1. i
Inoculation of Soil With Nitrogen-Fixing Bacteria Does Not Affect the Growth or Biomass
of Poa pratensis
A Thesis
Submitted on
the 21st of February, 2016
to the Faculty
of
Rose-Hulman Institute of Technology
In partial fulfillment of the requirements for the degree of
Bachelor of Science in Biology
by
Duane Lee Kristensen II
Approved by:_____________________________________
J. Peter Coppinger, Ph.D.
Research Advisor
3. iii
ABSTRACT
Kristensen II, Duane Lee
B.S. Biology, Rose-Hulman Institute of Technology
May 2016
Inoculation of Soil With Nitrogen-Fixing Bacteria Does Not Affect the Growth or Biomass of
Poa pratensis
Thesis Advisor: Dr. Peter Coppinger
Desertification is increasing at an alarming rate throughout the United States and other at risk
countries. This process is caused by over-grazing and poor farming techniques in endangered
areas. The use of microbes to re-enrich soil has been used as biofertilizers across the world.
Biofertilizers are used to prevent the over use of fertilizers and because it is more affordable for
farmers in under-developed countries. There are three aims to this experiment: 1) to discover if
introducing a foreign nitrogen fixing bacteria into the soil of a plant will increase the biomass of
that plant 2) to discover if introducing a foreign nitrogen fixing bacteria into foreign soil will
increase the nitrogen levels of the soil 3) to show that the foreign nitrogen-fixing bacteria can
successfully colonize in foreign soil and be extracted back out of its new environment. The
nitrogen fixing bacteria used was Azotobacter which was extracted from desert soil from White
Tank Mountain Regional Park near Phoenix, Arizona. The bacteria Azotobacter both fixes
nitrogen and stimulates the growth of plants. The plant tested was Poa pratensis, Kentucky
bluegrass, because of its common appearance throughout the contiguous United States of
America. Kentucky bluegrass also grows at a quicker grass than other grasses making it an ideal
grass to use in a greenhouse under time constriction. The experiment went for thirty days to see
what the initial growth of the grass would be.
5. v
TABLE OF CONTENTS
LIST OF FIGURES ............................................................................................vi
INTRODUCTION ...............................................................................................1
MATERIALS AND METHODS ........................................................................3
RESULTS............................................................................................................5
DISCUSSION......................................................................................................7
CONCLUSIONS ...............................................................................................10
BIBLOIGRAPHY..............................................................................................11
6. vi
LIST OF FIGURES
Figure 1. Photos of Azotobacter………………………………………………………….13
Figure 2. Population Growth of Azotobacter in Soil………….……………….………...14
Figure 3. Dry Mass of Grass After Harvesting..........................………………………...15
Figure 4. Nitrogen Levels in Soil………………………………………………….……..15
7. 1
INTRODUCTION
In the past fifty years many grasslands and pastures have been turned into desert
due to overgrazing, environmental changes, over usage of the land for crops and poor soil
nutrition (“Desertification”). Poor infrastructures for farming communities has led to
extreme degradation of land in various parts of the world (“Desertification”). Developing
countries and developed countries have economies that depend on both livestock and
agriculture. In 2010, researchers estimated that 38% of the world’s land was in danger of
becoming desert (Merchant).
Desert plants have proven to naturally survive in these new environments and are
being used to help reclaim lands turned to desert and revert them back into grasslands. At
the same time there are microbes that can be used to help return nutrients, such as
nitrogen, potassium, and phosphorus, into the soil. One approach currently being taken by
scientist is to take desert adapted legumes and reestablish colonies of these plants to help
curb the progression of desertification. A limiting factor in using legumes for land
reclamation is that there are only a few species of legumes that can survive the desert or
arid environment. The use of legumes has been widespread because of their symbiotic
relations with rhizobia, a nitrogen fixing microbe. The special characteristic about
rhizobia is that not only does it fix atmospheric nitrogen but it only creates symbiotic
relations with legumes. This is done by a series of biochemical signals between the
rhizobia and the legume resulting in root nodules that house the rhizobia so that it can
safely fix the nitrogen (Buchanan et al, 2000). Another genus of microbe that can fix
8. 2
nitrogen are Azotobacter. Unlike rhizobia, azotobacter do not create symbiotic
relationship with the plant’s roots but still can provide sufficient amounts of fixed NH3.
