Bacterial Interaction and Defenses Against Ecological Cheating In

Bacterial Interaction and Defenses against Ecological Cheating in Local Freshwater Ecosystems impacted by Climate Change and Pollution Conducted by: Brinda Suresh Introduction and Background 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 evolved 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 ofecological ‘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 essential EPS producers. 2 Rationale Why is this important? The preservation of vital remedial species is essential to the health of aquatic ecosystems. As anthropogenic disruptions—such as climate change and pollution—continue to plague freshwater bodies, ecological systems will be decimated from the microbial level upward.

3 Objective This research will explore the presence of a defense mechanism in essential EPS producing aerobes in an effort to predict their safety in the future and the resulting impact on freshwater quality.

4 Hypothesis The isolated EPS producer will possess a defense mechanism against cheater aerobes when placed in conditions of spatial competition or limited oxygen.

This will manifest as decreased growth when the EPS producer (P. reinekei) and the cheater aerobe (B. tropicus) are cultivated together in comparison to the cheater aerobe control plate. Without the defense mechanism, the EPS producer would be susceptible to death due to hypoxia. Methods

1 2 3

6 Results (GeneWiz 16S Sequencing)

GENEWIZ® - Solid science. Superior service.

Sequence Name BEST Genus & species BEST Complete Taxonomic Classification

1_1_16S_rRNA_Seq_CONSENSUS_TRIMMED Bacillus pseudomycoides Bacteria,Firmicutes,Bacilli,Bacillales,Bacillaceae,Bacillus,Bacillus pseudomycoides

1_3_16S_rRNA_Seq_CONSENSUS_TRIMMED Bacillus paranthracis Bacteria,Firmicutes,Bacilli,Bacillales,Bacillaceae,Bacillus,Bacillus paranthracis

1_4_16S_rRNA_Seq_CONSENSUS_TRIMMED Bacillus paranthracis Bacteria,Firmicutes,Bacilli,Bacillales,Bacillaceae,Bacillus,Bacillus paranthracis

2_1_16S_rRNA_Seq_CONSENSUS_TRIMMED Bacillus tropicus Bacteria,Firmicutes,Bacilli,Bacillales,Bacillaceae,Bacillus,Bacillus tropicus

3_1_16S_rRNA_Seq_CONSENSUS_TRIMMED Pseudomonas reinekei Bacteria,Proteobacteria,Gammaproteobacteria,Pseudomonadales,Pseudomonadaceae,Pseudomonas, Pseudomonas reinekei

3_3_16S_rRNA_Seq_CONSENSUS_TRIMMED Bacillus tropicus Bacteria,Firmicutes,Bacilli,Bacillales,Bacillaceae,Bacillus,Bacillus tropicus

3_5_16S_rRNA_Seq_CONSENSUS_TRIMMED Bacillus proteolyticus Bacteria,Firmicutes,Bacilli,Bacillales,Bacillaceae,Bacillus,Bacillus proteolyticus

Cheater Aerobe Ecologically essential EPS Producer 7 Results: Solid Media & ImageJ

ImageJ Analysis 2.1 (Bacillus tropicus) Control Plate Coverage: 76.8% P. reinekei and 2.1 (Bacillus tropicus) Experimental Plate Coverage: 78.3% 8 Results: Liquid Media and ALI Bioﬁlm Interaction

Start of ﬁlm collapse

P. reinekei Control: 24 P. reinekei Control: 72 P. reinekei and 2.1: 24 Hours in Saturated LB Hours in Saturated LB Hours in Saturated LB Broth Broth Broth 9 Analysis Upon examination, species Pseudomonas reinekei were unable to inhibit growth of other ‘cheater’ aerobes in their immediate proximity and were found to be defenseless in nutrient-rich environments with spatial competition (solid interfaces with limited surface area). Additionally, gradual collapse of the free bioﬁlms was observed when the competing species were cultured together in liquid media.

10 Results Contrary to the hypothesis, P. reinekei does not appear to possess a defense mechanism to ecological cheating. Extensions Possible research based off of this study can include: ⪢ Impact of oxygen depletion on EPS-cheater interaction ⪢ Effect of upregulation of existing defense mechanisms in P. reinekei on interaction with cheater aerobes ⪢ Transforming P. reinekei with evolved bacterial defense mechanisms

12 References 1. Schwefel, R., Müller, B., Boisgontier, H., & Wüest, A. (2019, April 29). Global warming affects nutrient upwelling in deep lakes.

2. Rainey, P. B., & Travisano, M. (1998, July 2). Adaptive radiation in a heterogeneous environment.

3. Nogales, J., García, J. L., & Díaz, E. (1970, January 1). Degradation of Aromatic Compounds in Pseudomonas: A Systems Biology View.

4. Ardré, M. B., Dufour, D. B., & Rainey, P. B. et al. (2019, September 15). Causes and Biophysical Consequences of Cellulose Production by Pseudomonas ﬂuorescens SBW25 at the Air-Liquid Interface.

5. Hibbing, M. E., Fuqua, C., Parsek, M. R., & Peterson, B. s. (2010, January 8). Bacterial competition: surviving and thriving in the microbial jungle.

Special thanks to my mentor, Mrs. Sbarro, my additional advisor, Mrs. Benegal, GeneWiz lab for their services, and to all previously cited researchers for their diligent work that I was fortunate enough to build upon. 13 Thank you for your time. All questions are welcome!