Materials and MethodsI. Preliminary microcosm optimization step
A. Preparation of Microcosms
We do not have data on the lab's microcosm resistance duration. Therefore, we have created three different batches of 200 grams of soil. The first batch (control) consists of soil sterilized in an autoclave at 121 °C for one hour. The second microcosm, called the natural microcosm, underwent no modifications. The last type is the microcosm enriched with a mineral solution to maintain the basic metabolism of the microcosm and promote the growth of slow-growing microorganisms, as shown in the experimental plan in Figure 1. Each of these three types of microcosms will be replicated four times, giving us a total of twelve microcosms for measurements after 1 week, 15 days, 21 days, and 30 days. All microcosms were incubated at 28 °C. All components used for the microcosms were sterilized in an autoclave for 30 minutes at 121 °C.
B. Screening of extraction methods
In the first step, we optimized the soil metabolome extraction protocol. To achieve this, we extracted 5 grams of soil using five extraction methods: M1, M2, M3, M4, and M5. Subsequently, the mixtures were allowed to rest for 2 hours, after which they underwent sonication at 37 kHz with a 100-pulse wave for 30 minutes at a temperature range of 30-60°C. Following this, the samples were centrifuged at 20,000× g for 15 minutes at 4°C. The supernatants were collected and stored at -20°C for further analysis.
Method 1: 100% methanol.
Method 2: 80%,20%, 0.001% (Methanol/ H2O/Formic acid).
Method 3: 100% DMSO.
Method 4: 4ml of ammonium bicarbonate + 10 ml methanol+ 5ml of chloroform.
Method 5: 4ml of ammonium bicarbonate + 10 ml ethyl acetate.
All the extractions have a final volume of 15 ml with 5 g of soil.
Additionally, 0.5 grams of the sample were diluted in 4 milliliters of sterile distilled water and vigorously shaken. A volume of 100 μL of the diluted sample was inoculated onto various culture media, including SCA supplemented with antimicrobials (nalidixic acid, cycloheximide, and nystatin), PDA, nutrient agar, and A4. The plates were incubated at 28°C, except for those inoculated on nutrient agar, which were incubated at 37°C.
Materials and Methods I. Preliminary microcosm optimization step
A. Preparation of Microcosms We do not have data on the lab's microcosm resistance duration. Therefore, we have created three different batches of 200 grams of soil. The first batch (control) consists of soil sterilized in an autoclave at 121 °C for one hour. The second microcosm, called the natural microcosm, underwent no modifications. The last type is the microcosm enriched with a mineral solution to maintain the basic metabolism of the microcosm and promote the growth of slow-growing microorganisms, as shown in the experimental plan in Figure 1. Each of these three types of microcosms will be replicated four times, giving us a total of twelve microcosms for measurements after 1 week, 15 days, 21 days, and 30 days. All microcosms were incubated at 28 °C. All components used for the microcosms were sterilized in an autoclave for 30 minutes at 121 °C.
B. Screening of extraction methods In the first step, we optimized the soil metabolome extraction protocol. To achieve this, we extracted 5 grams of soil using five extraction methods: M1, M2, M3, M4, and M5. Subsequently, the mixtures were allowed to rest for 2 hours, after which they underwent sonication at 37 kHz with a 100-pulse wave for 30 minutes at a temperature range of 30-60°C. Following this, the samples were centrifuged at 20,000× g for 15 minutes at 4°C. The supernatants were collected and stored at -20°C for further analysis. Method 1: 100% methanol. Method 2: 80%,20%, 0.001% (Methanol/ H2O/Formic acid). Method 3: 100% DMSO. Method 4: 4ml of ammonium bicarbonate + 10 ml methanol+ 5ml of chloroform. Method 5: 4ml of ammonium bicarbonate + 10 ml ethyl acetate. All the extractions have a final volume of 15 ml with 5 g of soil. Additionally, 0.5 grams of the sample were diluted in 4 milliliters of sterile distilled water and vigorously shaken. A volume of 100 μL of the diluted sample was inoculated onto various culture media, including SCA supplemented with antimicrobials (nalidixic acid, cycloheximide, and nystatin), PDA, nutrient agar, and A4. The plates were incubated at 28°C, except for those inoculated on nutrient agar, which were incubated at 37°C.