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Is Soil Microbial Diversity Impacted by Distance from Water in Prince Edward County?

Matthew Bowman

BIOL 250-50

Dr. Amorette Barber

April 16, 2017

Is Soil Microbial Diversity Impacted by Distance from Water in Prince Edward County? 2

Introduction

The environmental microbiome is being studied in various locations around the world

and in the United States; however, there are still various locations we have yet to study.

Scientists can formulate new medications, discover diverse sets of bacteria, and identify the

microbes in that specific location because of studying the environmental microbiome. Because of

studying different microbiomes, we are not only able to better the human microbiome, but evolve

environmental genomics. Not only are these microbes affecting the human microbiome, but the

environment we live in. Microbial communities are controlled solely by environmental factors

such as wind dispersion, runoff, amount of sunlight, and temperature (Heino et al. 2014).

These environmental factors can transfer microbes between distant environments

(Hooper et al. 2008). These microbes are effecting wildlife, especially the botanical life in distant

environments. The microbes are establishing new communities, embedding in the soil, and are

driving the classic plant diversity (Schnitzer 2011). Ecosystem productivity helps increase

asymptotically with plant species diversity, and determining these microbes are responsible in

determining species loss (Schnitzer 2011). Traces of certain microbial communities are being

found in soil due to water sources such as streams, rivers, and creeks. These water sources are

inputting high levels of nitrogen into the soil but low amounts of carbon (Heffernam and Fisher

2012). Microbes transport high levels of nitrogen, allowing plant roots to intake high levels of

nitrogen; however, plants are not receiving the amount of carbon required to grow properly

(Heffernan and Fisher 2012). Plant roots are now microengineering their environment and

habitats by altering the porosity and clustering properties of the soil pores (Feeney et al. 2006).


These studies sparked an interest for the microbiomes surrounding plant roots, and how

the microbes directly affect a plant’s water source. In this study, our objective is to determine

how microbial diversity changes the further a root system is from a water source. We assume

that plant roots close to a particular water source is benefitting from that microbial community,

while the plant roots further away from a particular water source is not benefitting from that

microbial community.

Materials and Methods

Environmental Sampling & Site Description

Samples of water and soil from a pond and creek was collected in February of 2017 at the

Environmental Education Center (EEC) at Lancer Park at Longwood University. Pond and creek

water was collected on the banks of the water. The soil was collected 5 and 10 meters away from

the creek and pond. The samples were then returned to the ECC and plated on the corresponding

plate at the following dilutions: 100μl of water and soil, 100μl of 1:10 dilution of water and soil

sample, and 100μl of 1:100 of water and soil sample. The water sample that was collected was

used for the direct count plate. 90μl of sterile nutrient broth was placed into the 1:10 and 1:100

tubes. 10μl of the water sample from the original tube was transferred to the 90μl of nutrient

broth in the 1:10 tube. 10μl of sample from the 1:10 dilution tube was also transferred to the 90μl

of nutrient broth in the 1:100 tube. Once samples were diluted and plated on corresponding

plates, the agar plates were incubated at room temperature.

Bacterial Growth & Characterization

Plate were checked 18-24 hours, 48 hours, and 7 days after sampling. Each day, pictures

were taken of the plates for data records. Any plate which had more than 200 colonies was

designated as “too many to count,” plates with fewer than 30 colonies were not taken into


consideration for analyzation. Plates were analyzed through number of colonies, color, size,

shape, texture, form, elevation, and margin.

DNA Isolation from Water & Soil Samples

One colony from each environmental sample was used to perform DNA analysis.

Microcentrifuge tubes were labeled for each colony and 300μl of microbead solution was added

to each tube and vortexed. The bacteria cells were then transferred to the labeled microbead tube

along with 50μl of solution MD1. The microbead tubes were heated to 65 degrees Celsius for 10

minutes and then transferred to a clean 2 mL collection tube.

