Valeria Guzmán, Janelle Macler, & Sandi Shahini

PURDUE UNVIERSITY

Purpose: During the last few years, rain gardens have been an active part of the management of stormwater runoff. The rain garden landscapes have proven to be attractive, useful, and sustainable stormwater management solutions for residential areas and urbanized green spaces. (Morash et al.). The ability to filter unwanted substances, like phosphorus, from stormwater, has helped the areas where people reside by adding ecological value. Microorganisms living in the soil have helped contribute to the degradation of organic materials into inorganic matter so they were able to supply nutrients to plants and help their growth (Hong et al.). Rain gardens show immense potential to be beneficial to both residential and academic areas on a university campus.

General Soil Collection Methods: 

Academic soil was collected west of and adjacent to Wetherill Building of Chemistry. Residential soil was collected in the area between the Honors College and Windsor Residence Hall. We collected several samples of soil each from academic and residential areas of campus. We used soil sampling tubes to collect the soil. We found various spots in the areas and stuck the tubes 3/4 deep and then twisted them a few times, then we proceeded to pull them out of the ground, with the soil, and then placed the collected soil into large Ziploc bags. Academic soil and residential soil were collected on the same day, January 23rd, 2024, with the academic soil being collected first, shortly followed by the residential soil. 

Method: We measured 2.95 grams of the soil for each of the conditions, added 12 mL of sterile deionized water to the soil, and closed the tube. The tube was vortexed for one minute and then incubated at room temperature for 30 minutes, being swirled every 5 minutes. Lastly, we used a pH probe to measure the concentration of hydrogen ions in each sample by placing the tip in the water.

Results:

The average soil pH for the academic condition is 8.056 with a standard deviation of 0.032, and the average soil pH for the residential condition is 7.97 with a standard deviation of 0.297. The average pH for the academic condition is 1.08% greater than that of the residential condition. The unpaired t-test assuming unequal variance gave a p-value of 0.554, which is greater than the critical threshold of 0.05, indicating that the results are statistically insignificant. 

In the soil samples for academic and residential areas, we were able to see cohesive values for the pH among all the groups. For the academic condition, the average was 8.056. The standard deviation was 0.03209. For the residential condition, the average was 7.970. The standard deviation was 0.2966. Since the p-value was 0.5536, there is no significant difference between the pH of the academic and residential conditions. These values are not unique. 

There are several explanations for the results of Figure 1. The relatively high pH values (> 7.6) for both variables, academic soil and residential soil, says something about what is happening at a chemical/molecular level. According to Smith, 2020, weathering of silicates and compounds containing Na+, Mg2+, K+ and Ca2+ is linked to silicates being hydrolyzed and subsequent OH- release, which increases soil pH. Another possible explanation for how results come from Smith, 2020. It says drought is another natural cause of soil alkalinity due to insufficient water to leach soluble salts, allowing their accumulation in the upper soil profile. Finally, it has been noted by Smith, 2020 that over liming also leads to the alkalization of soil. This is important, because Indiana is known for having a lot of limestone, and the abundance of limestone may be contributing to the higher pH values that are seen in Figure 1. 

Method: A sample of wet soil's initial mass was measured. After that, the dirt was placed in an oven to dry it out. To calculate the amount of water, the mass of the wet soil was subtracted from the total mass of the dry soil. To calculate the proportionate moisture content of the soil, the mass of the water was divided by the mass of the wet soil.

Results:

The average soil moisture content for the academic condition is 28.796 with a standard deviation of 9.058, while the average soil moisture content for the residential condition is 42.052 with a standard deviation of 19.339. The average soil moisture content of the residential condition is 45.96% greater than that of the academic condition. The unpaired t-test assuming unequal variance gave a p-value of 0.217, which is greater than the critical threshold of 0.05, indicating that the results are statistically insignificant.

In the soil samples for academic and residential areas, we were able to see cohesive values for the moisture content among all the groups. For the academic condition, the average was 28.80. The standard deviation was 9.058. For the residential condition, the average was 42.05. The standard deviation was 19.34. Since the p-value was 0.2172, there is no significant difference between the moisture content of the academic and residential conditions. These values are not unique. 

