Nutritional Sources and Salinity
In the laboratory, Serratia marcescens was utilized to test Growth and Prodigiosin production under various environmental factors. The environmental conditions were temperature, pH, oxygen availability, nutritional sources, and salinity. The temperatures tested were 4 degrees Celsius, 25 degrees Celsius, and 65 degrees Celsius, and the pH values were (3, 7, 11). The carbon source was maltose and glucose. The salinity measurements consisted of 0.1% NaCl, 1.0% NaCl, and 3.0% NaCl. Lastly, the oxygen requirement was tested by using two groups, one group was tested using aeration (shaking), and one group was tested with no aeration (no shaking). The results indicate that optimal growth was experienced at a temperature of 65 degrees Celsius, a pH of 7, NaCl concentration of 0.1% and no aeration. Prodigiosin production was affected under most of the same conditions, for example, it experienced optimal growth at a temperature of 65 degrees Celsius, a pH of 7, and the same carbon source. However, results differ because Prodigiosin production experiences a higher rate at a salt concentration of 3% NaCl, and, under conditions of no aeration.
Serratia marcescens is a pathogenic bacterium that invades humans and mimics the red color of blood making it hard to detect. S. marcescens was once considered a harmless saprophyte, which is a type of microorganism that lives on dead or decaying organic matter. However, S. marcescens is now recognized as a significant opportunistic pathogen which has a tendency to create healthcare related infection and antimicrobial resistance (Herra and Falkiner 1984). Prodigiosin is an antifungal red pigment produced by S. marcescens, it holds antibacterial, antimalarial, and antifungal activities, however, it is used primarily as a biochemical tool (NCBI 2014).
Although bacteria are highly adaptable in nature, which is why they are found in nearly every place on earth, certain environmental conditions affect optimal growth for different types of bacteria. For instance, most disease-causing bacteria thrive in warm temperatures, which is why the human body provides an ideal environment for many types of bacteria to grow. On the other hand, some bacteria are able to survive in freezing temperatures of 5 degrees Celsius, yet other types of bacteria would not survive in temperatures below 60 degrees Celsius (Wagner 2008). In another way, optimal growth of bacteria is affected by pH. pH is used to classify the hydrogen ion concentration in a solution, pH values range from 1.0 (very acidic) to 14.0 (very basic). Most bacterium grow best around a neutral pH value (7). However, some bacteria thrive in very basic conditions, yet some can tolerate a pH of 1.0 (Blamire 2000).
Carbon sources also play a significant role in the growth of bacteria. Carbon is known as the structural backbone of organic compounds, and each carbon source may support different bacterial growth rates. Salinity is the amount of salt dissolved in an amount of water, the percent of sodium chloride present in the solution used for this lab accounts for the salinity. Because bacteria are extraordinarily diverse, certain types of bacteria thrive in salty environments while others do not. For example, research suggests that S. Marcescens is highly adaptable to salty environments and can continue to reproduce under harsh conditions (classified as 80 g/L) and extreme conditions (classified as 100 g/L salt concentrations) (Ketola and Hiltunen 2014). On the other hand, oxygen requirements, also known as aeration or no aeration play an important role in the growth of bacteria. Aeration is believed to stimulate the growth of bacteria because it provides much needed oxygen to the environment for bacteria to use for growth and development (Mattick 1940). Such environmental conditions affect the metabolic functioning of bacteria, and as a result the growth of the cell.
S. marcescens is a bacterium which, like many others, requires a relatively warm temperature, neutral pH, and a low salt concentration to experience optimal growth. The objectives of this laboratory are to develop an experiment which will test the impact of environmental conditions on the growth of S. Marcescens and prodigiosin, to understand how different experimental conditions affect bacterial growth and prodigiosin production, and to gain a better understanding of the stages of the bacterial growth curve & the production of prodigiosin throughout the growth cycle of S. Marcescens. In order to address each objective, students were separated into groups and each group focused on one environmental condition. This allowed each group to fully test and experiment the possible implications of their environmental condition. For each group, a proper lab protocol was created and followed accordingly. After testing the condition through a series of experiments, each group recorded their data and made it available to all other groups.