I hypothesize that by combining both a nutrient fixing microbe and a plant used
for land reclamation will help to reverse and prevent desertification. More specifically, I
believe that introducing Azotobacter vinelandii to Achnatherum speciosa will increase the
biomass and help create more nitrogen for the soil.
9. 3
MATERIALS AND METHODS
Extraction and Isolation
In order to conduct my research I needed to extract the bacteria from the desert
soil. I did this by making a stock solution by taking half a gram of sand and placing it in a
milliliter of water and then vortexed for thirty seconds. I took 100 μL from the stock
solution and then place it in 900 μL of DI water. I then conducted three serial dilutions
and then plated each dilution on nitrogen selecting media. I used a protocol created by
Leah Cole which is used for making both plates and broth. After plating the dilutions, I
incubated the plates at 27˚C for 48 hours. I needed to incubate the plates for 48 hours
because the bacteria will grow slower on the selective media because the media is
nutrient deficient. The media is specialized to select for bacteria that can fix their own
nitrogen.
Identification of Bacteria
The bacteria was identified using microscopy. I used Carolina India Ink Stain to
identify the oval shape that is negatively stained. I cultured a plate of bacteria and then
took a loop of bacteria and smeared onto a glass microscope plate. I then placed a drop of
India Ink stain on the bacteria smear and added a cover slip. After that I added lens oil
and zoomed into 100x lens.
Sterilizing Soil
In order to have a control for the experiment, I needed to sterilize the soil. I took
the soil and placed it in a metal tray. I then placed the soil in an autoclave and ran it on
the soil cycle, the settings were at 120˚C for 2 hours. I kept the soil covered until I
planted the seeds in the soil and inoculated.
10. 4
Planting Seeds
Each pot that was planted had roughly 2.5 grams ± 0.5 grams of seed in each pot.
The seeds were spread evenly across the surface of the pot.
Measuring Dry Mass of Grass
The dirt was shaken from the roots of the grass into a garbage can and then set out
to dry for one week. It was set out to dry in order to dehydrate the grass so that I could
get an accurate measurement of the mass of the grass.
11. 5
Results
After thirty days of growth, there was no statistical difference from the grass
grown with Azotobacter compared to the grass without Azotobacter. However, the grass
with Azotobacter did have a higher yield compared to the grass without. In both plots
(Figure 3) that had Azotobacter had higher means than those without Azotobacter. These
results show that my initial hypothesis was wrong. Increasing the nitrogen in the soil, via
bacterial nitrogen fixing, does not have a statistical difference on the biomass of the
grass.
The bacteria in the soil was able to increase the nitrogen levels to twice the
starting value of nitrogen from the beginning of the experiment (Figure 4). The initial soil
levels of nitrogen were 2.5 on a 1 - 10 scale which was considered medium to low on the
scale provided by the sampling kit. The numbering is arbitrary because the soil sampling
kit didn’t have a numbering scale it had a low – saturated scale that I assigned numbers
to. It can be seen that in Figure 4 the nitrogen levels in the soils with bacteria rose to be
significantly higher than the soil without nitrogen fixing bacteria.
I was able to extract a nitrogen fixing bacteria from the environment, introduce it
to a new environment, have it successfully reproduce and then extract it from the new
environment. I was able to grow the bacteria in sterile soil and in soil with other
microbes. This shows that I have great skill in the lab and as a scientist. The sterile soil
had the largest count of bacteria compared to the soil of the non-sterilized pots. The final
12. 6
count of the sterile soil was 4.9 x 106 cfu/ml and the final count of the non-sterile soil was
2.8 x 106 cfu/ml. I saw that there was a significant drop from the initial inoculation at 1.0
x 106 cfu/ml in the sterile soil. The sharp decline was only recorded on day three post
inoculation and then the day six measurement showed that there was almost a complete
return to pre-inoculation levels (Figure 2). After that there were nothing but positive
gains throughout the experiment. Inoculating the non-sterile soil had less of a dramatic
decrease in colony count. The non-sterile count had a slow decline for nine days post
inoculation before starting to show any signs of positive growth (Figure 2).
