The Microbial Diversity of Wet and Dry Soil in Prince Edward County

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The Microbial Diversity of wet and dry soil in Prince Edward County

Debbie Pitt

Biology 250

April 2017

Introduction

Microbes are an unseen force of nature, that has recently been studied. Microbes can be found everywhere and originated long before any animals were created; they are living microscopic organisms,that are too small to be seen with the naked eye. Microbes play different roles in the environment and is anessential part in our universe. Microbes can be found just about everywhere else, particularly in soil (Huse et al. 2008).

Soil contains different sub-habitats, and these sub-habitats contain different beneficial microorganisms (Torsvik et al. 2002, Nannipieri et al. 2003). Microbial diversity in soil is important because these soil microorganisms have an essential role in regulating soil fertility, plant health, and the cycling of carbon, nitrogen, and other nutrients (Costello et al. 2006).

Many studies have been conducted by a range of different researchers on dry dirt, such as that conducted by Noah Fierer. His study examined the microbial diversity between 16 different types of environments. These environments consisted of cold deserts, hot deserts, forests, grasslands, and tundra. The study provided evidence to conclude that microbial diversity is greater in areas with high plant and species diversity. (Fierer et al. 2012)

These studies increase knowledge of microbial diversity in dry dirt, but there is a lack of information on microbial diversity in wet dirt.(Costello et al. 2006).

The purpose for this experiment was to determine the difference in microbial diversity between the wet soil and dry soil at Buffalo (fig. 8)Creekin Prince Edward county. Since the water in Buffalo Creek contains microorganisms and is a water base for many animals, it is safe to assume the wet soil will have high microbial diversity than dry soil. For this reason, it was hypothesized that the wet soil would have more microbial diversity than the dry soil.

Material and Methods

Collecting wet and dry samplesfor PCR purification and BLAST sequencing

Collecting soil samples- This study used two different soil samples: wet and dry. The wet soil sample was collected at the bank of a creek, while the dry soil sample was collected away from the water. One half gram of each sample soil was mixed with 25ml of sterile water. The samples were shaken vigorously before sitting for 3 to 5 minutes.

Plating samples- For each soil sample, two sterile tubes were used and labeled: 1:10 and 1:100. Both tubes consisted of 90µl of sterile nutrient brothbut the 1:10 tubes consisted of 10µl sample soil from the direct count tube, and the 1:100 tubes consisted of 10µl sample from the 1:10 tube. Both tubes were vortexed. For each soil sample, 3 LB plates were used and labeled as: direct count, 1:10, and 1:100. The shaken tubes were used as the direct count plate, 1:10 tubes as our 1:10 plates, and 1:100 tubes as our 1:100 plates. A pipette and the hockey stick method were used in the plating and transporting 100µl of sample to the plates. Each LB plate was incubated for 18-24 hours in a 25°C incubator. Once bacteria growth was visible, the shape, elevation, form, margin, and color of the colonies were counted.

Genomic DNA Extraction-Toidentify the species of one colony form each site, a sterile toothpick was used to collect one colony from the 1:100 plates. The toothpicks were then swirled for 15-35 seconds into microcentrifuge tubes that had 300uL of microbead solution.Three hundred microliters of the microbead solution was transferred to a microbead tube, and 50uL of MD1 solution was added. Each microbead tube was placed into a water bath set to 65°C for 10 minutes, vortexed for 10 minutes, and then centrifuged at 10,000 x g for 30 seconds. The supernatant in each tube was pipetted to a 2mL collection tube, 100uL of solution MD2 was added to each tube, vortexed for 5 seconds, then incubated for 5 minutes at 4°C. The tubes were then centrifuged for 1 minute at 10,000 x g, and the supernatant was pipetted into a 2mL collection tube. Nine hundred microliters of MD3 was added into the collection tube and vortexed for 5 seconds.The supernatant was applied to the spin filter column and centrifuged at 10,000 x g for 30 seconds: the flow through was discarded. The remaining supernatant was added to the spin filter, centrifuged at 10,000 x g for 30 seconds with flow through discarded again. Three hundred microlitersof solution MD4 was added and centrifuged for 30 seconds at 10,000 x g. Flow through was discarded. The spin filters were placed into separate 2mL collection tubes and 50uL of solution MD5 was added to the center of the white filter membrane. It was centrifuged at 10,000 x g for 30 seconds. Both spin filters were discarded and was stored in an incubator.

