Antibacterial Properties of Endophytic Fungi Cultured in Nutrient Supplemented Lunar and Mars Simulants

Description

These collective data are from projects at Davidson-Davie Community College (DDCC) in Thomasville, NC in 2022. All projects were conducted within a framework of endophytic fungi isolation, culturing in lunar or Mars regolith simulants supplemented by nutrient agar (potato dextrose or Sabouraud dextrose agar) and testing of organic extracts for antibiotic properties. Individual plant specimens representing 12 species were collected on the campus. Tissues were surface sterilized, and then plated on either potato dextrose, yeast-malt extract, or Sabouraud dextrose agar containing kanamycin (50 µg/mL) to inhibit bacteria followed by storage at room temperature. Plates were checked regularly for growth and subsampled and re-plated in an attempt to isolate endophytic fungi. Once isolated, sterilized scalpels were used to remove small strips of agar from a plate and placed in sterile 15mL conical centrifuge tubes containing sterile water (permanent water stock cultures) and are currently stored on the DDCC campus. Samples were then placed in autoclaved jars containing 16.5g of lunar or Mars regolith simulants supplemented with 10ml of PDA or SDA. Cultures were allowed to grow for one month as semi-solid fermentation cultures at room temperature followed by extraction. Organic extraction consisted of separation of organic material with ethyl acetate. The simulant-agar was removed to appropriate sized beakers and covered with twice the amount of ethyl acetate as semi-solid. A small amount of sterile deionized water was added to create two phases for separation in a separatory funnel. The resulting organic phase was placed in a modified distillation apparatus as there was no access to a rotary evaporator. The ethyl acetate was evaporated off and recollected in a receiving flask and the organic residue remaining from the extraction was collected and stored in methanol in a laboratory freezer until being tested for antibacterial properties. Extracts were tested against four species of bacteria (Escherichia coli, Staphylococcus epidermidis, Bacillus subtilis, and Klebsiella (Enterobacter) aerogenes) using the agar diffusion method in nutrient agar. Plates were inoculated by submerging sterile cotton swabs in fresh broth cultures of a species and subsequent swabbing of agar’s surface. Wells were constructed using sterilized 10mm diameter cuvettes and then filled with 25 µL of extract, or of control (+ kanamycin, - methanol) solutions. Plates were incubated at species appropriate temperatures for 48 hours followed by removal and the measurement (mm) of the zones of inhibition around each well. All testing was done in triplicate. Due to limited time and space, only six organic extractions were completed. Of the six, subsequent statistical analyses indicated five extracts had inhibitory effects against two species, B. subtilis and S. epidermidis while no inhibitory effects against E. coli or K. aerogenes were observed.

Steps to reproduce

Most methods followed or modified can be found in Bascom-Slack CA, Arnold AE, Strobel SA. 2012. Supplementary Materials for Student-Directed Discovery of the Plant Microbiome and Its Products. Science. 338 (October): Supplementary Materials. doi:10.1126/science1215227

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