13. 7
Discussion
The experiment shows that nitrogen levels in the soil increased with nitrogen-
fixing bacteria in the soil, but there was no significant difference between the biomass of
grass with nitrogen-fixing bacteria to grass without nitrogen-fixing bacteria. There is a
significant difference between the nitrogen levels in the soil. This can be attributed to the
nitrogen fixing bacteria in the soil. It was expected that the nitrogen levels in the soil that
was not inoculated would drop to negligible levels of nitrogen. This is expected to
happen because plants use nitrogen to grow. The nitrogen increased in the soil that was
inoculated to higher levels than expected as the final measurement was double that of the
original measurement (Figure 4).
There are a few possible reasons for why the nitrogen levels increased. The first
possible reason is that the increased bacteria count lead to the increased level of nitrogen
because of the degradation and death of the cells which contains nitrogen in themselves.
The second reason is that the bacteria are fixing nitrogen in significant amounts so that
there is a change in the nitrogen levels in the soil. One way to test this hypothesis would
be to culture a bacteria that did not fix nitrogen and inoculate the soil with the culture.
Measurements of nitrogen levels would have to be taken to see if there is any increase or
decreases. The third reason is a combination of both increased bacteria levels and
increased nitrogen levels.
I hypothesize that the reason the nitrogen levels are so high is due to the nitrogen-
fixing bacteria. The levels of bacteria in the soil provide an indicator towards how much
14. 8
nitrogen is being made in the soil. If the increased nitrogen levels are attributed to
number of bacteria in the soil then the non-sterile soil would have a higher nitrogen level
from degradation of bacteria because the non-sterile soil contains more bacteria in the
soil. It can be seen that the non-sterile soil without nitrogen fixing bacteria had
significantly lower levels of nitrogen than non-sterile soil with nitrogen fixing bacteria
(Figure 4). The small difference in biomass of the plants shows that the plants used the
same amount of nitrogen during their growing process. So in order for the nitrogen levels
to be significantly different is that the nitrogen-fixing bacteria must have been
contributing large amounts of fixed nitrogen to the soil.
The biomass of the grass with bacteria in the soil was greater than grass without
bacteria, but it was not a significant difference. The interest in biomass stems from the
benefits it can provide for land reclamation. Larger plants have more complex root
systems which lead to erosion prevention which is very important for areas that are prone
to strong winds. The increased biomass is good for the inhabitants of the environment.
Grass is used as a basic food provider for many large animals and is also used for shelter
for many smaller animals.
The initial decline in the bacteria levels is thought to be attributed to two possible
reasons. The first reason is the change in environment which may cause stress to the
Azotobacter. The change in environment may have had an adverse effect on the bacteria
due to the different nutrient levels or the environmental change from the lab to the
greenhouse. The second reason for the decline in the bacteria levels is the competition
15. 9
within the soil. The bacterial competition in the soil could have led to decrease
Azotobacter levels because the colony was grown in a lab and in a sterile environment
with no competition.
In areas that have become desert or areas that are susceptible to becoming a
desert, nutrients are limited and scarce. Plants use the scarce nutrients for growth and
there is little nutrient left for microbial growth. The minimum amounts of nutrients in the
soil are not enough to sustain a colony of microbes. Microbes, like plants, require the
basic nutrients to grow such as nitrogen, phosphorus, potassium, calcium, magnesium
and sulfur. This poses a problem for using microbes for replacing nutrients in the soil.
Due to the low nutrient levels in the soil, the bacteria would be unable to grow to produce
nutrients. The process of land reclamation would be hampered because the microbes
would be unable to grow and produce nutrients to fertilize the soil. Without the nutrients
from the microbes, the plants that are being planted to reclaim the lost land will not be
able to reclaim the land because they will not have the necessary nutrients to grow.
16. 10
CONCLUSION
In conclusion it can be seen that inoculation of the soil with Azototbacter does not
increase biomass but it does increase nitrogen levels in the soil. This means that
inoculation of soil with microbes is a viable option to re-enrich the soil. Using native
grasses would prevent invasive species from destroying the fragile ecosystem and
encourage the development of the ecosystem. One problem that would arise would be the
inability of the microbes to grow due to lack of nutrients in the soil. This would inhibit
the growth of the plants because without the nutrients the plants would not grow.
However, it might be possible to inoculate areas that are next to susceptible areas to
prevent the spread of desertification. Then through natural erosion the microbes would
spread to the affected areas. This would help spread the nutrients into the environment
and encourage the area to grow and flourish.
17. 11
BIBLIOGRAPHY
"Desertification." Scientific Facts on. Green Facts. Web. 11 Feb. 2015.
