The Efficiency of Denitrifying Woodchip Bioreactors

In todays society, everyone is concerned about the wellbeing of the environment. People
everywhere are trying to find new and different ways they can be environmentally friendly. Some
of the problems being addressed are how people are using products or processes which create an
excess of a harmful substances in the environment, endangering wildlife. For example, in order
for crops to grow larger in a shorter amount of time, farmers will use fertilizer on their crops.
However, many of these fertilizers contain large amounts of nitrates. Once it rains, the nitrates in
the fertilizer end up in rivers and lakes, raising the nitrate levels in these ecosystems, which
result in the death of the aquatic life. Scientists have come up with a solution: woodchip
bioreactors. These woodchip bioreactors contain anaerobic bacteria which convert the nitrates
into nitrogen gas and release the gas back into the atmosphere.
The purpose of this experiment was to determine the true percent difference of a
denitrifying bioreactor, a medium to remove nitrates from water. The hypothesis was that the
bioreactor would remove anywhere from 40 - 60% of the nitrates in the water, which would
bring the nitrates down to acceptable levels and prevent hypoxia, or oxygen starvation in plants.
In order to test this, an experimental design was created using 2-gallon buckets and a fabricated
peristaltic pump to pump fertilizer mixed in with water through the buckets, which were filled
with woodchips. The anaerobic bacteria in the woodchips converted the nitrates into nitrogen
gas, therefore lowering the nitrate levels. The results were consistent throughout the experiment,
with a 50% decrease for every trial. It was concluded that the hypothesis was accepted, since the
50% decrease received was within the hypothesized levels of 40 - 60%.

Table of Contents
Introduction..........................................................................................................................1
Review of Literature............................................................................................................3
Problem Statement...............................................................................................................9
Experimental Design..........................................................................................................10
Data and Observations.......................................................................................................14
Data Analysis and Interpretation........................................................................................21
Conclusion.........................................................................................................................26
Acknowledgements............................................................................................................30
Appendix A: Sample Calculations.....................................................................................31
Appendix B: Bucket Setup.................................................................................................35
Appendix C: Peristaltic Pump............................................................................................38
Appendix D: Nitrate Testing..............................................................................................43
Works Cited........................................................................................................................45

Auberle-Jacobs 1
Introduction
People of the world are always trying to find new and better ways to help the
environment and make the world a better place. There have been novel solutions in renewable
energy, such as the implication of wind turbines. Other solutions came from the creation of
systems to remove a harmful substance from the environment. An example of this is the
denitrifying bioreactor.
Nitrates in run-off water from farms and other agricultural fields can be harmful to the
surrounding plants and river ecosystems. The nitrates, if in one spot for a prolonged period of
time, surround the roots of plant life and prevent the plants from receiving oxygen and carbon,
which the plants need to survive, and cause the plants to suffocate to death. This phenomenon,
called hypoxia, is what products such as denitrifying bioreactors aim to prevent.
Denitrifying bioreactors, or woodchip bioreactors, are systems in which a trench in the
ground is filled with woodchips, which act as a carbon medium for denitrifying anaerobic
bacteria, and the water run-off from the fields around the bioreactor go through the woodchips.
The anaerobic bacteria in the woodchips convert the excess nitrates from fertilizers in the water
into nitrogen gas.
Several experiments and field studies have been conducted with bioreactors in order to
test their effectiveness in removing harmful nitrates from water run-off. These experiments or
studies range from models in a lab to an actual working bioreactor out in a cornfield in Iowa.
Some of these experiments were about determining the effects of changing different aspects of
the bioreactor, such as the carbon medium that was used, and observing the changes in the nitrate
levels of the water. However, it was found in most studies that the percent difference in nitrates

Auberle-Jacobs 2
was within a large range, generally a range of 30 - 70%. This large of a range does not tell how
efficient these bioreactors are.
The purpose of this experiment was to gain precision when determining the percent of
nitrates removed when nitrate-filled water goes through the woodchip bioreactor. In order to do
this, fertilizer was first mixed into a container of water. Next, the nitrate levels were measured
and then the nitrate rich water was pumped through the bioreactor. After the water went through
the woodchips, the nitrate levels were measured again. Once all the data was collected, the net
change in the nitrate levels was found and the percent difference between the two levels found.
Projects such as these can be used in the agriculture industry to help farmers and other
agriculturists make informed decisions concerning their crops. Such a large range for percent
difference as 30 - 70%, as seen in other studies (see Review of Literature), is too large a range to
determine whether or not a bioreactor is worth the cost of construction over time, since installing
one in a large scale requires an entire upheaval of a large portion of a field. Such a large range
also cannot be accounted for by environmental organizations, but a constant percent decrease
would aid those organizations in determining whether or not denitrifying bioreactors are
environmentally friendly in the long run, since the construction of one on a large scale can cause
short term damage to the surrounding area if not done correctly, if the soil is overturned
inexpertly or if the surrounding crops get buried by the soil.

