Monitoring:
1) After adding the culture, certain set-ups need to be monitored, as shown below :

Temperature – Constant ( blue line)
Stirred speed - Gradual increase (green line)
pO2 - Quite high, then decrease slowly (light blue line)
pH - constant 7.45 - 7.55 (red line)

(Since the machine was automated, nothing much needs to be done.)

2) As samples were taken at every hour, it is important to monitor any changes that occur in the fermentation.

Note:
There are a lot of spikes present in the graphs. This can be due to one of the 2 following causes, or a combination of both.

1) The probe was very sensitive and therefore could detect very minor changes in the parameters involved.

2) The bioprocess was conducted on a lab scale, so there was a low volume of culture. When the culture volume is low, small changes in the parameters can have a big effect on the graph.

pH
At start 7.45, constant for 6hrs aft inoculation, steep increase from 6th to 7th hr. ph increased from 7.45 to 7.60. PH remained constant throughout after 7th hour. The entire experiment lasted approximately 20hrs, inoculation to harvest. From the pH and time, we can tell that during the 1st 6 hrs, the bacterium was adjusting to its environment, there wasn’t any exponential growth observed within the 1st 6 hrs. The sudden increase in pH between the 6th and 7th hr, suggests that the growth phase occurred within that period. The pH then remained constant throughout, this is due to the fermentor system keeping the pH level constant and not that the bacteria had stopped multiplying

pO2
At start 77.0%. a steep drop was observed within the 1st 2hrs of inoculation. At 2hrs after inoculation the amount of saturated oxygen was 15.0%. The lowest saturated oxygen reading was 8.0%. Each time the oxygen level hit the range of 8.0 to 10.0%, it would then shoot up to a range of approximately 30.0 - 40.0%, this was observed from the 2nd hr of the experiment onwards till the end of the experiment. The rises and drops on pO2 percentage is caused by the fermentor system, as it tries to keep the consistency of the saturated oxygen level. Each time the level falls below a certain range, it compensates the drop by increasing the saturated oxygen level. This produces spikes in the graph. Spikes were observed along the gradient of the graph throughout the experiment. The saturated oxygen content is increased by increasing the stirrer speed (rpm).

Stirrer speed (rpm)
At start, 167rpm. Remained at this speed for 1st 2hrs, and then began to increase, its fastest speed observed was approximately 470rpm. The stirrer speed is tightly linked with the saturated oxygen content, each time the saturated oxygen content fell below a certain range. The stirrer speed would increase to have more oxygen saturated within the culture. The pattern of stirrer speed was opposite that of the oxygen saturation graph.

Record of Culture Absorbance and Fermenter Parameters

Discussion of graph of log _x/x0 against time

Log _x/x0 values were obtained by dividing the OD₆₀₀ (x) reading by the no. of hours into the fermentation. 10 such values were obtained by taking the OD₆₀₀ of samples taken hourly after the fermentation had begun, equivalent to growth rate against time.
OD₆₀₀ readings of the sample showed an increase over the hours, indicating an increase in cell density.

By plotting log _x/x0 against time, we can visualize the cell’s growth rate over that period of time. For the 1st hour, no cell growth was observed. During this period of time, the cells are probably adapting to their new environment, thus are not multiplying. From the 2nd to the 4th hour, exponential growth is observed. Fueled by media rich in nutrients, growth rate of the cells is increasing rapidly, the gradient of the graph is at its steepest. The cells have adapted to the environment conditions thus the optimal growth. From the 4th to the 6th hour, the curve’s gradient dropped and then increased again later, at the point where the graph hit before the slight drop is where optimal growth rate was observed, the slight drop in growth rate could be due to the fermentor reaching its maximum capacity of cells, thus the culture cannot keep increasing its growth rate but to maintain it, keeping their numbers. From the 6th hour on, the curve displays a gradual drop in its gradient, this shows a negative growth rate, which means, cells are dying faster then cells are cells are being produced, many factors could contribute to this, e.g. overcrowding and exhaustion of nutrients in media.

Further Questions:
1. Explain the control philosophy for pH, temperature and dissolved oxygen as was used in the fermentation process.

pH- as the bacteria culture grow, wastes are generated and the pH of the culture goes out of consistency. The pH may increase or decrease depending on the culture species. Acid and bases are added into the fermentor in certain amounts to keep the consistency of the pH. If the pH drops below a certain level (becomes too acidic), base is added in to return pH to its optimal level, if the pH rises above a certain limit (becomes too basic), acid is added in to get back optimal pH.

Temperature- there is an optimal temperature for the growth of the bacteria culture, fluctuations will not produce optimal growth. Therefore, the temperature has to be kept consistent (optimal temperature). Many processes emit heat, e.g. running motor of stirrer, and exothermic chemical reactions that occur within the fermentor etc. the temperature of the fermentor is kept constant with the help of a cooling jacket. The cooling jacket surrounds the fermentor. Water is pumped into the jacket from one end and out from another end, this way, water runs around the jacket surrouding the fermentor, carrying the heat emitted out of the system.

Dissolved oxygen- as the culture grows, oxygen content within the media is depleted. The culture will die off if the oxygen content is not replenished. Oxygen is dissolved into the media from the atmosphere, this is done with the help of a stirrer, as the stirrer spins the culture, oxygen from the atmosphere dissolves into the culture. The faster the stirrer speed the more oxygen is dissolved into the culture.

2. Describe the principle of the spectrophotometer which was used to determine the cell density (OD600). Why was 600 nm used?

A beam of light (of certain specificity e.g. wave-length) is emitted. The light passes through a cuvette where the sample is placed. Some of this light is absorbed by the sample and some pass through (transmitted light) hitting the detector on the other side. Transmitted light is then converted by a programme into absorbance values. The wave length of the light emitted by the spectrophotometer in this experiment is 600nm. 600nm is used as it is the most appropriate wave length to measure the bacteria. Bacteria fall under a certain range of sizes, this certain range of sizes can be picked up best by a wavelength of 600nm. If the wave length is too small, e.g. in the 200 range, the spectrophotometer may pick up other particles together with the bacteria, such as DNA and proteins, if the wavelength is too big, the spectrophotometer may not pick up the bacteria at all.

3. Is GFP a primary or secondary metabolite? At which phase should the product be harvested? At which phase was the product actually harvested?

GFP is a secondary metabolite. It is not directly involved in the cell’s growth, development and reproduction. The cell can still survive in the absence of GFP Trophophase is a period in cell culture where cell growth is still taking place and primary metabolites are being produced. Idiophase is the period in cell culture in which stationary growth is observed, this is the period where secondary metabolites are formed. GFP is produced in idiophase.

4. What are some advantages of using a computer control system? From the history chart (which will be given to you by your supervisor after the fermentation), comment on the effectiveness of the computer control.

Computer system can make visualizations, interpreting results so that it makes sense of the given mass of data, thus making it easier to understand (such as that of the history chart). A computer control system is also automated. Routines once vigourous to the lab technicians are made convenient by the programmes embedded within the computer control system e.g. temperature readings, maintenance of pH and saturated oxygen content. Also, errors are minimized as human error is eliminated almost completely (but they can still happen).