Figure 2 shows the moisture content in the 2 variables, academic and residential soil. It shows that there is not significantly more moisture content in the residential soil compared to the academic soil. It should be noted that the residential soil was collected from a garden of various healthy-looking plants that did not appear to be disturbed, while the academic soil was collected from a mostly barren garden with only a few plants in the area. A possible explanation for the difference in moisture content is the availability, or lack thereof, of plant roots and their vascular systems to intake water. Plants use water for important physiological processes and so the reasoning is that the more plants there are, the more water will be used. The reverse statement is also true. 

Figure 3: Soil Richness, Shannon Diversity Index, Evenness, Carbon Utilization

Figures 3A-D: Eco Plate Data Comparing Academic and Residential Conditions

Figures 3A-3C show the averages and respective standard deviations for species richness, Shannon Diversity Index, and species evenness for the academic and residential conditions. The unpaired t-tests assuming unequal variance for all three of these data points resulted in p-values that were greater than the critical threshold of 0.05, indicating that this data is statistically insignificant. Figure 3D shows carbon utilization efficiency by percent, divided up into the types of carbon sources. We used an EcoPlate with 96 wells and tested both conditions and a control group to observe carbon utilization and bacteria species in the samples. Featured on the graph is the indication that there is no significant statistical difference between the two conditions, denoted with "n.d.". 

Method: We used an EcoPlate with 96 wells. The EcoPlate is able to calculate how many of 31 different carbon sources were utilized in three different samples. The absorbance value found for the EcoPlate wells was used for the Species Richness, Shannon Diversity Index, Species Evenness, and Carbon Source Utilization.

Results: 

Figure 3A shows that the average species richness for the academic condition is 25.2 with a standard deviation of 2.387, and the average for the residential condition is 26.2 with a standard deviation of 2.588. The average for the residential condition is 3.968% higher than the average for the academic condition. The unpaired t-test assuming unequal variance gave a p-value of 0.5433, which is greater than the critical threshold of 0.05, indicating that the results are statistically insignificant. Figure 3B shows that the average Shannon Diversity Index for the academic condition is 3.153 with a standard deviation of 0.1140, and the average for the residential condition is 3.213 with a standard deviation of 0.0992. The average for the residential condition is 1.904% higher than the average for the academic condition. The unpaired t-test assuming unequal variance gave a p-value of 0.4011, which is greater than the critical threshold of 0.05, indicating that the results are statistically insignificant. Figure 3C shows that the average species evenness for the academic condition is 0.9781 with a standard deviation of 0.0091, and the average for the residential condition is 0.9853 with a standard deviation of 0.0033. The average for the residential condition is 0.736% higher than the average for the academic condition. The unpaired t-test assuming unequal variance gave a p-value of 0.1605, which is greater than the critical threshold of 0.05, indicating that the results are statistically insignificant. Figure 3D shows the percent carbon utilization separated by type of carbon source. The graphic shows that carbohydrates were the group that was most utilized, and amines were the least utilized.

In the soil samples for academic and residential areas, we were able to see cohesive values for species richness among all the groups. For the academic condition, the average was 25.2. The standard deviation was 2.388. For the residential condition, the average was 26.2. The standard deviation was 2.588. Since the p-value was 0.5433, there is no significant difference between the richness of the academic and residential conditions. These values are not unique. In the soil samples for academic and residential areas, we were able to see cohesive values for the Shannon Diversity Index among all the groups. For the academic condition, the average was 3.153. The standard deviation was 0.1140. For the residential condition, the average was 3.213. The standard deviation was 0.09923. Since the p-value was 0.4011, there is no significant difference between the richness of the academic and residential conditions. Therefore, we are confident that these values are not unique. In the soil samples for academic and residential areas, we were able to see cohesive values for the evenness among all the groups. For the academic condition, the average was 0.9781. The standard deviation was 0.009092. For the residential condition, the average was 0.9853. The standard deviation was 0.03315. Since the p-value was 0.1605, there is no significant difference between the richness of the academic and residential conditions. Therefore, we are confident that these values are not unique. In the carbon utilization samples, the highest percentage was about 30% for carbohydrates in both academic and residential conditions, while the lowest percentage was about 10% for amines in both conditions. We are confident that these values are not unique because the percentages are similar between the academic and residential conditions.

