15.3 Factors that influence the growth of microorganisms

Task 1: Help to plan an investigation to determine whether stomach acid (Ph 1.5-3.5) will kill the bacteria if ingested. [P4]

In order to identify in which environment a specific bacteria is able to survive we can subject different batches of said bacteria to various controlled environmental conditions. This would enable us to discover the conditions that are most or least favourable for growth.

Since we do not have a pH baseline to help guide us in our attempts, we can proceed by creating three different pH controlled environments that will establish a growth scale based on pH.

The most logical choices are the ones where all three environments differ enough for there to be a noticeable change in bacterial growth speed. Therefore we have chosen the pHs 4, 7 and 9 because we think that by being 2 integers apart we can establish a clear indication of growth in a specific direction.

Conduct the investigation and submit your data as well as a signed observation record. [P4]

Aim

  • Observe how varying pHs affect E. Coli.

Hypothesis

  • E.Coli has an optimum growth which can be identified by subjecting the bacteria to different environments.

Method

  • Disinfect working surface.
  • Using a sterilised loop*, gather a sample of E.Coli.
  • Insert the loop into a prepared broth of specific pH (4, 7 or 9) and temperature 30 ̊.
  • Leave the bacteria to spread throughout the broth (maintain temperature) for an hour.
  • Using a sterilised syringe insert a sample of the broth into an agar dish.
  • Spread the bacteria using a sterilised glass spreader* (lawn).
  • Incubate the agar dish at 30 ̊for 24 hours.

*Bunsen burner, along with alcohol, is used for sterilising.

Results

pH

 Observed growth

4

no growth

7

extensive growth

9

some growth

Conclusion

We can observe that there is no growth at pH 4, a clear indication that the E. Coli strain cannot survive in very acidic environments. At pH 7 however the bacteria proliferates strongly within a few hours to reach its lag phase. This is strongly indicative of the strains’ preference to neutral environments. At pH9 we can observe some growth but it is not as strong as pH 7 thus indicating that the strains’ optimum pH is not very alkaline. Further tests would need to be performed to establish an optimum as it could very well lie between 6 -7 to 7-8.

I would conclude that this strain of E. Coli is best suited to neutral or close to neutral environments and will therefore not survive within the stomach as it is an acidic environment.

Use the data provided to calculate the growth rate of E. Coli in broths of pH4, 7 and 9. Based on the growth rates; what is the optimum PH for the growth of E. Coli? [M3]

The growth rate of the bacteria started when we inoculated a sample into three broths of varying pH. Once we introduced these samples into the agar dishes their growth had already started.

Growth rate = Increase in population/ Time

At pH 4: 750 (cells per 1 cm3)/ 8 (hrs) = 93.75 cm3 per hrs.

At pH 4: 34/0 = 0 (no growth)

At pH 9: 765/12 = 63.75

Our experiment indicates that pH 7 was the most favourable environment for the growth of this particular strain of E. Coli. To further the accuracy of this statement we would need to perform more tests within the 6 -7 and 7- 8 pH levels.

Predict whether this strain of E. Coli will be able to survive in stomach acid and therefore poses a threat to humans. [D3]

It is clear that the strain of E.Coli is unable to survive below or at pH 4; therefore it would be correct to assume that it would not survive in the stomach as the acidity is below pH 4. This is in line with the established behaviour of bacteria, where very few can survive in such acidic environments.

Mutations are always a possibility and so although it is unlikely that the strain would survive, it is not at all impossible and precautions should be taken to avoid contact.

Task 2: Describe how industrial fermentation technology is used by biotechnology companies to grow E. Coli in order to produce insulin.

Insulin that can be used safely by humans is rather difficult to obtain as it only occurs in humans, other animals do secrete insulin but it is just as difficult to obtain and isn’t always accepted by the human body.

The solution to this problem came through the form of DNA recombination, a way to isolate the insulin producing gene and insert it into an organism that can be farmed en masse.