PCR amplification-(context 16srrna gene) In a PCR tube, 15 µl of Nuclease-free water, 25 µl of OneTaq 2X Master Mix, and 2.5µl of Primer mix (10uM Forward-5’GAGTTTGATYMTGGCTC-3’ and Reverse-5’-URGYTACCTTGTTACGACTT-3. The reactions in thePCR tubes were mixed by pipetting 7.5µl of the genomic DNA then added to the PCR tubes and then transferred to the thermocycling. The thermocycling had four steps with specific times and temperatures: initial denaturation at 94C for 4 minutes and 98C for 10 seconds, 30 cycles50C for 15 seconds and 72C for 20 seconds, final extension 72C for 5 minutes, and hold 4C.

Purification- Fiftymicrolitersof the of reaction was added to 250uL of binding buffer. The tubes were mixed well by pipetting. Each mixture was added to a spin filter and centrifuged for a minute at 16,000 x g. Flow through was discarded, then 200uL of DNA Wash Buffer was added to each spin filter. Once added, the spin filter was centrifuged for a minute at 16,000 x g. Flow through was discarded, and 200uL of DNA Wash Buffer was added to each spin filter and centrifuged for a minute at 16,000 x g again. Each spin filter column was placed in a clean 1.5ml. Twenty microliters of sterile water was added to the white filter. A nanadrop was used to measure the DNA concentration.

Restriction digest- Five microliters of PCR product and 10µl of MspI was mixed by gently pipetting it at 10µL. The tubes were incubated for 45 minutes at 37°C.

Agarose Gel Electrophoresis- In each tube 5 µL of 5X loading buffer was added and mixed by pipetting. The electrophoresis chamber was filled and the gel was covered with about 275 mL of 1X TAE buffer. The visibility and movement of the DNA was analyzed and taken for data.

BLAST (Basic Local Alignment Search Tool)- BLASTwas used in finding which type of bacteria was collected. Once on BLAST, “Target Loci” was clicked, and the sequence from purification was plugged into the search bar, then was ran through the database. The sequence from the PCR purification was matched based on similarities with hundreds of other sequences. The top 5 results were recorded and researched to identify the microbe in the chosen colony.

RESULTS

The Microbial Diversity in Wet plates

The goal of this experiment was to examine the microbial diversity between wet and dry

soil in Prince Edwards County and identify the bacteria that was isolated from each site.The sample was collected and placed on LB plates which allowed the bacteria to grow and reproduce in an aerobic environment. The Wet Direct Count (WDC) plate and the Wet 1:10 plate had the same number of colonies, with the Wet 1:100 plate having fewer. Intermingled colonies were more abundant in thethe WDC plate whiles the colonies from the Wet 1:10 and 1:100 were spaced out, but not evenly.All plates followed the same color pattern (fig 4) but not form scheme; the primary forms were: filamentous, and punctiform (fig 7). Each plate had margins that differed from the other, with entire being the similarity. Each colony on the plates were either large, medium, or small, with many medium sized colonies (fig 5). The Wet plates had a significantly higher number of colonies than the Dry plates.

The Microbial Diversity in Dry plates

The Dry Direct Count (DDC) plate and the Dry 1:10 were more similar in their form scheme with manybeing punctiforms, irregulars, circulars, and filamentous (fig. 7). The color pattern for the Dry plates were dark yellow or white with a slight few being light yellow( fig 4). The margins differed between plates, with The DDC plate having more of a selection unlike the 1:10 and 1:100. The plates had colonies that were small, small-medium, or medium (fig 6).

Identification of The Wet colony

A colony from the wet plate were sampled to identify the bacteria. The DNA

extraction provided weak results for the samples. The results for the wet dirt were

260/280= 1.19, and the concentration was 1.9 ng/uL.

