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Anthropogenic biguanide compounds are widely used in agriculture, industry, and medicine, making them prevalent in the environment. The extensive use of compounds like cyanoguanidine, guanylurea, and metformin has led to their accumulation as pollutants in waterways. This research focused on the microbial degradation of cyanoguanidine, a common biguanide compound used as an additive in agricultural products and frequently present as an impurity in the production of pharmaceuticals such as metformin. Due to its extensive use, cyanoguanidine is classified as a persistent and mobile pollutant commonly detected in wastewater. The presence of cyanoguanidine in wastewater is hypothesized to exert selective pressure on microbial communities, driving the evolution of bacteria capable of metabolizing this compound as their sole nitrogen source. We hypothesized that sludge-derived microbial cultures could serve as a reservoir for isolating cyanoguanidine-degrading bacteria. To test this, enrichment cultures were established using thick and return-activated sludge from a municipal wastewater treatment plant. Microbial media containing cyanoguanidine as the sole nitrogen source was used to support bacterial growth. Aliquots of enrichment cultures were plated on minimal media containing cyanoguanidine to isolate bacteria capable of its metabolism. HPLC analysis was employed to monitor and quantify cyanoguanidine degradation, revealing its conversion to guanylurea, CO2, and NH3 by two bacterial isolates. Genome sequencing identified these cyanoguanidine degraders as Pseudomonas stutzeri and Pseudomonas mendocina. Bioinformatic analyses identified candidate genes involved in the degradation pathway, including nitrile hydratase and guanylurea hydrolase enzymes in both bacterial genomes. Although further investigation is needed to confirm the role of nitrile hydratase in cyanoguanidine metabolism, this study advances our understanding of microbial biguanide degradation. These findings contribute to the development of biotechnological strategies for removing biguanide pollutants from the environment.
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Breaking the habit: isolating nicotine-degrading bacteria in undergraduate microbiology teaching labs
ABSTRACT Nicotine is a major alkaloid in tobacco plants and an addictive component of tobacco products. Some bacteria grow on tobacco plants and have evolved the ability to metabolize nicotine. As part of our microbiology teaching lab, we used minimal media with nicotine as the sole carbon source to isolate nicotine-degrading bacteria from tobacco leaves and commercial tobacco products. Students then identified these bacteria using 16S rRNA sequencing and biochemical assays and assessed their ability to catabolize nicotine using UV spectroscopy. Students were able to isolate and identify 14 distinct genera that can metabolize nicotine. This modification of the commonly used unknown project gave students firsthand experience using selective media, and students got the opportunity to work with largely uncharacterized microbes with a real-world connection to public health, which increased student engagement. Students had the opportunity to think critically about why nicotine-degrading microorganisms associate with tobacco plants, why there are different bacteria that use the same specialized metabolism, and how these organisms are isolated from other bacteria using selective media.
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- Award ID(s):
- 2236541
- PAR ID:
- 10513406
- Editor(s):
- Maloy, Stanley
- Publisher / Repository:
- American Society for Microbiology
- Date Published:
- Journal Name:
- Journal of Microbiology & Biology Education
- ISSN:
- 1935-7877
- Page Range / eLocation ID:
- e00152-23
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1128/jmbe.00152-23