<http://www.greenfacts.org/en/desertification/>.
Merchant, Brian. "38% of World's Land in Danger of Turning into Desert." TreeHugger.
10 Feb. 2010. Web. 11 Feb. 2015. <http://www.treehugger.com/natural-sciences/38-of-
worlds-land-in-danger-of-turning-into-desert.html>.
Buchanan, Bob B., Wilhelm Gruissem, and Russell L. Jones. "Nitrogen and
Sulfur." Biochemistry & Molecular Biology of PLants. Rockville: American Society of
Plant Physiologist, 2000. 796 - 814. Print.
Perkins, Steven, and Dan Ogle. "Desert Needlegrass." Plant Fact Sheet (2008). United
States Department of Agriculture Natural Resources Conservation Service. United States
Department of Agriculture. Web. <http://plants.usda.gov/factsheet/pdf/fs_acsp12.pdf>.
Wilson, Bert. "Video of Stipa Speciosa, Desert Needle Grass." Stipa Speciosa, Desert
Needle Grass. Las Pilitas Nursery, 8 Jan. 2012. Web. 11 Feb. 2015.
<http://www.laspilitas.com/nature-of-california/plants/stipa-speciosa>.
Agarwal, L., and H. J. Purohit. "Genome Sequence of Rhizobium Lupini HPC(L)
Isolated from Saline Desert Soil, Kutch (Gujarat)." Genome Announcements (2013):
E00071-12. Print.
"Azotobacter." Azotobacter. MicrobiologyBytes. Web. 11 Feb. 2015.
<http://www.microbiologybytes.com/video/Azotobacter.html>.
"Azotobacter Vinelandii." Azotobacter Vinelandii. John Innes Centre. Web. 11 Feb.
2015. <https://www.jic.ac.uk/SCIENCE/molmicro/Azot.html>.
"The Arid Environments." Arid Zone Forestry: A Guide for Field Technicians. Rome:
Food and Agriculture Organization of the United Nations, 1989. Print.
Leah, Cole. "Isolation of Azotobacter From Soil Samples." Print.
Zahran, H. H. "Rhizobium-Legume Symbiosis and Nitrogen Fixation under Severe
Conditions and in an Arid Climate." Microbiology and Molecular Biology Reviews 4.63
(1999): 968-89. Print.
"Azotobacter Vinelandii." Wikipedia. Wikimedia Foundation. Web. 11 Feb. 2015.
<http://en.wikipedia.org/wiki/Azotobacter_vinelandii>.
Schlesinger, W. H., J. F. Reynolds, G. L. Cunningham, L. F. Huenneke, W. M. Jarrell, R.
A. Virginia, and W. G. Whitford. "Biological Feedbacks In Global
Desertification." Science 247.4946 (1990): 1043-048. Print.
19. 13
FIGURES
Figure 1. Evidence supporting the isolation of a nitrogen-fixing bacteria Azotobacter
from the desert soil and from the lab soil. The polysaccharide coating (left) is a sign of
Azotobacter to protect the bacteria from oxygen so that the nitrogenase can work
successfully. The India Ink stain (right) shows negative staining around the bacteria. The
black marks are the ink stain attaching to the polysaccharide coating and the white marks
are the bacteria.
20. 14
0.00E+00
1.00E+06
2.00E+06
3.00E+06
4.00E+06
5.00E+06
6.00E+06
0 5 10 15 20 25 30 35
cfu/ml
Day post inoculation
Figure 2. Following the decrease after the inoculation, the bacteria count in the sterile soil
(solid line) increased by day six post inoculation. The bacteria count in the non-sterile
soil (dashed line) had a slow decrease in count but began rising after day nine
21. 150
1
2
3
4
5
6
7
8
9
10
0 5 10 15 20 25 30 35
Nitrogenlevelinsoil
Day Post Innoculation
soil - inoculantsoil + inoculantsterile - inoculantsterile + inoculant
20
15
10
5
0
Drymass(g)
Figure 3. There is no significant difference between dry mass of grass with or without
bacteria in both sterile and non-sterile soil. The mean for growth with inoculant is higher
than the growth without inoculant.
Figure 4. Nitrogen levels in the soil of both sterile soil and soil that was not sterilized.
Data shows that the levels of nitrogen increased in the soil with Azotobacter showing that
the Azotobacter did have an effect on the soil levels.