900μl of solution MD3 was added to the collection tube and vortexed. 700μl was loaded

into the spin filter and centrifuged at 10,000 x g for 30 seconds and room temperature. 300μl of

solution MD4 was then transferred to the collection tube and centrifuged. A new spin filter was

added in a new 2 mL collection tube, while adding 50μl of solution MD5 to release the bound

DNA. The collection tube was then centrifuged at 10,000 x g for 30 seconds at room

temperature. Analyzation of purity and concentration of DNA was then completed using a

Nanodrop.

PCR- 16srRNA – Amplification

6μl of DNA and 50μl of PCR reaction mix (1μl of Forward Primer, 1μl of Reverse

Primer, 25μl of OneTaq 2X Master Mix, and 17μl of Nuclease-free water) were added to the

PCR tube, the PCR was added to the tube first to simplify the process. The reactions were gently

mixed by pipetting up and down. 3μl of the genomic DNA that was isolated was inserted into the

PCR tubes. One PCR tube received 3μl of DNA from one of the targets of a single molecule of

DNA, and the second PCR tube received 3μl of DNA from the other target of a single molecule


of DNA. The PCR tubes were then transferred to a PCR machine to begin thermocycling. The

PCR tubes were exposed to initial denaturation, 30 cycles, final extension, and hold. The 16S

rDNA was then expressed through universal primers: Y (C, T), M (A, C), N (G, T, A, C) and R

(A, G). The universal primers that were used have the following sequences:

Forward – 5’ GAGTTTGATYMTGGCTC 3’

Reverse – 5’ NRGYTACCTTGTTACGACTT 3’

PCR Cleanup – Restriction Enzyme Digestion – Gel Electrophoresis

Restriction Enzyme Digestion was performed to cleave a functional piece of DNA. PCR

clean-up had to be performed before restriction enzyme digestion could occur. 100μl of a

reaction was transferred to a 1.5mL microcentrifuge tube along with 5 volumes of DF Buffer.

The microcentrifuge tube was then vortexed. A DF column was then placed in a 2mL collection

tube. The mixture was then transferred to the DF column and centrifuged, discarding the flow

through. 600μl of wash buffer was then placed into the center of the DF column and centrifuged

for 30 seconds, again discarding the flow through. The dried DF column was then transferred to

a new microcentrifuge tube, adding 25μl of Electron Buffer and centrifuging for two minutes.

Once the PCR products were cleaned, a tube of MspI premixed with the buffer was

retrieved to being the restriction enzyme digestion. For each PCR product, 5μl of PCR product

and 10μl of MspI was setup for each PCR product. The samples were then mixed by pipetting up

and down with the pipettor set to 10μl and incubated for 45 minutes at 37°C. A 1.5% agarose gel

electrophoresis was then casted. 10μl of each sample was then placed into separate wells in the

gel chamber. The gel was then developed at 120 V for 30 minutes and placed under a UV camera

for visualization.


DNA Sequencing

DNA was mixed with 3μl of deionized water, 5μl of clean PCR product, and 4μl of the

sequencing primer. The sequencing primer used follows: 5’-GAGTTTGATCCTGGCTCAG-3’.

Once the DNA was sequenced, SnapGene Viewer was used to analyze the sequences. Once the

DNA sequences were edited, a BLAST – Targeted Loci (16s ribosomal RNA) search was used to

identify the prokaryote.

Results

Number of Colonies

The number of colonies for each site were counted twice, the average was then taken

between the two numbers. The number of colonies from each sampling site did vary, as

represented in Figure 1. The tree 5 meters from Buffalo creek showed the highest growth rate of

colonies, while the pond at Lancer Park showed the slowest growth rate of colonies. Once the

tree 5 meters from the pond at Lancer Park reached the 48-hour growth mark, it stabilized and

did not show a dramatic increase nor a decrease of bacteria colonies.