The PCR extraction generated more copies of the 16S rRNA gene. The result from the wet dirt was 260/280= 5.89 while the concertation was -9 ng/ml.

The sequence of the wet dirt provided 1092 bases with over 800 good quality reads (Fig. 2). The PCR product reached about 600-700 bp while one Mspl block was about 100 bp and the second block was around 300-330 bp (Fig 3). The sequence was put into BLAST and Pseudomonas Koreensis Ps 9-14 closely resembled the original sequence with a 99% match (Fig 4).

Identification of The Dry colony

A colony from the dry plate was sampled to identify the bacteria. The results for the dry dirt were 260/280=1.30, and the concentration was 1.6 ng/ml. The Dry dirt did not produce any sequence that could be used in the SnapGene Viewer therefore it was thrown out of the experiment.

Discussion

The results from the plates showed the difference in the microbial diversity between wet and dry soil at Buffalo Creek. Based on the data provided form the plates, there is significant evidence to accept the hypothesis that, the wet dirt had more microbial diversity than the dry dirt.The microbial diversity was assessed by the bacterial morphology (i.e., color, form, abundancy, elevation, and margin). The colonies from both wet and dry either followed the punctiform, circular, or filamentous form, with little divergence. There were 3 primary colors: white, yellow and purple. All the colonies from the dry plates had white and yellow colonies while the 1:100 and 1:10 plates form the wet plates had few purple. This can provide evidence to show microbial diversity between plate, but to a point due to the limited number of purple colonies (Fig. 5).

The morphology of the colonies varied. This variation could be due to the limited amount of space on each plate, growth rate, and competition. The space given to these bacteria was limited which increased the need for survival. For survival, the bacteria needed to rapidly reproduce to keep the colony strong and many, which in turn will allow them to take in more space and food. The margin and elevation for many of the colonies were the same, and the amount colonies on each plate ranged from 30-70. These provided significant evidence to show the lack of microbial diversity in Prince Edward County but not between plates. The low levels of microbial diversity can be caused by the ecosystem these bacteria share.

The sequence of bacteria was identified by thier 16S rRNA gene. The identification sequence came from a wet plate colony, due to the poor results generated from the DNA and PCR extraction the dry dirt colony sequence could not be used.The genomic DNA extraction from the 16S rRNA gene, identified the bacteria present, because previous studies have shown, that the 16S rRNA generates better data due to its simplicity and ease to generate bacteria sequence. The results from this colony providedlittle to no evidence that there was more microbial diversity in the Wet plate versus the Dry plate.

The sequence provided many different DNA sequenceslike the original, but the top sequence was Pseudomonas Koreensis strain Ps 9-14. This bacterium sequence was

found in Korean soils that dealt with farming/agriculture. This bacterium is gram-negative with circular colonies; on a LB plate the bacterium consists of white and yellow coloring (Kwon W et al. 2003). Not many studies have been done on this bacterium but it is known to help and increase plant growth for consumption. The finding of this bacteria is not surprising because of its proximity to a farm. The purpose of different bacteria in surrounding areas can directly affect the bacteria at creek (Bonkowski M et al. 2005).

It was previously stated that the poor results generated from the PCR and DNA extractions produced data that could not be used. This is just one of the limitations that can render the results futile. Limitations, such as: using only one colony for the many extractions, not redoing the experiment, weathering, and accidently touching our face with the gloves can render results futile. Weathering played an important role in this experiment because soil temperature and water availability differs among seasons, and this can cause microbial diversity to change (Costello E et al. 2006). With the limitations to the side, these results showed soil richness at the expense of low of microbial diversity. Due to the limitations, there are steps that could clarify the experiment into greater detail (i.e., not only isolating the first choice of bacteria).

Figure 2. Wet DNA sequence had a total of 1092 bases(on top). The sequence had a rough count of 841 high quality reads. Figure 3. Lane 2 was of the wet PCR product at while lane 3 was the Mspl. The PCR product reached about 600-700 bp while one Mspl block was about 100 bp and the second was around 300-330 bp(left). Figure 4. The Wet PCR sequence had a 99% match with 2 errors. One mutation at base pair 460 and 553 the other at 516 and 610. The matched sequence has 1455 base pairs with a 0% gap(right).

