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Stress tolerance

Introduction

Microorganisms are ubiquitous organisms that are found in all environments around the world, from extreme environments such as hot springs, to more mundane environments such as our hands. Extremophiles are microorganisms that grow optimally in these extreme environments and are the minority. The reason microorganisms are widely researched around the world is because they contain enzymes that are functional under extreme conditions called extremozymes. These are useful for industrial use and research as they can remain alive and functional in the severe conditions used in many techniques (Gupta, Srivastava et al. 2014). The study of these organisms also provides insight into the possible origins of life on Earth billions of years ago when the environment was extremely anoxic. This also relates astro microbiology and research in to potential microbial life on other planets in the solar system which may potentially lead to the presence of other life forms (Davila 2010). Looking at microorganisms that can withstand various environmental stresses could lead to the discovery of a wide range of extremophiles.

Aim & Hypothesis

Aim: To isolate microbes from environmental samples and investigate their ecophysiology via studies of stress phenotypes.

Hypothesis: Microbes isolated from diverse habitats have specific adaptations to the environmental conditions in which they were found growing.

Methodology

Sample

The sample chosen for this experiment was unrefrigerated stale salami. This was chosen because the extremely dry, salty environment may display a good population of extremophiles. The salami was also chosen because it was readily available and easily stored and transported.

Figure 1. A drawing of the stale salami sample used to inoculate the agar plates at x1000 magnification

Media

Six different types of media were used (either MYA (20g malt extract, 20g yeast extract and 15g agar) or Paynes’s Medium (20 g MgSO4.7H2O, 10 g yeast extract, 7.5 g casamino acids, 3 g trisodium citrate.H2O, 2 g KCl and 15 g agar; pH adjusted to 7.6):

1. Control medium (MYA only, with no stressor added) – 6 plates (aw 0.998)

2. High-ethanol media:

MYA containing 2% w/v ethanol- 6 plates (aw 0.990)

MYA containing 4% w/v ethanol- 6 plates (aw 0.982)

3. High-fructose media:

MYA containing 55% w/v fructose- 6 plates (aw 0.890)

MYA containing 70% w/v fructose- 6 plates (aw 0.751)

4. High-MgCl2 media:

Payne’s containing 20 g NaCl l-1 plus 0.8 M MgCl2- 6 plates (aw 0.930)

Payne’s containing 20 g NaCl l-1 plus 1 M MgCl2- 6 plates (aw 0.916)

5. High-NaCl media:

Payne’s containing 2 M NaCl- 6 plates (aw 0.921)

Payne’s containing 3 M NaCl- 6 plates (aw 0.881)

6. High-sucrose media:

MYA containing 55% w/v sucrose- 6 plates (aw 0.834)

MYA containing 75% w/v sucrose- 6 plates (aw 0.780)

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Inoculation and Incubation

Each plate was labelled with the appropriate medium. In order to inoculate the medium with the sample in preparation for incubation, small pieces of the salami sample were cut using a scalpel. A set of metal tweezers were sterilised using a Bunsen burner flame and used to pick up a piece of the sample and evenly dot the medium between 25-30 times with the salami. This was repeated with a new piece of the sample for each of the agar plates. The plates were incubated for seven days at 37.5ºC.

Characterisation of Cultures and Cell Types

After incubation, the results were analysed. The types of cultures grown on the plates were characterised and recorded. This was done by observing the culture’s form, margin, elevation, texture, appearance, pigmentation and optical density. When identifying cell types the pigmentation and texture of the cultures were mainly observed at a macro level and further analysis was carried out at a micro level to determine the phylogeny and cell morphology. The stress biology of the cultures was identified by the medium within which they grew.

Growth measurement

In order to obtain a growth measurement of the cultures grown, rough estimates were made by counting the colony forming units (cfu). This involved counting the colonies formed and therefore, the viable number of bacteria in the sample.

Growth rate determination

The plates in this experiment were not reviewed at various time intervals, therefore growth was simply identified visually after the seven days of incubation. If the experiment were to be repeated this would be changed and growth would be recorded every day for the seven days.

Data Analysis and Presentation

The data collected was collated and inserted into a table as seen in the results section. Drawings were also done of the sample prior to the experiment and of the cultures at a macro level after incubation and of any observations of findings once analysed under a light microscope.

Results

The figures shown are taken from the plates that contained the largest amounts of growth and show the most interesting results. The other plates showed very little to no growth at all. The most growth occurred on the plates containing the control, 55% sucrose and 55% fructose medium. There was no growth however on the plate containing the 75% fructose agar. Both ethanol mediums show very little growth, but colonies did occur. The 2M NaCl media showed slight growth, however the 3M NaCl did not. The 0.8 MgCl₂ did not show any growth despite the 1M MgCl₂ showing slight growth. After observation at a microscopic level, it is likely that the colonies present are bacterium as opposed to fungi or archaea.

