coli are not fatal and the disease clears without treatment. (Kent, 2000)
Based on observations of the growth of E. coli at various glucose concentrations, one of the simplest models which include the effect of nutrient concentration was developed by Jacques Monod (Blanch and Clark, 1997). In the laboratory, E. coli can be grown in solid or liquid culture that contains nutrients (i.e. carbohydrates, proteins, nucleic acids, salts and vitamins). In this experiment, liquid culture was used to grow E. coli. The objectives of the experiment are to grow Escherichia coli in submerged culture and to monitor growth, dissolved oxygen, pH, and glucose utilization. As this experiment is a kind of bioprocess, so it should be carried out in a fermentation vessel. Safety rules
Safety goggles, which protect eyes from impact, spray, dust particles, must be worn all the times in the laboratory. Cotton knee length lab coats must be worn, which protect skin and clothing from dirt, inks, non-hazardous chemicals, biohazards without aerosol exposure. Every chemical container must be labelled to avoid mix-ups. Use of gloves is required for handling certain chemicals, so due to safety reasons gloves must be worn at all the times in the laboratory. Excess chemicals must never be returned to reagent bottles. Dispose of the excess chemicals in the proper waste container (Picot and Grenouillet 1995). Methods
3.1 Calibration of dissolved oxygen (DO2)
For the setting of the upper end of the scale 100% saturation is achieved with high agitation rate combined with adequate airflow in the absence of any appreciable oxygen demand. When 100% saturation was achieved DO2 meter was adjusted to read 100%. For the lower fixed (usually zero) point nitrogen gas passed through the sparger until a satisfactory stable zero was reached.
Air flow was changed back and 100% reading was achieved by using fermentation agitation rate 600r.p.m. and air flow rate 4L/min (lv/vm). This calibration was carried out at 37°C, because Escherichia coli (E. coli) is a normal resident of the human colon and therefore it grows ideally at 37 0C (Onyeaka, 2015).
3.2Calibration of pH electrode pH electrode was removed from the vessel carefully , clamped in the clamp stand, connected to meter and immersed in buffer pH7. The buffer control was adjusted to 7. After that electrode was washed in between water, blot dried and the process was repeated with buffer 4 and 10. As pH7 is neutral medium, zero was adjusted to 7 (Onyeaka, 2015).
3.3 Preparation of the media for 4L
Final concentration stock solution: KH2PO4 (13.6/L, 400ml), (NH4)2 SO4 (2g/L, 400ml), MgSO4 (0.2g/L, 40ml), yeast extract (5g/L, 20g), FeSO4 (0.0005 g/L, 4ml), peptone 40g. It was made up to 2 litres and pH was adjusted to 7 using KOH solution. Final volume was made up to 3200ml. 40g of glucose was separately weighed out and dissolved in hot tap water. The volume was made up to 400ml and it was poured into side arm flask provided, then it was plugged, foiled and sterilized. The final glucose concentration was 10g/L (Onyeaka, 2015).
3.4 Setting up of the fermenter
The vessel was set up in framework and air, pH, dissolved oxygen (DO2) electrodes, temperature sensor, assemble motor, fit heating jacket, cooling finger are connected. Air flow rate was set up and agitation and temperature controls were switched on. DO2 and pH were checked when temperature reached near set point. Glucose addition flask was connected by using ethanol, air was turned off and glucose was run in. 20ml sample was taken and inoculated with shaker flasks grown overnight-aseptically adding to side arm flask. Air was turned on. Initial set points for fermentation were as following: Temperature 37 0C pH 7 Speed 600r.p.m, DO2 100% Head pressure 2-3 p.s.i (Onyeaka, 2015).
Results
Time [hours] Optical Density [650 nm] Concentration of glucose [mM] Concentration of glucose [g/L] pH Dissolved Oxygen [%]
0 0.477 87 15.67 6.5 98.3
1 1.23 81 14.59 6.44 91.7
2 4.5 68 12.25 6.35 70.3
3 11 25.7 4.63 6.33 21.2
4 22.3 0 0 5.88 65.5
5 25.8 0 0 6.11 95.1
6 23.7 0 0 6.09 96.9
7 24.1 0 0 6.12 98
8 20.2 0 0 6.13 98.1
Dry Cell Weights [mg]
Time (hours) 0 8
Tube 1 1.97 11.17
Tube 2 2.37 10.87
Tube 3 2.47 11.47
Tube 4 2.17 10.17
Mean 2.24 10.92
Standard Deviation 0.19 0.48
Analysis of results and discussion
Analysis of graph 1 shows that the growth of E. coli can be plotted in several distinct phases (e.g., lag phase, exponential phase, etc.). The first phase, which is called lag phase, results from several factors. When cells are placed in fresh medium intracellular levels of cofactors (e.g., vitamins), amino acids and ions may be transported across the cell membrane and thus their concentration may decrease appreciably. Cell division occurs in the exponential phase, which lasts about 4 hours. In this phase, the rate of cell number is proportional to the number of cells. The exponential phase is followed by the stationary phase in which some cells may lyse; releasing nutrients that can be consumed by other cells and thus maintain the cell population. During the following phase, called death phase, it is thought that cell lysis occurs and the population decreases (Blanch and Clark, 1997). All these phases have been plotted in the graph 2 which shows the growth of E. coli in several distinct phases.