3A: Species richness is defined as a measure of the variety of species based simply on a count of the number of species in a particular sample (Species richness). It is a reasonable assumption that the reason why there’s no significant difference of species richness between both environments (residential and academic) is the similarity of conditions caused by abiotic factors. Factors such as pH, humidity, type of soil, etc impact how well microorganisms live in a given area (Species richness). Our group focused heavily on measuring the pH and so it is natural to discuss pH results as it pertains to average species richness. We found that the pH’s were very close to each other in both the residential and academic areas. So it’s reasonable that a possible explanation for why the average species richness values between academic and residential are not significantly different is because the similar pH allows the same number of microorganisms to thrive. 

3B: The Shannon diversity index yielded insignificant results for both academic and residential. The SDI estimates species diversity by taking into account richness and evenness (Measuring biodiversity). An explanation as to why the values between the 2 variables are insignificant follows the same logic as the explanation for A (average species richness). The similarity in pH between the two variables allows for a certain number of species to live in the soil (richness) as well as allowing for a certain variety of species to live in the soil (evenness). High pHs are more favorable to certain microorganisms, while lower pHs are more favorable to other microorganisms (The effects of ph on microbial growth). The relatively alkaline pH that we measured is more favorable to roughly the same number of species as well as the types of species and that is why there is an insignificant difference in shannon diversity index.  

3C: The average species evenness yielded insignificant results for both academic and residential. The similarity in pH between the two variables allows for a certain variety of species to live in the soil. High pHs are more favorable to certain microorganisms, while lower pHs are more favorable to other microorganisms (The effects of ph on microbial growth). The relatively alkaline pH that we measured is more favorable to roughly the same number of the types of species and that is why there is an insignificant difference in average species evenness. 

3D: There is an insignificant difference of carbon utilization among the academic and residential variables because of the similarity in species richness and evenness with both groups. Roughly equal numbers of each group would be expected to emit similar levels of carbon utilization. This is a possible explanation for why there is not a significant difference of carbon utilization between the 2 groups. 

Figure 4: Soil Richness, Shannon Diversity Index, Evenness, Taxonomy, and Taxonomic Occurrences

Figures 4A-4E – Genetic Biodiversity in Academic and Residential Areas of Purdue Campus: 

Sequencing was done by Rush University on the 16S rRNA gene, which was then processed through Nephele. Quality control, DADA2, and QIIME2 analysis were done using Nephele. Averages, standard deviations, and t-tests were done using Excel. Figures 4A-4C show the averages and respective standard deviations for Shannon Diversity Index, species richness, and species evenness for the academic and residential conditions. The unpaired t-tests assuming unequal variance resulted in p-values greater than the critical threshold of 0.05 for the Shannon Diversity Index and species richness, indicating that this data is statistically insignificant. This same t-test resulted in a p-value less than the critical threshold of 0.05 for species evenness, indicating that these results are statistically significant. Figure 4D is a taxa bar plot showing the distribution of the various phyla that were identified from the genetic evaluation of our samples for the academic and residential conditions for each sample group (4 total groups). Figure 4E shows bar graphs of the average taxonomic occurrences of two significant species, Pseudomonas and Sphingomonas, and the standard deviations. The unpaired t-test assuming unequal variance for both species occurrences resulted in a p-value less than the critical threshold of 0.05, indicating that this data is statistically significant. Featured on the graph is the indication that there is no significant statistical difference between the two conditions, denoted with "n.d.". The asterisk indicates that there was a significant difference in the data resulting from the t-test. 

Method: Genomic DNA was extracted using the ZymoBIOMICS kit and accompanying instructions. Next,  sequencing was done by Rush University on the 16S rRNA gene using the MiniSeq (Illumina) system, which was then processed through Nephele. Quality control, DADA2, and QIIME2 analysis were done using Nephele. Averages, standard deviations, and t-tests were done using Excel. 