Figure 1. Total Number of Colonies Present on Plates after 48 hours of growth.The number of colonies on each plate were counted at 24 hours and 48 hours. Plates that were counted were multiplied by their dilution factor.

Characterization

To correctly identify the bacteria isolated from our collections sites, the bacteria had to be

characterized. First, the colors present in our samples were yellow, purple, orange, and a white

color. The number of white colonies were dominant over all the other colors present at all sites

(Fig. 2). Figure 3 represents the shapes of the bacteria that were collected. Each site showed a

variety of shapes: punctiform, spindle, circular, filamentous, irregular, and rhizoid. The Lancer

Park pond showed a high amount of irregular and punctiform bacteria; however, Buffalo Creek

showed a mixture of all shapes. Plate sample pictures from Lancer Park are represented in Figure

4, Buffalo Creek samples are represented in Figure 5.

0 24 480

500

1000

1500

2000

2500

Buffalo Creek Buffalo Creek Tree (5m) Buffalo Creek Tree (10m) Lancer Park PondLancer Park Tree (5m) Lancer Park Tree (10m)

Hours

Num

ber o

f Col

onie

s


Figure 2. Characterization of bacteria colonies through color.Color of bacteria colonies were recorded. Colonies observed were light yellow, purple, orange, and white.

Figure 3. Shape of Colonies from Collection Sites. Six different shapes were observed and recorded. Plates chosen were those that had DNA isolated from them. Diluted plates were accounted for through multiplying by the dilution factor.

punctiform spindle circular filamentous irregular Rhizoid0

200400600800

1000120014001600

Lancer Park Pond Lancer Park Tree 5m Lancer Park Tree 10mBuffalo Creek Buffalo Creek Tree 5m Buffalo Creek Tree 10m

Num

ber o

f Col

onie

s

Lancer Park Pond

Lancer Park Tree 5m

Lancer Park Tree 10m

Buffalo Creek Buffalo Creek Tree 5m

Buffalo Creek Tree 10m

0

20

40

60

80

100

120

Yellow white purple orange

Num

ber o

f Col

onie

s


Figure 4. Colonies taken from Lancer Park pond and nearby treesTop row: Pseudomonas koreensis, Flavobacterium Sp. R30-9,Bottom row: Bacillus cereus, Pseudomonas frederiksbergensis

Figure 5. Colonies take from the Buffalo Creek and nearby trees1st: Janthinobacterium lividum2nd: Pseudomonas trivialis 3rd: Pseudomonas extremoustralis

DNA Purity/Gel Electrophoresis

DNA was extracted from our isolated colonies. To send the DNA for testing we were

required to have substantial concentrations of DNA and relativity good quality DNA, which

ranges from A260 and A280. The purity of DNA collected in this study ranged from 1.71 and 2.23,

with an overall average of 1.94 (Table 1). The second pond water had a significant difference in

the concentration of DNA (26.7 ng/mL) compared to the other locations. For example, pond


water two indicated a 26.7 ng/mL concentration of DNA while Buffalo creek two indicated a 7.8

ng/mL concentration of DNA.

The sequenced PCR product for the 16s rRNA gene generated 857 bases of high-quality

base pairs that were used to identify the genus and species of the bacteria colonies. This allowed

us to separate and analyze the DNA and their fragments, based on their size and charge. Figure 6

shows the nucleic acid molecules separated by bands. The shorter molecules moved faster and

traveled farther than the longer ones because the shorter molecules traveled through the pores of

the gel. As you can clearly see in Figure 6 bands lining up, which represents a protein that

compromises that band is highly abundant.

Location Concentration of DNA (ng/mL) Purity of DNA (260/280)Pond Water 1 22.8 1.98Pond Water 2 26.7 1.95

Pond Tree, 5 meters 17.2 2.23Pond Tree, 10 meters 14.3 1.71

Buffalo Creek Water 1 15.1 1.96Buffalo Creek Water 2 7.8 1.98

Buffalo Creek Tree, 10 meters 12.3 1.76

Table 1. Concentrations and Purity of DNA Genomic DNA extraction efficiencies of the eight sampling sites. The sites were assessed by transferring up to 100 μl of a reaction produce to a 1.5 microcentrifuge tube.


