Comparison of Soil Properties and Bacterial Biodiversity Between Soil With Straw Placed Down and Soil Without Straw Placed Down at Erie Street Garden in West Lafayette, IN

Payton Gault, Anna Kavanaugh, and Carly Steen

Purdue University

Purpose

Food deserts, defined by the local low-income population having limited access to healthy food, can have tangible negative health effects on the residents living in them (Kelli). It was found that “those without known cardiovascular disease that live in a [food desert] is independently associated with a higher prevalence of cardiovascular risk factors, inflammation, oxidative stress, and arterial stiffness” (Kelli). Community gardens can provide access to healthy food to local communities, relieving the negative effects of food deserts on the community. Therefore, it is important to focus on gardening strategies to maximize the amount of healthy food that is produced in the garden. Putting down straw on the soil on and around the crop is one of these strategies. It has been shown that putting down straw significantly increased the soil organic carbon storage (Zhang). This can be significant for stimulating plant growth because, increased soil organic carbon storage “provides a source of nutrients through mineralisation, helps to aggregate soil particles (structure) to provide resilience to physical degradation, increases microbial activity, increases water storage and availability to plants, and protects soil from erosion” ("Soils and Carbon for Reduced Emissions"). This study will further explore the effect putting down straw has on the soil properties and bacterial biodiversity.

Soil Collection Method

The aim of this study was to find differences in the microbiomes of plants growing in straw versus plants not growing in straw. Using samples from one type of plant growing with a layer of straw and another type of plant growing without straw we compared pH, soil moisture, and the bacteria diversity to determine the impacts of straw. We collected soil samples from the Erie Street Garden in West Lafayette in early September. One sample was soil without straw placed down and the other sample was soil with straw placed down. The soil was collected by taking soil cores from each area which were taken back to the lab and frozen. We then ran four tests, where the independent variable was soil with No Straw versus soil with Straw and the dependent variables were the microbiomes, pH, and moisture content. To find out the differences in microbiomes we looked at the pH, soil moisture, and the biodiversity of the bacteria.

Soil pH

Method: We compared the soil pH of the plants growing with Straw to plants growing without Straw to see the acidity of the soil. We diluted three grams of each soil sample with deionozed water and vortexed the sample for one minute. After allowing it to sit for thirty minutes we tested the pH with a pH probe to get each samples pH values.

Analysis: The data shows that the values are very similar, the no straw condition is only 0.37% smaller than the straw condition. The straw conditions had a standard deviation of 0.27679 and the no straw conditions had a standard deviation of 0.299607. Such low values mean the data is clustered around the mean which indicates all the trials data was close to the average (“Finding and Using Health Statistics”). With the p-value of 0.766095, greater than 0.05, it indicates there is evidence our data is not statistically significant, meaning our pH data is similar for both conditions. With a p-value indicating that the data is similar and standard deviations indicating that our data is closely clustered around the average we can confidently say that there is no major pH difference between soil with straw on top vs soil without straw on top. As soil pH is impacted by climate, mineral content, soil texture, rainfall and weathering of the soils (“Soil pH”). When gathering soil samples, we noticed that the soil under the straw was more clay-like and dense while the soil without a straw layer was more crumbly. It had rained before we got to the garden so the sample without straw was compacted due to the moisture in the soil. As the rainfall and amount of water in the soil can impact the pH, these two samples may have had similar pH levels because the sample without straw took in more water than the sample protected by straw. This may have led to the sample without straw having a change in the pH making it more similar to the pH of the soil with a straw layer. One study found the areas with a straw layer held in more water than the non soil areas. When they tested the pH of the control groups, which had straw and no straw, both had a change in pH that was less than 0.001 (Zhao). It also may be that while straw has benefits on crop yield and growth, it does not impact the soils pH and will not cause a change. The slight change we saw may be due to the rain earlier in the day which is what led us to see a small difference between the pH’s, but if we monitored it over time we might see they are about the same.

Soil pH Test Results For No Straw vs. Straw:

Mean pH data of No Straw and Straw is shown with their standard deviations. We tested the pH of each sample with a pH meter, and with the data collected we found the mean pH and standard deviation of No Straw samples and Straw samples, and ran an unpaired t-test. The p-value found for the 8 trials was 0.766095 which is greater than 0.05, indicated on the graphs by n.d. (no difference).

