Due to the extensive urbanization trend in the United States, much of the environment will be and has been impacted. To analyze differences in urban and rural environments, we tested the presence of microbes on Maple tree surface roots in an urban environment, the University of Tennessee- Knoxville, and in a rural environment, Dean’s Woods. For the testing, five specified Maple trees were sampled at each location three separate times. The trees were swabbed along the soil line, the swabs were plated in agar, and the plates were incubated for seven days. After the incubation period, bacteria colonies were counted and compared between the two locations. We found statistically significant evidence that microbial presence on Maple tree surface roots differs between urban and rural setting. Our findings are significant because the results emphasize the effect of the massive urbanization trend on microbial presence as well as the effect that humans have on the environment.
Introduction:
According to the Environmental Protection Agency, the urban population has seen an almost five hundred percent increase since 1910 and the rural population has increased by only nineteen percent. Additionally, the EPA reports that eighty-one percent of the United States population was considered urban by 2010 (ROE 2018). The tremendous increase has lead to much experimentation regarding the effect of this mass urbanization on the environment. To name a few, researchers have tested association with urban stress and variation in microbial species composition and richness in Manhattan, the relationship between soil microbes and plants, why bacteria is attracted to tree roots, and comparisons between urban and rural microbial activity in the human gut (Reese 2015; Baltrus 2017; Currier 1976).
Uniquely, roots of plants come into direct contact with with soil and therefore come in direct contact with multiple microbes. Thus, plant roots represent microbe diversity in soil in different environments. Notably, plants in a particular area will build their phytobiomes with the flora that is found within soil, which could lead to urban plants selecting different types of bacteria than plants in forested areas (Baltrus 2017). Because phytobiomes encompass the plant, the environment and all organisms living in and around it, plant roots display a representation of the microbial presence in an area.
Additionally, bacteria undergo chemotaxis when choosing which roots the bacteria are attracted to; This is significant to the understanding of plants because it shows that plants can evolve to attract bacteria they need by producing chemicals (Currier 1976). Since plant roots attract bacteria, they are representative of microbial presence in their ecosystem. Additionally, due to attraction from chemotaxis, bacteria attach to certain plant roots, leading to root surface colonization. Therefore, by testing the surface root, many microbial communities can be detected.
Also, many studies have been conducted contrasting urban and rural settings. Richness of microbes between more urbanized and less urbanized environments in NYC don’t differ, but the community compositions do (Reese 2015). This reveals that humans have a significant impact on all sorts of ecological communities: even those that are less obvious such as communities in the soil. Because the microbial presence was found to not differ between the rural and urban setting in NYC in 2015, a different finding by our study would show how the continuing urbanization has potentially led to differing microbial richness. The study informs that biological diversity affects human health and ecological processes, so knowledge of factors that affect microbial biodiversity is important. Generally, biological diversity is limited by habitat size, which is often reduced in developed areas like cities, and by stressors or disturbances. Additionally, the study found that microbes may be able to withstand the negative effects of urbanization better than previously believed (Reese 2015). Since the University of Tennessee- Knoxville is located in a city and endures many disturbance as a college campus with thousands of students and fertilizers used to keep the campus beautiful, the study suggests that biodiversity should be restricted. Also, regarding richness of gut bacteria, more beneficial microbiota communities have been discovered in rural Russians compared to urban Russian populations (Tyakht 2014); This study reveals a difference in microbial composition between urban and rural populations.
These studies helped to answer why bacteria attach to tree roots, how bacteria varies in urban and rural settings, and interactions of microbes. Interconnecting and adding to these studies, we researched differences in microbial presence on Maple tree surface roots in rural versus urban settings. We predicted that the microbial presence in forested areas will differ from urban settings due to the higher population of diverse organisms in the forested environment, less contamination from humans in the forested environment, and more natural organisms in the forested environment. Thus, our alternate hypothesis was that there is a difference in diversity of microbial presence in urban and forested environments.