Figure 6. Gel Electrophoresis results of PCR amplification of Msp1 digestion. The digested gels are 3, 5, 7, and 9 with the top being Buffalo Creek tree at 5m, tree at 10m, and the two water samples. Each had a digested and undigested form. The bottom was the Lancer Park Pond tree at 5m, tree at 10m, and the two water samples also with a digested and undigested form of each.

Genus and species classification

The prokaryotes were then identified based on the sequence of their 16s rDNA, as shown in table

two. The first set of samples came from Buffalo Creek. The prokaryote collected from creek one

was identified as Janthinobacterium lividium. This prokaryote showed a 99% identity with 863/865

bases matching and 1/865 gaps in alignment. Janthinobacterium lividium is primarily isolated from

the soils of maritime Antarctica/the Peninsula of Antarctica. The prokaryote collected from creek

two was identified as Pseudomonas trivialis. This prokaryote had a 99% identity with 954/961

bases matching and 1/961 gaps in alignment. Pseudomonas trivialis is thought to be isolated from

the rhizosphere of Hippophae rhamnoides. The final prokaryote identified at the Buffalo Creek site

was collected at a tree located 10 meters from the creek, which was identified as Pseudomonas


extremoustralis. This prokaryote had a 99% identity with 897/905 bases matching and 2/905 gaps

in alignment. Pseudomonas extremoustralis is also thought to be isolated from cold environments,

such as the Antarctica.

The second set of samples came from the pond at Lancer Park. The prokaryote collected

from pond one was identified as Flavobacterium sacchorophilum. This prokaryote had a 99%

identity with 984/993 bases matching and 1/993 gaps in alignment. Flavobacterium

sacchorophilum is commonly isolated from the artic sea waters and cold environments. The

prokaryote collected from pond two was classified as Pseudomonas koreensis. This prokaryote

had a 99% identity with 942/949 bases matching and 2/905 gaps in alignment. Pseudomonas

koreensis is native to Korea and usually found in colder environments. The third sample site at

Lancer Park was a tree 5 meters from the pond and the prokaryote found here was identified as

Pseudomonas frederiksbergensis. This prokaryote had a 100% identity with 862/862 bases

matching and 1/862 gaps in alignment. This prokaryote has only been isolated from a coal site

located in Fredericksburg, VA. The final prokaryote identified was from the fourth sampling site

at Lancer Park, the tree 10 meters from the pond. The prokaryote identified here is known as

Bacillus cereus. This prokaryote showed a 100% identity with 887/887 bases matching and

0/887 gaps in alignment. This prokaryote is a soil bacterium found near plant roots causing food

poisoning (Table 2).

Sample Site Closest BLAST % identity Gaps in Environment


Match Alignment

Buffalo Creek 1

Janthinobacterium lividum

863/865(99%) 1/865

Isolated from the soils of maritime

Antarctica/the Peninsula of Antarctica

Buffalo Creek 2

Pseudomonastrivialis

954/961(99%) 1/961

Isolated from the rhizosphere of

Hippophae rhamnoides

growing in the Lahaul-Spiti district, India

Lancer Park Pond 1

Flavobacterium sacchorophilum

984/993(99%) 1/993

Found in arctic sea water and

cold environment

Lancer Park Pond 2

Pseudomonas koreensis

942/949(99%) 1/949

Native to Korea and found in

generally colder environments

Buffalo Creek Tree (10 m)

Pseudomonas extremoustralis 897/905 (99%) 2/905

Isolated from a temporary pond

in Antarctica/cold environments

Lancer Park Pond Tree (5

m)

Pseudomonas frederiksbergensis

862/862(100%) 1/862

Isolated from a coal site in

Fredericksburg

Lancer Park Pond Tree (10

m) Bacillus cereus887/887(100%) 0/887

Soil bacterium found near roots and can cause food poisoning

Table 2. Genus and species classification using 16S rRNA sequencing The taxanomic affiliation of representative isolates was determined using 16S rRNA gene sequencing and is classified through the bacteria’s percent identity and gaps in the sequencing alignment.


