The Effect of Rain Acidification on the Growth of Freshwater Bacteria

Table: ENV1502

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Abstract:

The Effect of Rain Acidification on the Growth of Freshwater Bacteria

Acid rain is caused by increasing pollution in the atmosphere and has been impacting the pH of aquatic ecosystems around the globe. Slight variations to pH levels have notable impacts on microbial growth which are important components of ecological systems. Data collected by the United States Environmental Protection Agency shows that normal rainfall and precipitation has a pH of 6 while acid rain typically has a pH of about 4 (USEPA). In this experiment, three different species of freshwater bacteria (Bacillus pseudomycoides, Bacillus paranthracis, and Bacillus tropicus) were assigned to three different simulated acidic environments. Each of the three experimental groups were characterized by the change in concentration of sulfuric acid and quantified using the pH scale. The pH levels utilized were 4.0, 3.0, and 2.0, respectively. The microbial plates were compared based on the density of the bacterial growth. All three Bacillus freshwater bacteria are known to thrive at a pH range of about 5-7 and the majority of the species behaved accordingly. However, the Bacillus paranthracis showed more growth at the pH of 2.0 than at 4.0. The Bacillus pseudomycoides and Bacillus tropicus showed decreased growth at lower pH levels but featured the same amount of growth at the higher pH levels. Therefore, of the species studied, Bacillus paranthracis is most likely to survive at the lower pH levels.

As the pH levels in freshwater bodies begin to decrease due to acid rain, Bacillus paranthracis demonstrate increased reproductive fitness and survivorship. Conversely, Bacillus pseudomycoides and Bacillus tropicus are likely to die off as the pH in aquatic environments continue to decrease. These changes will lead to a shift in microbial communities and population density. Further research could examine the role of Bacillus paranthracis in the environment to determine if their increased presence would provide a benefit or disadvantage to aquatic ecosystems.

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Research Plan:

 

  1. The Effect of Acid Rain on the Growth of Freshwater Bacteria

  2. The hypothesis is that as pollution increases, this will cause acid rain to decrease the pH in aquatic ecosystems. In turn,freshwater bacteria will be less likely to survive, hence negatively impacting the environment by changing the species of bacteria which live in aquatic environments.

  3. Procedures: Detail all procedures and experimental design to be used for data collection   

  1. Obtain fresh water sample from local lake (Mercer County Lake) 

    1. Collect water in sterile cups

    2. Record the water temperature and pH 

  2. Plate 200 μL of the samples onto separate nutrient agar plates

    1. Incubate the plates at room temperature for two days

  3. Isolate pure colonies

    1. Use a sterile inoculating loop to gently scrape off a colony to isolate it

    2. Swish off bacteria from the inoculating loop in 10 mL of nutrient (luria) broth 

    3. Incubate the cultures in a shaking incubator at room temperature for two days

  4. Repeat step 2 and step 3 until qualitative data shows pure colonies 

  5. Send the plate with the isolated colonies to GENEWIZ for sequencing

  6. (While plates are being sequenced) Create nutrient (luria) broth solutions to mimic a pH of 7.5, 4.0, 3.0, and 2.0

    1. Obtain 16 sterile culture tubes and fill each with 10 mL of nutrient (luria) broth

    2. To 4 of the tubes, add 2 μL of sulfuric acid and invert two times (creates a pH of 4.0)

    3. To 4 of the tubes, add 20 μL of sulfuric acid and invert two times (creates a pH of 3.0)

    4. To 4 of the tubes, add 200 μL of sulfuric acid and invert two times (creates a pH of 2.0)

  7. Culture isolated bacteria in treatment

    1. For each identified (sequenced by GENEWIZ) bacterium, culture the bacteria in one of each of the broth solutions. For example, for Strain A, culture in one tube of plain nutrient broth, one tube with a pH of 4.0, one tube with a pH of 3.0, and one tube with a pH of 2.0

      1. Use a sterile inoculating loop to gently scrape off a colony to isolate it

      2. Swish off bacteria from the inoculating loop in 10 mL of nutrient (luria) broth 

      3. Incubate the cultures in a shaking incubator at room temperature for two days

  8. Plate each of the cultures on nutrient agar plates and allow them to incubate at room temperature (25C) for one day. Observe the growth and compare the growth at each different pH level. 

Data Analysis: Describe the procedures you will use to analyze the data/results that answer research questions or hypotheses

  • The bacterial colonies were sent on nutrient agar plates to a lab (GENEWIZ) to be sequenced. The sequences were then compared to the NCBI database to determine the ecological niche of each bacterium. 

  • Qualitative data was collected for analysis based on bacterial colony morphology including growth and density  

  1. Bibliography

Bach, Lennart T., et al. “Simulated Ocean Acidification Reveals Winners and Losers in Coastal Phytoplankton.” Plos One, vol. 12, no. 11, 2017, doi:10.1371/journal.pone.0188198.

“Figure 2f from: Irimia R, Gottschling M (2016) Taxonomic Revision of Rochefortia Sw. (Ehretiaceae, Boraginales). Biodiversity Data Journal 4: e7720. Https://Doi.org/10.3897/BDJ.4.e7720.” doi:10.3897/bdj.4.e7720.figure2f.

Kumar, Parul. “Biological Nitrogen Fixation and Its Genetic Engineering: A Close Look.” Biology Discussion, 16 Oct. 2015, http://www.biologydiscussion.com/nitrogen-fixation/biological-nitrogen-fixation-and-its-genetic-engineering-a-close-look/11857.

Wakefield, Petra. “How Do I Isolate Bacteria From Soil?” Sciencing, 2 Mar. 2019, https://sciencing.com/do-isolate-bacteria-soil-6918955.html.

Webster, N. S., et al. “Host-Associated Coral Reef Microbes Respond to the Cumulative Pressures of Ocean Warming and Ocean Acidification.” Scientific Reports, vol. 6, no. 1, 2016, doi:10.1038/srep19324.

Why is Acid Rain Harmful? (n.d.). Retrieved from https://www3.epa.gov/acidrain/education/site_students/whyharmful.html 

  1. (5) Potentially Hazardous Biological Agents

Since the bacteria we are investigating are unknown, they could potentially be hazardous or dangerous. To keep these hazardous bacteria contained, the bacteria will be grown on sterile agar plates and kept isolated from the rest of the environment using sterile technique. After using the bacteria, the plates will be taped shut and discarded in the trash.