In order to obtain the most thorough results possible, and to ensure the experiment was carried out as accurately as possible, it is important to generate hypotheses which can be compared to the acquired end results. For instance, the null hypothesis for temperature expects that temperature will not have an effect on the growth of S. marcescens or Prodigiosin production. While the alternative hypothesis for temperature expects that temperature will have an effect on the growth of S. marcescens and/or Prodigiosin production. The null hypothesis for pH expects that pH values will not have an effect on the growth of S. marcescens or Prodigiosin production. And, the alternative hypothesis claims pH values will have an effect on the growth of S. marcescens and/or Prodigiosin production. The null hypothesis for the carbon source condition expects that different carbon sources will not have an effect on the growth of S. marcescens or Prodigiosin production. While the alternative hypothesis for the carbon source condition expects that carbon sources will cause an increase or decrease on the growth of S. marcescens and/or Prodigiosin production. The null hypothesis for the NaCl concentration condition, also known as salinity, expects that sodium chloride concentrations will not affect the growth of S. marcescens or Prodigiosin production. However, the alternative hypothesis expects that differing sodium chloride concentrations will cause and increase/decrease on the growth of S. marcescens and/or Prodigiosin production.
The null hypothesis for the oxygen requirement
Suggests that there will be no difference in the growth of S. marcescens and Prodigiosin production due to aeration or no aeration. While the alternative hypothesis suggests that depending on the condition, aeration or no aeration, an effect on the growth of S. marcescens and/or Prodigiosin production will occur. The null hypothesis in each condition suggests that no change will occur, regardless of the environmental condition. The primary purpose of the null hypothesis is for scientists to reject or disprove it, it is impossible to ""accept"" the null hypothesis, in a scenario where the null hypothesis is not rejected, it would simply be said that it failed to be rejected. And, the alterative hypothesis is simply the opposite of the null hypothesis.
Materials and Methods
The environmental condition tested in this experiment was oxygen requirement. The oxygen requirement refers to the practice of using aeration (shaking) or no aeration (no shaking) during the experiment. Aeration is important because it provides oxygen to bacteria, and it is commonly believed that aeration helps to increase optimal growth/prodigiosin production.
In order to carry out this experiment and to test the effects of aeration/no aeration, the lab group had to utilize a machine called a Spectrophotometer. Sterile technique was used to remove 2mL of fresh media which was then pipetted into two cuvettes and set as blanks. Sterile technique was used again to remove 2 ml of each of the S. Marcescens cultures, Culture a which was shaken at 30 C, and Culture b which was not shaken at 30 C, into a cuvette. The spectrophotometer was set to 499 wavelength and measurements were recorded for each culture group after blanking. Next, the spectrophotometer was set to read absorbance's at 600 wavelength and measurements were recorded for each culture after blanking. These steps were repeated every 30 minutes for the course of 180 minutes. Finally, after obtaining all necessary measurements, each S. Marcescens culture was returned to their respective incubators.
The full dataset obtained through this group can be found in Figures 5 & 10. This group found that bacterial growth was higher when no aeration was implemented, and prodigiosin production experienced a higher rate during no aeration as well. The equation A499/(OD600 x 5.8x108 cell/ml x 2 ml)= units of prodigiosin per cell was utilized to calculate prodigiosin production.
The graphs pictured above show a complete summary of all the data collected throughout the experiment. Each graph shows data points that were collected and recorded every 30 minutes throughout the testing period, for both Growth and Prodigiosin Production. For temperature, the graph (Figure 1) indicates that optimal bacterial growth was reached at a temperature of 25 degrees Celsius. Yet, growth was more consistent at a temperature of 65 degrees Celsius, this in agreement with the idea that most bacteria tend to thrive in environments of higher temperatures. However, consistency alone is not effective at drawing a proper conclusion, so due to the growth curve, 25 degrees provides for optimal growth. Likewise, optimal growth was reached when the pH stayed at 7 consistently. This result was expected as many bacteria, including S. marcescens, thrive in neutral conditions. And, S. marcescens remained constant throughout the carbon source experiment, there was no rapid increase/decrease of S. marcescens, this can be expected due to the fact that carbon is a substance found in all organic matter and is the backbone of all living organisms. Next, it was revealed that optimal growth was reached when the concentration of NaCl was at 0.1% NaCl, again, this is expected as most bacteria are not able to survive in environments that are high in NaCl. Lastly, it was determined that S. marcescens reached optimal growth under conditions of no aeration, this is different than what was expected as it is believed that aeration provides stimulation for growing bacteria.
