The study is about the bench-scale agitation experiment which was conducted as a portion of the cell mills faculty. The aim of the survey was to put up a 5l capacity batch vas and to turn Escherichia coli in submersed civilization to supervise growing, dissolved Oxygen, pH and glucose use.
Escherichia coli has been used in lab graduated table surveies for a long clip as it is one of the most studied and good known micro-organism. The growing features for this bacteria are really simple, doing it an of import species in biotechnology and microbiology. The most common lab strains used are K-12 and B. Escherichia coli is a facultative anaerobe and besides the doubling clip for Escherichia coli is really less doing it one of the favorite micro-organism for bench graduated table survey ( 2 ) .
For a bench graduated table agitation survey, the most normally used fermenter is the 5l fermenter controlled by a little programmable logic accountant which helps in commanding the assorted parametric quantities used in the agitation. It has a temperature, pH and dissolved O control. The commixture in the reactor is normally achieved by the usage of impellers connected to a top thrust motor. The agitation rate can be controlled by PLC accountant. The recess and issue gases are sterilized through air filters of appropriate size. All the chief measurings like pH, temperature and dissolved O can be controlled manually or with the PLC accountant which in bend is connected to the computing machine.
The micro-organism used for the survey was Escherichia coli and was propagated at 37 & A ; deg ; C.The medium used for the agitation consisted of barm infusion ( 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 & A ; deg ; 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 vas and was kept for sterilization at 121 & A ; deg ; C at 15 pounds per square inch
Glucose flask was connected and fermenter was run in glucose.
Sample of 20 milliliter was taken
Innoculum was added aseptically and air was turned on
Samples were taken hourly
Growth was measured by supervising the OD at 650 nanometers
Glucose depletion was measured utilizing glucose metre
pH and DO2 was monitored continuously from computing machine
RQ and other parametric quantities were monitored continuously from Mass spectrophotometer
At the terminal of agitation 10 milliliter sample was taken and dry weight was measured.
RESULTS AND DISCUSSION
Table 1: Doctor of optometry and Cell count
( H )
Dilution to try
OD at 650nm
Dilution factor for plating
ln cell count
Fig 1: OD vs. Time
Fig 2: Cell number vs. Time
Fig 3: ln Cell count vs. Time
Fig 4: ln OD vs. Time
Table 2: Glucose depletion with clip.
Time ( H )
Glucose depletion ( g/l )
Fig 5: Glucose Depletion vs. Time
Fig 6: pH vs. Time
Fig 7: DO2 VS Time
Specific Growth Rate
From the secret plan of Figure 4 we can acquire the incline which will be equal to specific growing rate
Slope of the graph is 0.776582 h-1
It can besides be found from the Figure 3
Slope of the graph is 1.08 h-1
It ‘s derived from the expression, x = x0eµt
The value shows that the growing is go oning at good rate inside the fermenter.
Mean Doubling clip
td = ln 2/µ
so td = ln 2/0.776582
= 53.55 proceedingss
td = ln 2/1.08
= 38.5 proceedingss
The doubling clip for E.coli under optimal conditions is 20 proceedingss and this consequence gives us a determination that the difference may be due to the food used and besides due to the fermenter features.
Final cell output
Sum of sample taken = 10 milliliter
Dry cell weight= 5.0463 – 5.0077 =0.0386g
Initial concentration of Glucose= 10g/l
Concluding concentration of Glucose= 0.3267g/l
Sum 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 prohibitionist 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 growing by mensurating the optical denseness at 650 nanometers and the figure of cells by first serially thining the sample and so plating them to happen the figure of feasible cells. Figure 1 clearly shows the growing dynamicss of E.coli with four typical stages. The slowdown stage, where there is no addition in cells followed by an exponential stage where cell Numberss increase exponentially with clip and so a stationary stage at which the population reached its maximal size. Finally there is the decease stage where a diminution in figure of cells is seen. In this experiment it was seen that the slowdown stage and stationary stage was non really typical and this may be attributed to the samples taken and the mistake in consecutive 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 so eventually stabilized at around 6.7.This can be attributed to the formation of Acetate in instance of E.coli. This is due to the mechanism of overflow metamorphosis where the initial concentration of glucose is high and as a consequence the being uses is it for the aerophilic ethanoate formation and the system alterations from pure respiratory to assorted respiratory and fermentative resulting in decrease in pH and at the ulterior phase there is the ammonia production and therefore the pH increased ( 2 ) .
On rating of Dissolved O vs. Time it was seen that the value decreased during the initial phases and maintain on fluctuating and at the terminal of the agitation got steady. It can be attributed to the growing of the micro-organism in the frementer. The micro-organism uses the O in the medium efficaciously at first and after sometime and as the DO2 became restricting the formation of secondary metabolites started and besides the over flow metamorphosis caused the fluctuating usage of the dissolved O. The alteration can besides be attributed to the fact that a batch reactor is used.
On rating of glucose depletion vs. Time, it was seen that there was changeless alteration in glucose degree in the medium demoing that the being used the glucose efficaciously and one value was contradictory which might hold been due to trying error.
The major job of overflow metamorphosis which causes the cells to bring forth by-products and utilize up energy inefficiently could be avoided by different mechanism of glucose initiation ( 2 ) . The provender initiation should be done through exponential provender, which would diminish the high inflow of glucose at the start and thereby forestalling the overflow metamorphosis and increasing the cell outputs.
The operator mistakes that happened in the experiment had a major consequence on the consequences that were drawn and if this could be avoided the overall effectivity can be increased. Use of flow cytometry can greatly increase the quality of the consequences as it can acquire the feasible cells count and besides accurate consequences better than the measuring of optical denseness and cell count by plating.
Good and Bad points of the Fermenter used
Sterilization is easy
Aseptic conditions can be maintained
Shear job is negligible
Low hazard of taint
Easy to command
High fomenter tonss cause stableness jobs
Changeless monitoring needed
Incomplete control of metamorphosis
Probes have to calibrated often
The survey was done on the growing of E.coli in a fermenter. The growing features were studied efficaciously and the specific growing rate, duplicating clip and concluding cell output was found out. The doubling clip was found to be above the optimal doubling clip for E.coli which might hold happened due to the conditions that prevailed inside the fermenter and besides the medium used. It was observed that acerb formation occurred by supervising the pH and it was attributed to the overflow metamorphosis that occurred due to the high inflow of glucose at the start. The jobs with utilizing a batch reactor was analysed and besides alterations were suggested.