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Discussion of Results

Sucrose and Fructose Plates

The results obtained from this experiment have shown that it is possible to isolate stress tolerant microbes. It is clear that the microbes grown during this process have a specific tolerance against the sucrose environment as growth occurred at the high and low concentration media. These microbes are likely to be xerophiles with a preference for sucrose. This is because the culture was able to grow in conditions with a low water activity (aw 0.780) which is not typically optimal for microbial growth, and high sucrose levels (Pitt, Hocking 1997). From visual analysis the cell morphology is a cocci shape, with small colonies spread throughout the culture, indicating a bacterial species (figure 5). There was also growth on the 55% fructose culture, it shows the cells having a long rod shape, indicating a Bacillus species. However this species is unlikely to be an extremophile as it did not grow on the more concentrated fructose media. As the salami sample has high salt and sugar content and any microbe growing in this environment must adapt accordingly, this result is what was expected.

Control Plate

The control plate also showed significant growth, this indicates the presence of mesophiles. The control plate did not contain a stressor and therefore any species grown here will not adapted to extreme environments. Figure 5 shows a non-extremophile bacterium with a cocci morphology. From this it could be concluded that the bacteria Staphylococcus xylosus is present. This species is commonly found in dried, salted meat products and was expected to be found in this experiment (Leroy, Vermassen et al. 2017).

Magnesium Chloride Plate

Another interesting result is the presence of microbial growth in the 1M MgCl₂ and not in the 0.8M agar. The magnesium chloride creates a chaotropic stress environment in which macromolecules become disrupted and unstable (Cray, Stevenson et al. 2015). It is possible that the small growth on this plate contains a potential extremophile as it required the extreme environment of 1M MgCl₂ in order to grow. This is not surprising as previously stated the salami creates a highly salty environment and any microbes growing here are adapted to these conditions and therefore have the ability to withstand and grow optimally in the chaotropic environment.

Results in relation to Aims and Hypothesis

Based on these results it can be concluded that the aims of this experiment were achieved because microbes were successful isolated from a sample and analysed in relation to environmental stressors. The majority of plates showed growth. However, three plates did not show any growth and seven plates showed very little growth. This may be due to the sample type and to the fact during the production of salami a lot of focus is directed at food safety and ensuring harmful microbes cannot grow and cause illness when consumed. Therefore, the microbial diversity is very minimal.

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Due to the growth of some species during this experiment it can be said that microbes found in extreme environments do have adaptations that allow them to grow and thrive in these conditions and therefore the hypothesis is accepted.

Discussion of Experimental Approach/Design

The strong aspects of this experiment were the use of aseptic technique, ensuring minimal contamination and invalid results. Also, the experiment was relatively cheap and simple, allowing replications to be easily carried out in the future. The experiment included a range of agar medium, allowing for a range of potential extremophiles to be grown and analysed. However, there could have been other extreme conditions tested for example thermophiles cannot be identified through this experiment. The main drawback of this experiment was the sample chosen. If a more extreme and diverse sample were chosen, it is likely that much more microbial growth would have been observed and potentially multiple species would have grown in each medium.

Conclusion & Suggested Further Work

This experiment has shown that with the sample used it is possible to isolate and grow specific stress tolerant microbes in various environmental conditions. Xerophilic microbes were found with a preference for sucrose. However, non-stress tolerant mesophiles were also found in the control media, indicating the potential for more extreme and diverse samples to be tested. In the future to see more specific and interesting results a different sample source may be used. For example, more natural sources such as fermenting fruit may present a wider range of xerophilic species. A soil sample will also show a range of difference species that have adapted to extreme environments such as different fungal species, none of which were found using the salami sample.

References

CRAY, J.A., STEVENSON, A., BALL, P., BANKAR, S.B., ELEUTHERIO, E.C., EZEJI, T.C., SINGHAL, R.S., THEVELEIN, J.M., TIMSON, D.J. and HALLSWORTH, J.E., 2015. Chaotropicity: a key factor in product tolerance of biofuel-producing microorganisms.

DAVILA, A.F., 2010. Astromicrobiology.

GUPTA, G.N., SRIVASTAVA, S., KHARE, S.K. and PRAKASH, V., 2014. Extremophiles: An Overview of Microorganism from Extreme Environment. International Journal of Agriculture, Environment and Biotechnology, 7(2), pp. 371-380.

LEROY, S., VERMASSEN, A., RAS, G. and TALON, R., 2017. Insight into the Genome of Staphylococcus xylosus, a Ubiquitous Species Well Adapted to Meat Products. Microorganisms, 5(3), pp. 52.

PITT, J.I. and HOCKING, A.D., 1997. Xerophiles. In: J.I. PITT and A.D. HOCKING, eds, Fungi and Food Spoilage. Boston, MA: Springer US, pp. 417-437.

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