Purpose

This study aimed to investigate the difference between the soil of young and old rain gardens in their microbial biodiversity and therefore the health and effectiveness of the rain gardens. The microbial biodiversity of the soil is important to the ability of the soil to collect and filter runoff to deposit into our waterways (Steeb). Therefore, by analyzing the difference between the microbial biodiversity and soil conditions of the two soil conditions, we gained insight into whether the age of the rain garden impacts its effectiveness.

Method

The soil was collected by inserting a sampling probe 6 inches deep into the dirt of the rain garden. The probe was then pulled out and the soil collected was gathered in a plastic bag and taken back to the lab. Soil samples were gather on January 20, 2024 in two separate locations on the campus of Purdue University.  The first location was  the young rain garden near Memorial Mall. The second location was the old rain garden near the Armory. From these samples, we will compare the soil properties and microbial biodiversity of each young and old. 

Experimental Plan

In this experiment, the independent variable we are manipulating is the age of the rain garden. We will be testing different conditions of soils from rain gardens that are relatively old and new. We will then compare the dependent variable, which in this scenario, is the amount microbial biodiversity. To measure these variables, we will be testing the pH and moisture of the soils and determining the bacterial diversity using an Ecoplate as well as genomic DNA 16S rRNA microbiome sequencing.  An Ecoplate is an instrument used to assess the stability and changes of a microbial population. pH can be referred to as the Hydrogen ion (H+) activity in a solution at equilibrium. Based on the soil's acidity or alkalinity, certain nutrients are less available. The pH is the "main parameter influencing [...] richness and communities. Soil pH also differentiated the structure of microbial communities”. (Mark Tibbett, Catena) Testing the soil's pH will provide information about the amount of microbial biodiversity present (Westerman, soil testing and plant analysis). In lab, we tested the pH of the soils by using an instrument (the vortex) to thoroughly mix soils with DI water, then used a pH probe. 

Soil moisture refers to the amount of water that is retained within the soil (Acurite GDPR Notice). The presence of water in soil is crucial to many ecological and physiological processes and is “greatly dependent on precipitation (rainfall), temperature, humidity, and soil type” (Meena and Datta, Acurite GDPR Notice). Therefore, determining the soil moisture at each rain garden will give insight into how equipped the soil is to carry out these important natural processes. To measure our samples' soil moisture, we will take the mass of an empty glass vial and then the soil samples in the vial. We will then bake the samples in a laboratory oven set at 105–110 °C and dry the sample for 1 week so that the water dries off the sample. The end mass will then be taken and subtracted from the beginning mass of the soil and vial, and then the mass of the empty vial to determine the amount of moisture that left the sample.

EcoPlates are used to detect the stability of the microbial population in soils as well as changes in an environment following the onset of a variable. The collected soil samples from the old and new rain gardens will be inserted into the instrument which will scan at a certain wavelength and collect their average colors through monitored absorptions. This instrument will provide insight into certain characteristics such as pattern development (similarity), rate of color change (activity), and richness of response (diversity). A study done in Antarctica used EcoPlates to determine the “microbial metabolic diversity” which led to “community-level physiological profiling of microbes in soil and ice from maritime and peninsular Antarctica” (Nagata, Application of Biolog EcoPlate). This example provides evidence that Ecoplates are an effective method and can be used to observe and measure biodiversity. 

Genetic biodiversity of bacteria species using genomic DNA 16S rRNA microbiome sequencing can be used for a wide range of microbiomes to “identify the different types of bacteria or archaea within a biological sample” (Robertson). For use in ecological microbiomes, like the rain gardens, 16S rRNA sequencing will help provide identification and classification of the microorganisms found in the soil samples gathered from the sites. Gaining insight into the diversity of the microorganisms present will help us assess the microbiome profile and the degree of pollution and contamination at each site. The 16S rRNA gene is used for sequencing due to its presence in all bacteria and archaea, high conservation, and possession of highly variable regions (Robertson). This allows for the identification of specific microorganisms by cross-referencing the 16S sequence with a molecular assay or database (Robertson, Patel et al.). For our experiment, the DNA will be extracted from the soil by mixing enzymes into the soil, adding salt and alcohol to separate the soil, and then filtering out the rest of the soil to leave behind the purified DNA. To analyze the soil we will compare the markers present in the soil with known markers for common microorganisms.