Initial Research Plan

1. Title: Bacterial Interaction & Defenses against Ecological Cheating in Local Freshwater Ecosystems
2. Rationale: Low concentrations of dissolved oxygen remain a global concern regarding the ecological health of lakes and reservoirs. In addition to high nutrient loads, climate‐induced changes to lake stratification and mixing represent further ramifications of anthropogenic activity,  resulting in decreased deep water oxygen levels. Due to these changes, various ecologically essential aerobic bacteria have developed the ability to produce extracellular polymeric substances (EPSs) and generate free biofilms on or adjacent to the air-liquid interface (ALI) to receive the maximum oxygen uptake.  However, these organisms remain susceptible to the effects of ecological ‘cheaters,’ or aerobic bacteria which adhere to the free biofilms but cannot produce EPS. These bacteria alter the buoyant properties of the film and can lead to collapse of the film and eventual death of EPS producers. This research will explore the presence of a defense mechanism. The preservation of vital remedial species is essential to the health of aquatic ecosystems. As unnatural disruptions—such as climate change and pollution—continue to plague freshwater bodies, the diversity of ecological systems will be decimated from the microbial level upward. 
3. Hypothesis: The isolated EPS producer will possess a defense mechanism against cheater aerobes when placed in conditions of spatial competition or limited oxygen. This mechanism will be represented by decreased growth when cultivated together when compared to the cheater aerobe control plate. Without the defense mechanism, the EPS producer would be susceptible to death due to hypoxia. 
4. Procedure:

1. Make nutrient agar plates
   1. Dissolve agarose powder in TBE buffer and boil until clear and smooth. 
   2. Cool slightly before pouring plates with clamshell technique. 
2. Obtain water samples from Mercer County Lake. 
   1. Collect samples in sterile cups from multiple locations. (Marshy, green, deep, clear, etc.)
   2. Record temperature and pH around location of collection at multiple depths and areas. 
   3. Store samples until further use. 
      1. Place each of the samples adjacent to a window where sunlight is readily available. 
   4. Pour fresh water from each location into labelled sterile tubes. 
3. Plate and incubate
   1. Pipette 200 μL of each sample onto agar plates. Utilize clamshell technique and neutralize any spills with bleach. 
   2. Incubate at 37 C until growth is visible but not lawn. 
4. Isolate pure colonies
   1. Use inoculating loop to isolate a morphologically unique colony. 
   2. Suspend colony in nutrient broth in a numerically-labelled subculture tube. 
   3. Store tubes in a shaking incubator at 37 C overnight. 
   4. Plate 10 μL of each subculture and incubate at 37 C until colonial growth is visible. 
   5. Gram Stain to ensure pure colonies in 3 locations on the plate. 
   6. If colonies prove to be impure, repeat subculture. 
5. Send gene for Sanger gene sequencing at GeneWIZ Lab.
   1. Utilize the highly conserved 16S gene for analysis and identification of the species. 
   2. Analyze sequencing reports and research species return to identify EPS producer. 
6. Plate and compare interspecies competitive growth between prospective cheater aerobes and the EPS producer. 
7. Incubate liquid culture tubes of EPS producer alone and EPS producer with cheater aerobe and analyze film production and collapse. 

Data Analysis: 

• Bacterial DNA barcodes will be sent to the GeneWIZ lab to be sequenced and sent back to us. Then, we will compare the sequence reports to the NCBI and BOLD databases to identify the bacterial species. 
• ImageJ analysis will be used to quantify growth 
• Once the experimental variables have been applied, distinct changes in density or general appearance will also be recorded as a method of qualitative analysis, and then another protein assay will be run to quantify any changes in expression.

5. Bibliography

Bach, L. T., Alvarez-Fernandez, S., Hornick, T., Stuhr, A., & Riebesell, U. (2017, November 30). Simulated ocean acidification reveals winners and losers in coastal phytoplankton. Retrieved November 11, 2019, from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5708705/.

Bauer, C., & Bird, T. (1996, April 5). Regulatory Circuits Controlling Photosynthesis Gene Expression. Retrieved from https://www.cell.com/fulltext/S0092-8674(00)81074-0.

Boundless. (n.d.). Boundless Microbiology. Retrieved from https://courses.lumenlearning.com/boundless-microbiology/chapter/nonproteobacteria-gram-negative-bacteria/.

Marxsen Jürgen. (2016). Climate change and microbial ecology: current research and future trends. Norfolk, UK: Caister Academic Press.

Santos, H. F., Carmo, F. L., Duarte, G., Dini-Andreote, F., Castro, C. B., Rosado, A. S., … Peixoto, R. S. (2014, November). Climate change affects key nitrogen-fixing bacterial populations on coral reefs. Retrieved from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4992079/.

6. Potentially Hazardous Biological Agents:

Since the bacteria being investigated are unknown to start, 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. Once the research is finished, the plates will be taped shut and discarded in biohazard bags.