Discussion

In this study, our objective was to determine how microbial diversity changes the farther

a root system is from a water source. We assumed that plant roots close to a particular water

source is benefitting from that microbial community, while the plant roots further away from a

particular water source is not benefitting from that microbial community.

The samples collected from both sites did show similarities. All the bacteria showing a

light-yellow color came from the genus, Pseudomonas. There were multiple types of this

bacteria, and each site contained at least one type of Pseudomonas. This shade of pseudomonas

was also present in other studies that have been conducted (Tribelli et al. 2015). Also, our study

did align with other studies supporting the environment of Janthinobacterium lividum (Valdes et

al. 2015). Valdes et al. 2015 found that Janthinobacterium lividum was isolated from the soils of

maritime Antarctica/the Peninsula of Antarctica, and we were also able to isolate this bacterium

from the soil. Based on this finding, we can conclude that there are microbes establishing new

communities, embedding in the soil, and are driving the classic plant diversity (Schnitzer 2011).

Also, according the Feeney et al. plants are now micro engineering their environment and

habitats by altering the porosity and clustering properties of the soils pore. We can support that

through our finding of the Bacillus cereus. This prokaryote is found near plant roots and can

cause food poison or even poison the plant. We can assume that the plants are micro engineering

their selves to be able to support this bacterium and live in a habitat with this bacteria present.

Pseudomonas trivialis has been isolated from the rhizosphere of Hippophae rhamnoides

(Mejri, Gamalero, & Souissi et al. 2012). The rhizosphere is a soil bacterium, and we also found

this bacterium in the soil; which can also support our hypothesis of species diversity (Schnitzer

2011). This bacterium also consumes nitrogen, which is a macronutrient. This supports the


theory of microbes transporting high levels of nitrogen, allowing plant roots to intake high levels

of nitrogen; however, plants are not receiving the amount of nitrogen/carbon needed to grow

properly (Heffernan and Fisher 2012).

From the bacterium, we isolated in this study, four of the seven were found to thrive in a

colder environment. When we collected our samples, and isolated them, the environment was

cold. If we had collected our samples when the environment was warm we may not have found

Flavobacterium sacchorophilum, which is a prokaryote that thrives in artic sea water and cold

environments.

Is this study, we can now conclude the microbial diversity does change the farther a root

system is from a water source. We can also conclude that some plant roots are intaking the

required number of nutrients needed to survive due to certain prokaryotes. This study does need

to be extended to fully conclude our hypothesis and rule out some of the possibilities. For

example, bacteria samples should be isolated during each season of the year. This will allow us

to see if diversity of bacteria occurs due to the season or not. Also, other sites should be sampled

as well. Sites should be extended away from each other, within a few miles’ radius. By doing

this, we will also be able to see if certain bacteria are found in other areas and see if that

influences the environment.

References


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Heffernan JB, Fisher SG. 2010. Plant–microbe interactions and nitrogen dynamics during

wetland establishment in a desert stream. Biogeochemistry. 107(1-3):379–391.

Heino J, Tolkkinen M, Pirttilä AM, Aisala H, Mykrä H. 2008. Microbial diversity and

community-environment relationships in boreal streams. Journal of Biogeography.

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microbe transfer between distant environments. PLoS ONE. 3(7):e2607.

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productivity and maintain species diversity. Plant Signaling & Behavior. 6(8):1240–1243.

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One. 10(12)

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