Results: 

Figure 4A shows that the average Shannon Diversity Index for the academic condition is 4.406 with a standard deviation of 0.1703, and the average for the residential condition is 4.580 with a standard deviation of 0.2271. The average for the residential condition is 3.95% higher than the average for the academic condition. The unpaired t-test assuming unequal variance gave a p-value of 0.2700, which is greater than the critical threshold of 0.05, indicating that the results are statistically insignificant. Figure 4B shows that the average species richness for the academic condition is 133.5 with a standard deviation of 26.01, and the average for the residential condition is 149.8 with a standard deviation of 35.94. The average for the residential condition is 12.21% higher than the average for the academic condition. The unpaired t-test assuming unequal variance gave a p-value of 0.4940, which is greater than the critical threshold of 0.05, indicating that the results are statistically insignificant. Figure 4C shows that the average species evenness for the academic condition is 0.9030 with a standard deviation of 0.0051, and the average for the residential condition is 0.9178 with a standard deviation of 0.0048. The average for the residential condition is 1.64% higher than the average for the academic condition. The unpaired t-test assuming unequal variance gave a p-value of 0.005642, which is lower than the critical threshold of 0.05, which indicates that these results are in fact statistically significant. Figure 4D shows the frequencies of the various phyla of bacteria that were discovered in our sample. There are 43 different phyla observed in the sample, within two domains, Archaea and Bacteria. Figure 4E shows that the average taxonomic occurrences for the academic and residential conditions for Pseudomonas are 6.5 and 24.5 respectively, with standard deviations of 6.028 and 7.047. The average for the residential condition is 277% greater than the average for the academic condition. The unpaired t-test assuming unequal variance resulted in a p-value of 0.0085 which is less than the critical threshold of 0.05, indicating that this data is statistically significant. The average taxonomic occurrences for the academic and residential conditions for Sphingomonas are 9.25 and 25.5 respectively, with standard deviations of 4.193 and 7.853. The average for the residential condition is 176% greater than the average for the academic condition. The unpaired t-test assuming unequal variance resulted in a p-value of 0.0172, indicating that this data is statistically significant.

In the soil samples for academic and residential areas, we were able to see cohesive values for species richness among all the groups. For the academic condition, the average was 133.5. The standard deviation was 26.0064095. For the residential condition, the average was 149.75. The standard deviation was 35.94. Since the p-value was 0.4940, there is no significant difference between the richness of the academic and residential conditions. These values are not unique. In the soil samples for academic and residential areas, we were able to see cohesive values for the Shannon Diversity Index among all the groups. For the academic condition, the average was 4.406. The standard deviation was 0.1703. For the residential condition, the average was 5.580. The standard deviation was 4.580. Since the p-value was 0.2700, there is no significant difference between the richness of the academic and residential conditions. Therefore, we are confident that these values are not unique. In the soil samples for academic and residential areas, we were able to see cohesive values for the evenness among all the groups. For the academic condition, the average was 0.9030. The standard deviation was 0.005081. For the residential condition, the average was 0.9178. The standard deviation was 0.004832. Since the p-value was 0.005641, there is a significant difference between the richness of the academic and residential conditions. Therefore, we are confident that these values are unique. Proteobacteria and Acidobacteriota had the highest relative frequency percentage. The species of bacteria that had the highest number of occurrences in the academic and residential soil conditions were Sphingomonas and Pseudomonas. In the academic condition, Sphingomonas appeared an average of 9.25 times. The standard deviation was 4.193. In the residential condition, it appeared an average of 25.5 times.The standard deviation was 7.853. In the academic condition, Pseudomonas appeared an average of 6.5 times. The standard deviation was 6.028. In the residential condition, Pseudomonas appeared an average of 24.5 times. The standard deviation was 7.047. Therefore, we can say that Sphingomonas is more common in the residential condition and Pseudomonas is more common in the academic condition because both of their p-values are lower than 0.05, making their differences statistically significant.