Methods and Materials:
We tested microbial presence on the surface of Maple tree roots at two different locations at the same time. The study will be conducted in the year 2018 in Tennessee during the fall season. During the testing months, September through November, the weather averages around 65 degrees Fahrenheit.
Our testing was repeated three separate times. First, we identified five Maple trees at each of the two locations and marked them with flagging tape as well as numbered them from one to five at each site. Wearing sterile gloves, we swabbed the surface of the tree root along the soil line with sterile cotton swabs. Once swabbed, we placed the swab in a transfer tube and cut the wooden stick portion, so that the cotton end of the swab fit into the transfer tube and the transfer tube was then closed to prevent contamination. Then, we platted the swab in petri dishes that were filled with Potato Dextrose Agar, which promotes bacterial growth, by gently rubbing the cotton end of the swab across the agar plate while rotating the swab. While plating the swabs, one agar plate was left open to observe bacteria in the air that could compromise the integrity of the Maple tree swabs. Once plated and labeled using a sharpie, we taped the petri dish closed with parafilm. After, we incubated the agar plates for seven days in an incubator set at 25 C. Following the seven day incubation period, we looked for colonies of bacteria and identified the various species by different physical appearances like color and texture. Once all data was gathered, a t-test was conducted using JMP Pro 13 because we were comparing the colony means between urban and rural settings.
Results:
Once the natural log of all data points were taken and the Shapiro-Wilk W test gave us a p-value greater than .05, we did not have significant evidence to reject the null, meaning that the data was normally distributed. A One-way Analysis of ln of data by location was performed. The mean ln number of colonies at the University of Tennessee-Knoxville was 3.5835 with a ln of data range from 2.1972 to 4.4188. Whereas the mean for the ln of colonies at Dean’s Woods was only 3.1781 with a natural log of data range from 1.9459 to 4.9836. The 4.9836 data point is an extreme outlier for Deans Woods. The urban setting, the University of Tennessee-Knoxville had a smaller range and a higher average number of colonies in comparison to Dean’s Woods. A t-test was performed that resulted in p-value of .0473. The graphed results are shown in figure 1.
Figure 1:
A boxplot of microbial presence at Dean’s Woods and the University of Tennessee- Knoxville. Microbial presence on Maple tree surface roots between the urban and rural environment significantly differed ( p = 0.0473).
Discussion:
We found a statistically significant difference in microbial presence between Dean’s Woods and the University of Tennessee- Knoxville. Our findings are significant because they emphasize the stress that urbanization is said to cause on microbial presence. The Maple tree surface roots provided a representation of the microbial presences due to the encompassing of their phytobiome as well as the chemotaxis that leads to root- bacteria attraction (Currier 1976; Baltrus 2017). However, unlike “Urban stress is associated with variation in microbial species composition-but not richness-in Manhattan”, we did found that microbes between more and less urbanized environments due differ and were not found to be limited by the stressors of a city (Reese 2015). Notably, the difference in finding could be caused by the increased urbanization since 2015 and confounding variables such as fertilizer could prevent the stressors of a city from affecting the microbial presence. Additionally, unlike “Rural and urban microbiota: To be or not to be?”, which found a greater richness of human gut bacteria in rural Russians compared to urban Russian populations, our experiment yeilded results that suggest the more urban setting tended to have a higher microbial presence (Tyakht 2014). The difference in microbial presence between urban and rural settings suggest urbanization and human effects on microbial richness. However, due to the close proximity of Dean’s Woods to a highway in the future, testing forested areas that are not as close to heavily traveled roads and comparing to the chosen urban area could reveal differing results. Additionally, repeating the experiment could yield variation from our findings. Because the testing was only conducted in one urban location and one rural location and was only repeated three times, the results could differ by further testing the difference in microbial presence on Maple tree surface roots in urban versus rural settings. Also, testing should be repeated because the fertilizer used by the University acts as a confounding variable toward true urbanization effects.