Prodigiosin production experienced a more significant increase at a temperature of 65 degrees Celsius and a pH of 7, the same temperature and pH which was consistent with optimal bacterial growth. And, carbon source also remained the same throughout this part of the experiment. However, salinity experienced a significant shift, for instance, growth was more optimal at an NaCl concentration of 0.1%, yet prodigiosin production experienced a higher rate at 3.0% NaCl. While prodigiosin production did decrease with aeration, there was a more significant decrease in no aeration, this is contradictive to the ""Aeration vs. Growth"" graph, and in agreement with scientific research.
The objective of this experiment was to test the effect of various environmental conditions and on the growth and Prodigiosin production of S. Marcescens. In order to efficiently and accurately detect any possible change that these environmental conditions had we composed two different hypotheses, a null and an alternative. The null hypotheses suggest that the various factors would not significantly alter the amount of growth or Prodigiosin production. On the other hand, the alternative hypothesis speculated that each of the environmental conditions would significantly change the growth or Prodigiosin production of the S. Marcescens in comparison to the bacterial scientific conventional wisdom. While this numerical change in the alternative hypothesis was not initially mentioned, it would later be calculated following the completion of the experiment. Following the experiment and analysis of data, the results were both revealing and expected. In the end, each of the environmental conditions had an effect on growth and Prodigiosin production, some of these effects were expected, while others came as a surprise, such as the observation that no aeration provided more optimal growth for S. Marcescens.
As it was aforementioned in the results paragraph, some of the manipulated variables yielded results parallel to the umbrella beliefs on optimal bacteria growth. The S. Marcescens had the highest optimal growth when exposed to a temperature of 65 degrees Celsius (Figure 1). It was revealed that optimal growth occurred at 65 degrees Celsius, and at a pH of 7. This is consistent with scientific data that suggests a neutral pH and high temperature are optimal conditions for bacterial growth (Herra and Falkiner 1984). In a similar sense, results showed that optimal growth was reached at an NaCl concentration of 0.1%, this is in agreement with scientific research which states that bacteria grow best in an environment with a low salt concentration. On the other hand, it was revealed that optimal growth was reached during conditions of no aeration, which is contradictive to the idea that aeration provides important stimulation to bacteria (Mattick 1940).
As for Prodigiosin production, results stayed in agreement with optimal growth, with the exception of aeration. It was revealed that the rate of Prodigiosin production experienced an increase during conditions of aeration.
Difficulties that may have impacted the results of this experiment were kept at a minimum, and if any occurred, they were minor. For example, groups relied on each other to relay correct information throughout the experiment, and since each group had a different environmental condition, it was nearly impossible to indicate whether the information being relayed was correct. If a group made a slight mistake or accidentally relayed the wrong information, it is unlikely that the error would have been caught and corrected, which could result in possible errors and inaccuracies in the end.
With the exception of the oxygen requirement, the growth results obtained in this experiment support the findings by others based on the literature research. As mentioned, temperature, pH, carbon sources, and salinity are in agreement with the results of others. The Prodigiosin production results are the most supportive of the findings of others, as every environmental factor is in agreement with that of other scientific data, while in the growth section, aeration was the only one that separated from a common trend in thought.
In order to enhance Prodigiosin production, it would be important to increase the amount of S. Marcescens used in this laboratory experiment. The more S. Marcescens we are able to use, the more Prodigiosin production will occur, and likely the results will become more accurate.
In order to continue working on this project, it would be wise to consider adding more environmental factors and to further test the ones that have already been used. For instance, it would be a good idea to test more temperatures other than the three that are being used, and to test more pH values than the current three. All conditions can be expanded on, and it would provide more information and accurate details to expand more on each one. Other nutritional requirements such as energy source, nitrogen source, and mineral source would be interesting to look at since they all provide important chemical nutrients, and by doing this, we would be able to determine more accurately the effects of the current nutritional requirements.
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