A: The Shannon diversity index yielded insignificant results for both academic and residential. The SDI estimates species diversity by taking into account richness and evenness (Measuring biodiversity). An explanation as to why the values between the 2 variables are insignificant follows the same logic as the explanation for A (average species richness). The similarity in pH between the two variables allows for a certain number of species to live in the soil (richness) as well as allowing for a certain variety of species to live in the soil (evenness). High pHs are more favorable to certain microorganisms, while lower pHs are more favorable to other microorganisms (Measuring biodiversity). The relatively alkaline pH that we measured is more favorable to roughly the same number of species as well as the types of species and that is why there is an insignificant difference in shannon diversity index.  

B: Species richness is defined as a measure of the variety of species based simply on a count of the number of species in a particular sample (Species richness). It is a reasonable assumption that the reason why there’s no significant difference of species richness between both environments (residential and academic) is the similarity of conditions caused by abiotic factors. Factors such as pH, humidity, type of soil, etc impact how well microorganisms live in a given area (Measuring biodiversity). Our group focused heavily on measuring the pH and so it is natural to discuss pH results as it pertains to average species richness. We found that the pH’s were very close to each other in both the residential and academic areas. So it’s reasonable that a possible explanation for why the average species richness values between academic and residential are not significantly different is because the similar pH allows the same number of microorganisms to thrive. 

C: The average species evenness yielded significant results for both academic and residential. The results from the data and graph show that the residential condition contained significantly higher levels of evenness compared to the academic condition. A significantly higher evenness score indicates that the residential area is a healthier area than the academic area. This can be attested to the fact that there was visually significant plant life and growth in the residential area and hardly any in the academic area. A healthier ecosystem, no matter how large or small, generally has high evenness levels, with little to no domination from a few groups of species (NIH). This would then stand to reason that the residential area maintained more necessary abiotic factors compared to the academic area. Examples include: temperature, precipitation, humidity, soil type, pH, salinization, etc.

D: The 3 dominating species of microorganisms found in the sample were: Proteobacteria, Acidobacteriota, and Actinobacteriota. These were all roughly evenly distributed among the various sites and collection attempts. This makes sense if the findings are cross referenced to the scientific literature. Proteobacteria is a major phylum that encompasses a diverse group of organisms. It includes various shapes, sizes, and lifestyles. They are found in diverse habitats. So it stands to reason that this would be commonly found in the samples (Proteobacteria). The Acidobacteriota is widespread in various environments and are among the most abundant bacteria in soil ecosystems (Huber, K. J.). So this too stands to reason that this species would also be commonly found in the samples. The Actinobacteriota are widely distributed in various environments (Roda-Garcia, J. J.) and because of that, it also stands to reason that this would also be commonly found in the samples. 

E: It makes sense that Pseudomonas and Sphingomonas were commonly observed in the various locations. This group is known for their metabolic diversity and adaptability to diverse habitats, which means that they are suitable for both academic and residential conditions. Sphingomonas are known for their metabolic diversity, biodegradation, plant-microbe interactions, and biofilm formation (Team, M.). All of these are logical explanations as to why they are found in both conditions. 

Discussion: The overarching question that we are trying to answer is how rain gardens can be used to improve overall ecological quality in West Lafayette, Indiana. Microorganisms help supply nutrients to plants (Hong et al.), which in turn leads to a steady cycle involving increased animal health and human well-being. The sub question we are trying to answer, in hopes of getting more insight into the overarching question, is: what effect do residential and academic soil have on microbial biodiversity? Our findings showed very similar results for both variables. The evenness between Pseudomonas and Sphingomonas was about equal. This implies that both soil groups had similar biodiversity and similar abiotic conditions.  Practical use of these findings would involve use of either of these soils for widespread rain garden plantation and maintenance throughout the city of West Lafayette in order to increase the utility of the local environment, both for local residents and flora and fauna. 

Acknowledgments: Special thank you to our graduate TA, Madi Reid, for overseeing our data collection, analysis, and reports, as well as to Dr. Adler for being the Principle Investigator of the entire class-wide project for Biology 13500. This project is funded by Purdue University.  

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