In this scenario bacteria are very well suited to this application as they can be easily produced in large quantities, just as long as certain details of production are followed.

In industrial fermentation organisms such as bacteria are produced by following multiple steps. The very same steps are followed to produce insulin on a large scale:

  • The bacteria samples that have been modified to produce insulin are isolated within a small area and subject to environmental conditions that will affect growth (i.e. nutrients, pH, temp, etc…). The growth speed is calculated and adjustments are made to the controlled conditions to increase growth to its optimum. This step will also tell them at what stage of the growth curve the insulin will be produced.

  • The bacteria are inserted into a much larger containment area this time, a small bioreactor. The optimum conditions recorded in the first step will be used to grow the bacteria, this time on a larger scale, to observe if they produce the same results as before. If this is the case then the production of the bacteria moves onto step 3, if not then the process goes to back to step 1.

  • This last stage involves the production of the bacteria using the recorded optimum environmental conditions on an industrial scale. This stage will also involve designing bioreactors capable of handling the production of such large quantities of bacteria.

If all the steps have been performed correctly insulin will be extracted from the bacteria and purified to treat diabetes.

Compare the aseptic techniques used during the investigation with the techniques used with bioreactors.

In our investigation we disinfected our work surfaces with chemicals in order to remove the chance of anything contaminating our microorganisms through contact. This is also performed in industry when cultivating microorganisms because the same risks exist.

Because of the large scale of experiments or cultivation in industry, the techniques used are much more in depth than in our investigation.

A salt based steriliser is used to clean the fermenters at extremely high temperatures (around 1000-1300 Celsius). This is radically different from how small scale fermenters are cleaned (usually only with high pressure steam) and highlights the difference between industrial and small scale aseptic techniques.

Fouling, which is the accumulation of unwanted products is a threat to safety and the integrity of the cultivation process and so it must be regularly removed. Entire rooms are sometimes disinfected completely and kept under quarantine, while the equipment itself is regularly cleaned using extreme temperatures and steam to eliminate potential contamination threats. (Fouling)

The appropriate handling of vials in our experiments is similar to what is performed in industry, especially in the early stages of bacterial cultivation. However once the initial stages of establishing optimum growth conditions are completed then the similarities end. The bioreactors themselves are cleaned as explained above using a combination of steam and extremely hot temperatures. Steam and heat is used because they are chemically inert in that they will not react with the materials used to build bioreactors and will kill any microorganisms.

All the equipment used to transfer microorganisms or chemicals from or to a bioreactor are completely are disinfected regularly and thus kept sterile to avoid contamination. This is similar to our use of apparatus that has been either cleaned thoroughly or comes pre-packaged and sterilised e.g. syringes.

Predict whether this strain of E. Coli will be able to survive in stomach acid and therefore poses a threat to humans. [D3]

It is clear that the strain of E.Coli is unable to survive below or at pH 4; therefore it would be correct to assume that it would not survive in the stomach as the acidity is below pH 4. This is in line with the established behaviour of bacteria, where very few can survive in such acidic environments.

Mutations are always a possibility and so although it is unlikely that the strain would survive, it is not at all impossible and precautions should be taken to avoid contact.

The bacteria exhibit some growth at pH 9 and this could potentially be a problem depending on the alkalinity of certain areas in the body. The blood and lymph fluid are slightly alkaline in a healthy human being and so it is possible for someone whose diet is composed of very little acidic nutrients to tend to be higher in pH then the norm. This could potentially result in favourable conditions for bacterial growth. However this is hypothetical and I am unsure if this entirely possible. Very few areas of the body have a pH higher than 7 and so this isn’t a natural occurrence, thus not a real threat to humans.

Bibliography

Bioreactor. (n.d.). Retrieved March 2012, from Wikipedia: http://en.wikipedia.org/wiki/Bioreactor

Fouling. (n.d.). Retrieved March 2012, from Wikipedia: http://en.wikipedia.org/wiki/Fouling

Class notes

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