Percent Moisture Content

Method: For soil moisture we tested the moisture content to see what straw does to soil content. Ideally plants should have between 41% to 80% moisture content (AcuRite). The soil moisture content is found by drying the soil and comparing the dried mass to the initial mass to see how much liquid was lost. If the moisture content is too low or too high it prevents the plant from thriving, seeing how the straw impacts the soil content can determine how it impacts the moisture content ("Soil Survey Standard Test Method Soil Moisture Content").

Analysis: The percent moisture of No Straw and Straw turned out to be fairly similar. The soil without straw had a percent moisture content of 29.16% and the soil with straw had a percent moisture content of 24.88%, meaning they had a difference of 4.28%. We also found that our standard deviation for No Straw was 0.055336 and Straw had a standard deviation of 0.054000. There are two ways in which we can confirm our data was similar throughout both conditions. First, after running an unpaired t-test, we found that our p-value was 0.168489854. A p-value below 0.05 indicates that there was a statistically significant difference in data while a p-value above 0.05 indicates that there was not a statistically significant difference in data. As stated earlier, our p-value was 0.168489854, meaning there was not a statistically significant difference in our data. This concludes that our data was similar for both conditions. This can be proved in another way as well, with our standard deviation values. Standard deviation is “a measure of how dispersed the data is in relation to the mean” (“Finding and Using Health Statistics”). Essentially, the smaller the values, the closer our data was to the standard deviation averages, therefore meaning our data was more similar and had minimal, if any, outliers. By using these two methods, we are confident that it can be concluded there was not much of a difference in soil moisture content between the soil with straw vs. the soil without straw. “Mulching: A Soil and Water Conservation Practice”, claims that the use of straw can significantly increase the amount of moisture retention in soil, meaning that the percent moisture content should have been higher in the soil with straw (Shirish). However, as stated earlier, we had found that there was not much of a difference in soil moisture content between the soil with straw vs. the soil without straw. The lack of difference in soil moisture may have been due to the fact that it had rained within about the last 48 hours of us taking the samples, so this makes it a bit difficult to determine whether or not having straw on the soil does actually help it retain moisture or not. A better time to take samples of the soil would most likely be about a week after it had rained in order to give the soil ample time to be rid of some of the moisture and let the moisture content levels balance out.

Percent Soil Moisture Content of No Straw vs. Straw:

Visual representations of moisture content in both conditions is shown. We also included standard deviation values as well. To gather this data, we placed a sample of soil from each condition into an oven for one week, recording masses along the way. The samples were re-measured after a week and with our measurements we calculated our standard deviation and ran an unpaired t-test, which resulted in a p-value of 0.168489854 which is greater than 0.05, indicated on the graphs by n.d. (no difference).

Functional Biodiversiy and Carbon Utilization

Method:An EcoPlate was used to analyze the makeup of the microbial community in our soil samples. We diluted the soil samples from both the No Straw soil and Straw soil and pipetted them onto the EcoPlate. One third of the EcoPlate used as a control, one third of the EcoPlate was used for the No Straw soil, and one third of the EcoPlate was used for the Straw soil. The microbes in the soil then began to consume the carbon sources in the wells of the EcoPlate. According to the directions of the manufacturer of the EcoPlate, Biolog, “formation of purple color occurs when the microbes can utilize the carbon source and begin to respire” (Biolog). The EcoPlate was put into a spectrophotometer to measure how much the wells turned purple, indicated by absorbance, and how much the microbes used up the carbon source. These values were measured before and after incubation.

The absorbance values were used to calculate the Evenness, Richness, and Shannon Diversity Index values for the No Straw and Straw soil samples. The initial values before incubation and the control readings from the sterile water were subtracted from the final incubation readings to get the corrected absorbance values. If the corrected value of a well is less than 0.25, then it is changed to zero. Richness is calculated by adding up the total amount of positive wells (corrected wells with absorbance values greater than 0.25). Shannon Diversity Index values are calculated by dividing each corrected well value by the sum of the corrected well values. The natural log is taken of each value, and then, they are multiplied by the corrected well value divided by the sum of the corrected well values to get the Shannon Diversity Index values. Evenness is calculated by dividing the Shannon Diversity Index value by the Richness value for the soil condition.

The EcoPlate has various categories of carbon sources depending on the well, including Carboxylic Acids, Amino Acids, Carbohydrates, Polymers, and Amines. If a well has an absorbance value greater than or equal to 0.25, then the well counts as 1 and counts towards the total for the carbon source type it contains. The sums for each carbon source type for each condition are added up, and then, each category is divided by the total of all the categories to get the relative utilization efficiency percent for each category. These percentages are put in a bar graph with the two bars being the two conditions, No Straw and Straw. Each color on the bar graph is a different carbon source, as indicated by the legend below the graph. If a color takes up more y-axis space, then that carbon source is more prominently utilized for the condition.

