Charline Cauley

Ecology Research Project

Quantification of Soil Microbes on Eight Prairie Sites versus Eight Deciduous Tree Sites

Abstract

A comparison was tested between soil surrounding deciduous trees and soil in the Alverno prairie. This was done so by taking eight different samples per site type and growing microbes onto nutrient agar plates from the soil samples. To determine if nutrients had an effect on microbe type, nutrient tests for Phosphorus, Potassium and Nitrogen were performed. It was determined that there were more microbial community types in the prairie soil as compared to the soil surrounding the trees. Also, the only variance in nutrient amount was found to be in Phosphorus which had a lower level in the soils surrounding the trees than it did in the prairie soils.

Introduction

My hypothesis was that the number of soil microbial colony variety will be fewer under deciduous trees than on prairie sites. Knowing that microbes need specific nutrients to grow, it seems logical to deduce that fewer microbes would live where there are fewer nutrients, like in soil under trees. Reason being, as plants begin to grow, the surrounding soil is deficient in both Carbon and Nitrogen, so, the plants present would need to perform photosynthesis and nitrogen fixation (this is done by microbes) (Paul 1989). As trees begin to grow their need for nutrients increase and are not easily accessible in their immediate surrounding areas. In order for the tree, then to be provided with an adequate amount of nutrients, the roots extend in search of the desired amount. The tree has already absorbed the nutrients in its surrounding area and will grow to leech them out of further and deeper soil deposits. This means that the nutrient level surrounding the trees should be lower, which in turn means, fewer microbial species. The dominating microbial population will be that of nitrogen fixation microbes like Rhizobium, Azorhizobium or Bradyrhizobium bacteria (Atlas 1998).

Theoretical Basis for the Hypothesis

The hypothesis will be refuted or supported based on the p value and the nutritional data. For example, if the nutrient levels are found to be high under trees and the microbial species types are similar in number to that in the prairie, then it is not the nutrient level, alone which accounts for the growth. Ideally, the p value found in the comparison of data found from all sites will yield a value lower than one denoting a significant difference in colonization. Also, a lower nutrient level under trees coinciding with a lower colony population will as well be supportive of the initial hypothesis. Finally, a higher nitrogen level with fewer microbial species types will probably yield a dominating Rhizobium bacteria growth sample.

Research Methods

All samples were collected on the same day so that weather was a consistent factor with sample type; also soil was collected from the same depth and topographical area so as to ensure unbiased data collection.

  1. Soil samples were collected randomly from eight areas of each site type (Prairie and Under Tree). The soil collected from each site was collected 8 cm down from soil surface.
  2. The nutrient tests for Phosphorus, Potassium and Nitrogen were performed using the Lamar soil sampling kits.
  3. Nutrient agar plates were made (using nutrient agar bottle specifications), and were inoculated with all soil sample cultures and incubated at 27 degrees Celsius for 24 hours. These plates were inoculated directly from the soil samples, no dilutions were performed.
  4. The incubated cultures were checked and then colony types were isolated onto separate nutrient agar plates.
  5. A species count table was prepared combining the given sites and a chi-square test was performed using Microsoft Excel to determine its significance.

Results

I was surprised to find that the phenotypic characteristics of the microbes were clear enough to isolate the various varieties. I had originally thought that several microorganism tests would have to be performed to determine variety. When the original plates were grown, it was clear the varieties of microbes as well as the growth amount were more on the prairie plates than the tree plates, See Plate 1.1 1.16 below. Plate 1.1 indicates the highest growth plate of the prairie sites whereas Plate 1.2 indicates the highest growth plate of the tree sites (T = tree, P = prairie and the number indicates the site number).

From the above plates, the types of microbes were seen and isolated onto new plates of their own. These plates can be seen below, five different types were isolated from the prairie plate, P6, whereas four different types were isolated from the tree plate, T7.