Conclusion
It was observed that the growth of E. coli displayed a normal growth curve for bacterium. The availability of glucose and dissolved oxygen had noticeable impact on the growth of E. coli. Lack of DO₂ caused acetic acid to be produced, lowering the pH of the mixture. The anomalous readings discovered suggest room for improvement in collecting data and the agitation process. The yield coefficient (Y_(C⁄S)=0.554) could be improved by increasing the supply of glucose and DO₂, and converting the process to a fed-batch one. The method of heating the bacterium leaves room for development. The majority of the equipment was simple and easy to use and process. References
Blanch. W. H. and Clark. S .D. (1997) Biochemical Engineering New York: Marcel Dekker, Inc.
Onyeaka, H. (2015) Growth of Escherichia coli in a 5 Litre Batch Fermentation Vessel [online] University of Birmingham – School of Chemical Engineering Available from https://canvas.bham.ac.uk/courses/10000/pages/fermentation [Accessed 12 April 2015]
Kent, M. (2000) Advanced Biology. Oxford: Oxford University Press
Picot, A. and Grenouillet, P. (1995) Safety in the Chemistry and Biochemistry Laboratory 2nd ed. Paris: Lavoisier TEC&DOC Appendix
8.1 Calculations
8.1.1 Specific growth rate
The values for E. coli cell concentration at certain times are needed to calculate specific growth rate. But they are not provided in this experiment. As cell concentration and optical density are directly proportional to each other, so the values for optical density can used to calculate using the following equation:
C=C_o e^μt or μ=ln(C/C_o )/t (Blanch and Clark, 1997) (1) (Where C [g/L] is the cell concentration of E. coli at time t, C0 [g/L] is the initial cell concentration of E. coli, t is time in hours and µ [h-1] is specific growth rate)
From Graph 1, it can be clearly seen that after t=5 hours, the growth of the E. coli starts to decrease because of exhaustion of “limiting substrate” and the stationary phase starts at t= 5 hours. So t=5 hours is to calculate specific growth rate. μ=ln(25.8/0.477)/5=0.798 [h^(-1) ] (3 s.f.)
8.1.2 Mean doubling time
Mean doubling time can be derived from equation (1). At t=td, C=2C0 then the equation 1 for t become as following t_d=ln(C/C_o )/μ=ln(〖2C〗_o/C_o )/μ=ln(2)/μ (2) (where td [h] is the doubling time and µ [h-1] is specific growth rate). So td can be calculated using the value for µ obtained in the equation (1). t_d=ln(2)/0.798=0.868 [h]
8.1.3 Growth rate
The Monod equation to calculate growth rate is as following: rG= µCc (3) (where rG [gL-1h-1] is the growth rate, µ [h-1] is specific growth rate and Cc [gL-1] accumulation of cell mass)
There is a relation between the accumulation of cell mass, the mean net dry cell growth of E. coli and the volume of the mixture, which can be used to calculate the accumulation of cell mass (Cc).
C_(C )=(Final mean dry cell weight-Initial mean dry cell weight)/(volume of mixture) (Blanch and Clark,1997) (4)
C_C =(10.92- 2.24)/4=2.17[ gL¯^1]
Now rG can be calculated by using the value for specific growth rate (µ) from equation (1) and that for accumulation of cell mass (Cc) from equation (4).
r_G=µCc= 0.798×2.17=1.73 [gL¯^1 h¯¹] (3s.f.)
8.1.4 Cell yield coefficient
The cell yield coefficient can be calculated with the help of the following equation.
Y_(C⁄S)=(Mass of new cells formed)/(Mass of substrate consumed) =-( ΔCc )/ΔCs(Blanch and Clark,1997) (5)
Mass of substrate (glucose) consumed = (Initial concentration –Final concentration) × volume
Mass of substrate (glucose) consumed = (15.67-0) ×4=62.68 [g]
Y_(C⁄S)=(8.68×4 )/62.68=0.554 (3s.f.) or 55.4%
8.2 Possible improvements of fermentation cell yield
In this experiment, the fermentation process was carried out in a batch reactor, in which the fermentation cell yield coefficient 〖(Y〗_(C⁄S)) obtained was around 55%.
In the batch reactor the concentration of glucose decreased from certain amount to o g/L within first 4 hours of the process. However, the fermentation cell yield coefficient could be improved by using fed batch reactor, in which the substrate (i.e., glucose) is added into the mixture at desired times. The level of dissolved oxygen (DO2) was also one of the major factors that affect the fermentation cell yield coefficient. The decrease in the amount of DO2 supplied prevents E. coli from continuing to increase exponentially. If DO2 was provided continuously it would prevent the cells from respiring anaerobically and producing acetic acid. The growth of the cells was inhibited by the acetic acid which lowers the pH, so the fermentation cell yield could be raised by preventing this from
occurring.
8.3 Good and bad design points of the equipment
One of the good design points of the equipment used in this experiment is that it had an electronic display which presented values for variables (e.g., temperature, pH) at any given time. On the other hand, there is also a little need to improve this equipment. The temperature was not consistent throughout the culture, because heating jacket was only wrapped around the fermenting vessel. Besides this, as discussed before, this equipment could be more efficient in terms of fermentation cell yield by converting it from batch system to fed-batch one.