Analysis:The Richness value of the No Straw soil was 31. The Richness value of the Straw soil was 30. Therefore, the Richness value of the Straw soil was about 4% smaller than that of the No Straw soil. Because the Richness value of the No Straw soil was larger than the Richness value of the Straw soil, more carbon sources of the No Straw soil were being used in comparison to the Straw soil. The Shannon Diversity Index value for the No Straw soil was 3.3837042. The Shannon Diversity Index value for the Straw soil was 3.3731732. Therefore, the Shannon Diversity Index value for the Straw soil was about 1% smaller than that of the No Straw soil. Because our Shannon Diversity Index value was larger for our No Straw soil than our Straw soil, we had more diverse biodiversity in our No Straw soil. The Evenness value for No Straw soil was 0.9853572. The Evenness value for Straw soil was 0.9917605. Therefore, the Evenness value for the Straw soil was about 1% larger than that of the No Straw soil. Because our Evenness value for our Straw soil was larger than our Evenness value for our No Straw soil, there was a more similar abundance of different species in our Straw soil in comparison to our No Straw soil. Our p value for Richness (S) was 0.67908248. Because this value is greater than 0.05, there is not a statistically significant difference between the Richness of the microbiome of the soil with the straw and without the straw. Our p value for the Shannon Diversity Index (H) was 0.57933809. Because this value is greater than 0.05, there is not a statistically significant difference between the Shannon Diversity Indexes of the microbiome of the soil with the straw and without the straw. Our p value for the Evenness (E) was 0.61451948. Because this value is greater than 0.05, there is not a statistically significant difference between the Evenness of the microbiome of the soil with the straw and without the straw. The No Straw soil had a Relative Utilization Efficiency percentage of 29.03226% for Carboxylic Acid while the Straw soil was 26.6667%. This was the largest noticeable difference in the categories of carbon utilization with No Straw utilizing 3% relative more carboxylic acid than the Straw soil. The No Straw soil had a Relative Utilization Efficiency percentage of 19.35484% for Amino Acids while the Straw soil was 20%. The No Straw soil had a Relative Utilization Efficiency percentage of 32.25806% for Carbohydrates while the Straw soil was 33.3333%. The No Straw soil had a Relative Utilization Efficiency percentage of 12.90323% for Polymers while the Straw soil was 13.3333%. The No Straw soil had a Relative Utilization Efficiency percentage of 6.451613% for Amines while the Straw soil was 6.66667%. The Carboxylic Acid was the only category with a difference larger than 1%. The No Straw and Straw soil samples did not provide a unique soil microbiome carbon utilization in comparison to each other, with the exception of the decreased relative Carboxylic Acid utilization in the Straw soil. I am relatively certain of these results because the p values for Evenness, Richness, and Shannon Diversity Index values were all nearly 10 times that of 0.05, indicating a significant lack of a statistically significant difference between the two soil sample carbon utilization. Therefore, we are confident in this conclusion that there is not a unique soil microbiome carbon utilization between the No Straw and Straw soil samples. While gathering soil samples in the garden, there was not an observable difference in the quality of the produce being grown in the No Straw soil in comparison to the quality of the produce being grown in the Straw soil. An article titled “Fungal-bacterial diversity and microbiome complexity predict ecosystem functioning” claims that “microbial diversity is linked to ecosystem functioning, implying that communities with higher microbial richness perform better” (Wagg). Because differences in microbial diversity can cause a difference in the success of a soil community and the produce in it, the similarities in the quality of produce being grown indicates a similarity in the microbial diversity. This was verified by the lack of statistically significant differences in our experiment between the carbon utilization measures between the No Straw and Straw soil samples.

Figure A. Mean Richness of No Straw vs. Straw

Figure B. Mean Evenness of No Straw vs. Straw

Figure C. Mean Shannon Index of No Straw vs. Straw

Figure D. Relative Utilization Efficiency of No Straw vs. Straw

The mean Richness values (Figure A), the mean Evenness values (Figure B), the mean Shannon Index (Figure C) and the breakdown of the sources in which the bacteria in the samples obtained the carbon they used for their biological processes (Figure D) are shown for the No Straw and Straw soil samples . We diluted the soil samples from each condition and placed the diluted soil on the EcoPlate, and collected carbon utilization data from the plate by putting it in the plate spectrophotometer. We found the mean and standard deviations of and ran and unpaired t-test for the Richness, Evenness, and Shannon Index values. Figure A had a p value of 0.67908248, Figure B had a p value of 0.61451948, and Figure C had a p value of 0.57933809, all of which were greater than 0.05, indicated on the graphs by n.d. (no difference). For Figure D, Each of the colors indicate a different source of carbon. A larger block of color on the graph indicates more bacteria utilizing that source of carbon relative to other sources of carbon. The key for the carbon source colors are at the bottom of the graph.

