Figure 4: Genetic Biodiversity
Figure 4.1: Phyla distribution
From the taxa barplot of phyla that we constructed on Nephele using the QIIME pipeline of the 16s gene, all the phyla are quite evenly distributed across the two conditions, meaning that there is not much variation between the phyla of bacteria. The most commonly appearing bacterial phyla across 92% of soil samples globally are Proteobacteria, Acidobacteriota, Actinobacteriota, Bacteroidota, Planctomycetota, Chloroflexi, Verrucomicrobiota, Firmicutes, and Gemmatimonadota. In our samples, we have Proteobacteria, Acidobacteriota, Actinobacteria, Bacteriodota, Crenarchaeota, Planctomycetota, Chloroflexi, and Myxococcota.
Figure 4.1a
Our samples have Myxococcota and Crenarchaeota while most soil does not have these two phyla. This is what barplot 4.1a is demonstrating. Even though these phyla are present, the distribution is quite even between both conditions. This is true of all the phyla in both conditions, so there are no distinctive trends that differentiate the Straw and No Straw phyla biodiversity.
The rarefaction curve from the DADA2 pipeline does not give us much information to differentiate between the two conditions because the sequencing depths are not the same between the two conditions so there will automatically be more readings of species with the conditions that have a higher sequencing depth, so we have not included the rarefaction curve.
Figure 4.2: PCoA Plot with Binomial Distance
The PCoA plot with binomial distance shows a cluster of both conditions together with one outlier from each condition, meaning that the beta biodiversity is very similar in straw and no straw soil, so there is not any unique information from this plot. It reinforces what we already know about the beta biodiversity; it is very similar across the two conditions.
Table 4.3: Shannon Biodiversity Index
Table 4.4: Richness
Table 4.5: Evenness
To gather the Shannon Biodiversity Index (H), Richness (S), and Evenness (E) values, we used the DADA2 pipeline results from the four samples from each condition, calculated mean values, standard deviations, and performed a two tailed t-test assuming unequal variance.
The H value's t-test gave us a p-value of 0.992, which is far greater than the threshold of p=0.05 which would indicate statistical significance. The information we can deduce from this is that there is no significant difference between the two conditions' Shannon biodiversity (H) values.
The Richness (S) of the soil conditions reflects the amount of different species of microbes in the soil. A higher richness value is correlated with more species present. Our p-value was 0.957, and similarly to the H value's t-test, is much greater than p=0.05, so there is no statistical significance of these results.
The Evenness (E) of the soil shows how similar the two conditions' biodiversity is to each other. From the t-test, there is again no statistical significance because the p-value = 0.973, which is greater than 0.05.
Method
1) We began our procedure by extracting and purifying the DNA from our samples using a ZymoBIOMICS TM DNA MiniPrep kit from Zymo Resarch. The samples were lysed physically and chemically in order to disrupt the cell membrane so we can access the DNA in the nucleus, first with a bashing bead lysis tube and vortex machine, and then with two different Wash Buffers. After the initial lysis with the bashing beads, we used a centrifuge to separate the pellet from the DNA-containing supernatant. The supernatant was then run through two Zymo-Spin III-F Filters, the second time with a binding buffer added so the DNA would adhere to the second filter. Then, two rounds of wash buffers were applied to the column, centrifuged between each wash buffer. The next step was to use the ZymoBIOMICS DNase/RNase Free Water to replace the purified DNA from the column back into solution. Finally, Zymo-Spin III-HRC Columns and ZymoBIOMICS HRC Prep Solution completed the DNA collection and purification.
2) Once the purified DNA was obtained, the 16s rRNA gene was isolated using polymerase chain reaction (PCR). We did this by adding Master Mix, Forward and Reverse Primersl, DNase/RNase free water, and DNA template to each vial (one for each condition plus a control with no DNA). Once these samples went through all the replication cycles using the thermocycler, we conducted a gel electrophoresis to double check that we were successful. However, this information was not used in our final data analysis, because the DNA samples were sent to Rush University for more extensive and quicker replication, and we received the results of their larger-scale PCR using the Nephele program.
3) We used Nephele analysis to upload the sequencing files we received from Rush University and analyze alpha biodiversity, beta biodiversity, and taxonomic barplots of the phyla of microorganisms from the samples.
Evidence
The Shannon Diversity Index (H) of condition one, soil with straw, had an average (mean) of 4.54 which was the same as the average of condition two, soil without straw. Despite the average for the two conditions being the same, the standard deviation was not. Condition one had a standard deviation of 0.055 and condition two had a standard deviation of 0.234. This means that the standard deviation of condition one is lower than the standard deviation of condition two and therefore there was less variability in the H values from condition one data. The p-value from the t-test performed was p = 0.992 which is greater than the threshold value p = 0.05 meaning there is no statistical significance. Therefore, there is no statistically significant difference between the two H conditions.
The Richness (S) of condition one, soil with straw, had an average of 140.5, and condition two, soil without straw, had an average of 141.25. While similar in value the average of condition two was roughly 0.54% greater than the average of condition one. The standard deviation of condition one was 9.75 and the standard deviation of condition two was 24.35. Condition one has a lower standard deviation than condition two and therefore there is less variability in S values from condition one data. The p-value from the t-test performed was p = 0.957 which is greater than the threshold value p = 0.05 meaning there is no statistical significance. Therefore, there is no statistically significant difference between the two S conditions.
The Evenness (E) of condition one, soil with straw, had an average of 0.9191, and condition two, soil without straw had an average of 0.9194. While both have very similar average values the average of condition two was roughly 0.99% greater than condition one. The standard deviation of condition one was 0.006259 and the standard deviation of condition two was 0.015691. Condition one has a lower standard deviation than condition two and therefore there is less variability in E values from condition one data. The p-value from the t-test performed was p = 0.973 which is greater than the threshold value p = 0.05 meaning there is no statistical significance. Therefore, there is no statistically significant difference between the two E conditions.
There are no notable differences in the phyla of both conditions because the distributions of different phyla between the two conditions are very similar and there are no significant outliers.
Conclusion
The biodiversity of the soil that we measured between our two conditions (presence of straw or no straw) was calculated to be the same, with a non-significant value. Meaning, that whether or not there was a presence of straw with the soil, the soil composition was the same overall. Originally there was confidence that there would be some biodiversity within the two soil types but after performing these tests through Nephele, that has been proven wrong.
Explanation
Given that the biological composition of the soil whether there was a presence of straw or not was conclusively the same, we can confidently say that straw was not a factor in biodiversity. However, many other studies have shown that presence of straw is enough to cause significant changes in the biodiversity of the soil. (B. Liu et al.) Research shows that it is the degradation of the straw that leads to increased biodiversity. (L. Liu et al.) As such, we can infer that the straw in the soil of the Erie Street Community Garden has not been in place long enough for the straw to degrade and provide the benefit of increased biodiversity, and this is the reason why the composition of the soil are the same.