Figure 4. Compares the microbial diversity between the wet and dry plate through color. Color is one way to show microbial diversity between bacteria. Blue is purple, orange is white, gray is yellow, and yellow is dark yellow. The left column is the wet and the right column are the dry plates. The order the plates follow is DC on top. 1:10 plate in the middle, and 1:100 plate on the bottom.

Figure 5. The plates shown is the number of colonies that were produced after being incubated for 2-3 days. The picture on the left is after 1 day of being incubated and the picture on the right is after 3 days. The plate allowed the colonies to rapidly produce and divide.

Figure 6. In the dry dirt direct plate, the large colonies occupied 5% of the plate while medium 90% and small 5%. The dry 1:10 plate had 10% large colonies, 85% medium colonies, and 5% small colonies. The dry 1:100 had 25% large colonies, 10% medium colonies, and 65% small colonies. In the wet dirt direct plate, the large colonies occupied 70% of the plate while medium 20% and small 10%. The wet 1:10 plate had 0 large colonies, 10% medium colonies, and 90% small colonies. The wet 1:100 had 5% large colonies, 80% medium colonies, and 15% small colonies.

Figure 7. The wet direct count plate had 47 irregulars, 2 punctiforms, and 1 filamentous. The wet 1:10 plate had 35 punctiforms, 10 circulars, and 3filamentous. The wet 1:100 plate had 32 punctiforms, 6 filamentous, and 2 irregulars.The dry dirt direct count plate had 2 punctiform, 10 irregulars, 7 circular, and 8 filamentous. The dry 1:10 plate had 15 irregulars, 5 circular, and 5 filamentous. The dry 1:100 had 2 punctiform, 6 irregulars, and 33 circular colonies.

Figure 8. sample collection location, Buffalo Creek.

Citations

Costello EK, Schmidt SK. 2006. Microbial diversity in alpine tundra wet meadow soil: novel

Chloroflexi from a cold, water-saturated environment. Society for Applied Microbiology and Blackwell Publishing Ltd.;8(8).

Huse SM, Dethlefsen L, Huber JA, Welch DM, Relman DA, Sogin LM. 2008. Exploring

Microbial Diversity and Taxonomy Using SSU rRNA Hypervariable Tag Sequencing. PLoS genetics; 4(11).

Noah F, Jonathan WL, Byron JA, Uffe NN, Scott TB, Christian LL, Sarah O, Jack AG, Diana

HW, Gregory C. 2102. Cross-biome metagenomic analyses of soil microbial communities and their functional attributes. PNAS; 109(52).

Nannipieri P, Ascher J, Ceccherini MT, Landi L, Pietramellara G, Renella G. 2003. Microbial

diversity and soil functions. European Journal of Soil Science; 54(4).

Torsvik V, Øvreås L. 2002. Microbial diversity and function in soil: from genes to ecosystems.

Elsevier Science Ltd ;5(240).

Bonkowski, M., Roy, J. (2005). Soil Microbial Diversity and Soil Functioning Affect

Competition among Grasses in Experimental Microcosms. Springer in cooperation with International Association for Ecology, 143(2)232-240

Costello., E. K., Schmidt., S. K. (2006). Microbial diversity in alpine tundra wet meadow

soil: novel Chloroflexi from a cold, water-saturated environment.Society for Applied Microbiology and Blackwell Publishing Ltd, 8(8), 1471–1486

Fierer,. N. (2012).Cross-biome metagenomic analyses of soil microbial communities and their

functional attributes. PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 109(52),21390-21395

Kwon, W., S., Kim, S., J., Park, C., I., Yoon, H., S., Park, H., D., Lim, K., C., Go, J., S.

(2003). Pseudomonas koreensis sp. nov., Pseudomonas umsongensis sp. nov. and Pseudomonas jinjuensis sp. nov., novel species from farm soils in Korea. International Journal of Systematic and Evolutionary Microbiology, (53), 21-27

Torsvik., V., Øvreås., L. (2002). Microbial diversity and function in soil: from genes to

ecosystems. Elsevier Science Ltd, (5)240–245