Genetic Biodiversity- 16S rRNA Sequencing

Method: We used DNA 16S rRNA genomic sequencing to determine the types of bacteria present in our soil samples. To Sequence the No Straw and Straw soil samples we initially had to extract the DNA and ensure it was purified. Using ZymoBIOMICS™ DNA MiniPrep kit we lysed the cells and filtered the DNA out. We vortexed and centrifuged each soil sample in a ZR BashingBead lysis tube to break the sample up. Then they were transfered to a collection tube that had a Zymo-Spin III-F filter, which ensured only the DNA passed through, we added buffers and centrifuged. Then we transfered the sample to a Zymo-Spin IICR Column to ensure purification, and added DNase/RNase free water. We then repeated the process witn Zymo-Spin III-HRC colunm and had purified DNA (“ZymoBIOMICS™ DNA Miniprep Kit.”).

To isolate and sequence the 16S rRNA DNA we sent our samples to Rush University where they were ran through PCR tests. A second PCR was run and put into the MiniSeq system than analyzed the genetics in our soil sample. The results were sent back for us to analyze.

We then analysed the results on Nephele to get alpha biodiversity, beta biodiverity, taxonomic bar graphs, and rarefaction curves. This data allowed us to see what the makeup ouf our samples were and how similar or different each sample was from one another. Using these findings, we used the data to get Shannon Diversity Index and richness values which allowed us to calculate the evenness.

Analysis: The Shannon Diversity, Richness, and Evenness all turned out to be fairly similar. The mean Shannon index value for No Straw was 1.37% smaller than the areas with Straw. This small variation indicates that they are very similar and the diversity is about the same in both conditions. The No Straw condition had a standard deviation of 0.17719971 while Straw conditions had a standard deviation of 0.13180483. As the standard deviations are so close together it indicates that the conditions are similar and may not be very different. With a p-value of 0.597228869, greater than 0.05. This indicates that there is evidence our data is not statistically significant. This means that the diversity is similar within No Straw and Straw samples. With such a large p-value and very low standard deviations that are within 0.04 of each other it shows that the data is close to the average Shannon Diversity. Due to this, we can confidently say that there is not much difference between each conditions Shannon Diversity.

The mean richness for No Straw was 136.5 while for Straw it was 150, making the Straw samples about 10% greater than No Straw samples. The Standard deviations were less than two away from each other but fairly large with No Straws standard deviation at 21.43789 and Straws standard deviation at 23.30951. The higher the value the more unique species there are in the soil. Our data indicates that there are more species in the Straw sample than in the No Straw sample. With such large standard deviations it indicates that the samples greatly varied among types of species. The p-value, from running an unpaired t-test, was 0.418293 which is greater than 0.05. With a value greater than 0.05 we can confidently say that the conditions data is not statistically significant and the samples are similar.

The evenness was calculated by dividing the Shannon Index by the natural log of the richness value. The mean evenness of the No Straw samples was 0.921074 and the mean evenness of the Straw samples was 0.913357. No Straw conditions are only 0.84% greater than the Straw conditions. The standard deviation of No Straw was 0.009544 and the standard deviation of Straw was 0.003374. We then ran an unpaired t-test and got a p-value of 0.206901. With a p-value larger than 0.05 and such small standard deviations we can confidently conclude that the conditions are not statistically significant. Both samples are very close in size and with such small standard deviations it indicates that the evenness is very close to the mean evenness and both samples are very close to each other.

When looking at the phyla chart we can see that No Straw conditions and Straw conditions have very similar results. By finding the 16S rRNA gene, we were able to look into particular regions and gather more information from those regions (Robertson, Ruairi). This included information regarding the specific species and strains of bacteria or archaea that existed in our soil samples. Both have an abundance of Proteobacteria and Acidobacteria with a majority of Proteobacteria. Combined the two phyla make up about half of every sample indicating that both No Straw and Straw have very similar phyla make ups.