 

As seen in the plates above, there is a clear phenotypic distinction between the diversity of the species. Even though five different ones were found in the prairie, three of the five were found under the trees as well. This means there were two different species types in the prairie than existed under the trees and there was one different type under the trees than in the prairie. To determine the significance of this variation, a Chi-square test was performed comparing the data of the species number per plate and the data listing the nutrient levels per site sample (see Table 1.1 and Table 1.2).

Nutrient Test Results per sample site

( P = Phosphorus, K= Potassium, N = Nitrogen)

Sample Prairie

P (4 high)

K (18 low)

N (4 high)

Sample Trees

P (4 high)

K (18 low)

N (4 high)

1

4

16

1

1

1

14

1

2

3

14

1

2

1

16

1

3

3

16

1

3

2

14

1

4

4

16

1

4

2

14

1

5

4

16

1

5

2

16

1

6

3

16

1

6

2

18

1

7

4

14

1

7

2

12

1

8

3

14

1

8

1

14

1

(Table 1.1)

The Chi- square test performed comparing each nutrient type of Prairie plates vs. Tree plates resulted in the p-values listed below:

P = 0.284, K = 0.976, N = 1

Species diversity per initial plate growth

Sample P

Species #

Sample T

Species #

1

4

1

2

2

4

2

3

3

4

3

4

4

3

4

3

5

4

5

2

6

5

6

3

7

3

7

4

8

4

8

2

(Table 1.2)

The Chi-square test performed comparing the species number per plate type resulted in:

P value = 0.4376.

Discussion

The resulted p-values do not necessarily mean the difference is significant because the number is close to one. Had the numbers been further away from one, the significance would be great. However, the plate species results are based on the phenotypic characteristics only, meaning that if more tests were performed more distinct characteristics could be made resulting in more species types. I would have liked to perform more identification tests for the microbial communities however, time was the limiting factor for this project. The plates have been stored so that in the future the actual identification can be made and future plates can be grown to further study.

The growth of the species per plate did bring up interesting support for the initial hypothesis, though, the fact that there was a more diverse population of species in the prairie versus that under the trees. Also, the nutrient variations for phosphorus led to future possibilities for study. It was surprising, based on the background information about nitrogen that higher levels were not found, especially under the trees. More information alluded to why this might be. For instance, when reading about nitrogen fixing bacteria more in depth, it was found that possibly the microbes were closer to the roots of the trees instead of the surrounding soil. There is an area clumped around roots called the "Rhizosphere" where these types of bacteria generally thrive (Atlas 1998). They could have a closer relationship with the roots making the nitrogen necessary for the tree to be absorbed directly into the roots and not in the surrounding soil. It was also found that the zone just behind the root tip is the site of maximum root exudation (where water-soluble compounds such as: sugars, amino acids, organic compounds, vitamins etc. are released) (Curl 1986). You would still expect seepage into the surrounding soil, but quit possibly it could have been to dry surrounding the tree environments to move compounds more fluidly. Another thought about the nitrogen-fixing bacteria, could be the fact that no nitrogen existed in the general area, meaning none to fix. Since the tree roots extend to absorb more nutrients from further sources, the nitrogen-fixing microbes could be at those further or deeper root tips. Finally, some nitrogen bacteria are programmed to die after differentiating (Saier 2000). Possibly the nitrogen fixers were not caught at an opportune growth time.

In general, my initial experiments did prove that there is a hypothesis to expand on which could lead to further evaluations of soil and its microbial communities.

 

Works Cited

Atlas, R.M. & Bartha, R. Microbial Ecology: Fundamentals and Applications 4th Edition. Benjamin/Cummings Publishing Co: Menlo Park, 1998.

Curl, E.A. & Truelove, B. The Rhizosphere. Springer-Verlang: Berlin, 1986.

Paul, E.A. and Clark, F.E. Soil Microbiology and Biochemistry. Academic Press, Inc.: San Diego, 1989.

Saier, M.H. Jr. Bacterial Diversity and the Evolution of Differentiation. ASM News: Vol 66. Number 6, 2000.