We used a rarefaction curve as a control, which allowed us to see if the conditions were similar or if there were differences. All of the sample's rarefaction curves were in a similar area regardless of if they were No Straw or Straw. This indicates that there is similar amounts of diversity as the curves level out, they are all in the same area. There were slightly less species observed in the No Straw samples than in the Straw samples but not a major difference.

Based on the large p-values for Shannon Diversity, richness, and evenness we can confidently conclude that there are not statistically significant differences between No Straw and Straw conditions. While there is slightly more variety of species in the Straw samples overall everything is fairly similar as the samples all clustered around the mean condition and the mean values are typically very close to each other.

The similarity in both No Straw and Straw samples could be due to the fact that both samples were taken from nearby each other. By taking samples near each other it could be that the diversity is similar as they are in a similar area. A study performed, looking at the genetic makeup of soil with straw, found that their straw and no straw samples had about the same make up when they tested the soil samples. They found that the Straw condition had little change compared to the No Straw condition, they were very similar (Yuan). It may be that the straw helps retain some moisture but it does not impact the biodiversity. As bacteria may move and shift in the soil to find the areas best suited for them. Their movement could explain why the soil samples were similar even with the difference in the straw layer.

Figure A. Shannon Diversity of No Straw vs. Straw

Figure B. Richness of No Straw vs. Straw

Figure C. Evenness of No Straw vs. Straw

Figure D. Biodiversity Bar Chart of No Straw vs. Straw

The mean Shannon Diversity Index (Figure A), mean richness values (Figure B), mean evenness values (Figure C), and breakdown of Phyla in the soil samples based on the genetic information (Figure D) and their standard deviations are shown for soil with No Straw and Straw. We had the samples tested in Rush Universities lab and ran the results through Nephele which analyzed the genetic biodiversity to get richness and Shannon Index, and found the phyla makeup. We calculated the evenness with the Shannon Index and richness values. We then found the mean, standard deviation, and ran an unpaired t-test on the Shannon Index, richness, and evenness. Figure A had a p-value of 0.59728869, Figure B had a p-value of 0.418793, and Figure C had a p-value of 0.206901, all of which were greater than 0.05, indicated on the graphs by n.d. (no difference). We used the Figure D to look at similarities and differences in each condition.

Discussion

Community gardens are a useful resource in working to increase access to healthy food in food deserts. Having access to healthy food options will help decrease the risk of cardiovascular disease by risk factors such as inflammation and arterial stiffness (Kelli). Optimizing the growth of plants in the gardens is important as it will increase the positive benefits of healthy food options to those in food deserts.

Each of our studies concluded with p-values of over 0.05, meaning there was no statistically significant difference in any of our data. This means our data was of fairly good quality, allowing us to analyze each study and discuss why we may have gotten the results we did.

Our pH analysis concluded that each sample had similar pH levels, which we found to be a bit unusual. However, after concluding from the percent moisture content analysis that the moisture levels were relatively even, we realized that these results may have been due to rainfall as it had rained a couple of days prior to us taking our samples. One study showed that while soil retained more moisture when covered with straw there is not much difference in the pH (Zhao). As it rained before we collected our samples the similarity in moisture levels could be due to the recent rain which would show why our study found similar moisture levels while other studies expect us to see a higher moisture content in samples of soil with Straw. However, similar to this study we found that there was a similar pH level which is expected when looking at other studies on pH levels of soil with Straw versus No Straw.

While applying straw to soil can increase nutrient availability and therefore increase the amount of biodiversity present in the soil (Liu), our functional biodiversity and carbon utilization analyses concluded that there was more biodiversity in our No Straw sample and a more similar abundance of different species in our Straw sample. These results can also tie into our results from our 16S rRNA sequencing analysis. From this analysis, we concluded that our Shannon Diversity, Richness, and Evenness were all similar to one another. It was also concluded that the diversity was about the same for both Straw and No Straw, and lastly, we concluded from the richness section of this analysis that there are more species in the Straw sample than the No Straw sample.

With these findings, the next step would be looking at the soil composition over time. Seeing if there are more noticeable differences over time by collecting samples throughout a variety of seasons and weathers may indicate more differences in the pH, Moisture Content, and Biodiversity of soil with No Straw when compared to soil with Straw.

Acknowledgements

This study would not have been possible with out Dr. Jacob Adler, Lisa Stillman, Madilyn Reid, and the teaching team; Rohan Aryan, Reagan Long, Ella Robinson, and Ethan Szajko. Thank you to Purdue University for the funding.

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