Auberle-Jacobs 3
Review of Literature
Many large farms nowadays use chemical fertilizers to keep their crops healthy. The
fertilizer also helps crops grow faster and larger, which creates more produce quickly. It also
keeps the chemical levels in the soil steady. However, many kinds of fertilizer contain nitrates.
When it rains and the fertilizer gets into the water due to water run-off, so do the high levels of
nitrates that are in the fertilizer. These nitrates are harmful to other plant and marine life,
resulting in a condition called hypoxia, or a severe oxygen deficiency (Joyce). Woodchip
bioreactors have become a solution to this problem. These systems are a growing trend in the
agriculture industry. Generally, in an agricultural setting, the bioreactor would be created by
digging large trenches in the ground near fields of crops and filling them with wood chips so that
the water run-off would run through the wood chips and enter the local stream or lake with less
nitrates than it went in with (Denitrifying). The reason these bioreactors are effective is that the
wood chips contain bacteria called facultative anaerobes, which gain energy from oxygen
respiration. When oxygen is not present, however, the anaerobes resort to anaerobic respiration
or fermentation. In anaerobic respiration, the bacteria utilize other, less oxygen rich electrodes to
gain energy, such as sulfate, nitrate, or sulfur (Knowles).

Figure 1. Bioreactor Set-up

Auberle-Jacobs 4
Figure 1 above shows a typical set-up of a woodchip bioreactor. The woodchips are
contained under the soil, divesting them of oxygen. Normally, in a field setting, there is a built in
capacity control structure in order to control water flow, but in this experiment that job is
delegated to the peristaltic pump, so that structure is unnecessary (Buckner).
In a bioreactor, the system is deprived of oxygen and flooded with nitrates, forcing the
anaerobic bacteria to use anaerobic respiration and convert the nitrates to nitrogen gas, illustrated
by the chemical equation below (Powers). The anaerobic bacteria use carbon, C, nitrates, NO3,
and water, H2O, to produce nitrogen gas, N, along with bicarbonate, HCO3 -, and carbon dioxide
gas, CO2, through respiration.
+CO 2
+ 2 H 2 O 2 N 2+ 4 HCO3
5 C + NO3
Another element to the wood chip bioreactor is the rate of water flow, also known as the
hydraulic retention time. If the water moves too quickly, or the retention time is too fast, the
denitrification process has no time to occur and the container will overflow, but if it moves too
slowly, or the retention time is too low, the wood chips will become saturated and will not be
able to access all the water flowing through. In order to balance the timing correctly, the equation
below was used.
Hydraulic RetentionTime=

Bioreactor Volume
Volume
Flow Rate(
)
Time

Figure 2. Hydraulic Retention Equation

Figure 2 shows the equation used to find the hydraulic retention time for the bioreactor.
As shown, Hydraulic retention time is equal to the bioreactor volume divided by the flow rate in
volume over time (Powers 10).

Auberle-Jacobs 5
There have been multiple field studies involving bioreactors and their effectiveness.
Researchers will study actual working bioreactors by collecting data over years to analyze how
well they continue to work after being used for so many years. However, research testing these
bioreactors in a lab setting is more relevant to the experiment than the larger scale field studies or
experiments.
One research project was conducted by a graduate student, Siobhan Powers, as part of
their fulfillment for a Masters in Engineering Degree for Cornell University. This student tested
for both nitrate and phosphorus removal. Powers method for his research was to take six 12.5
gallon plastic containers, which were to act as the bioreactors. Wood chips were used to fill three
of the containers, and a mix of wood chips and biochar, a charcoal soil amendment, was used to
fill the remaining three containers. The lids were sealed and the inflow pumps were connected
and sealed to the containers.
Woodchip containers

Inflow pump

Figure 3. Powers Setup

Figure 3 is the picture Powers used in their research paper to show how they setup their
experiment (Powers). As can be seen, there are two blue pumps attached to three bioreactors
each. These pumps control the inflow rate of the water into the containers in order to ensure that
the nitrates were not filtered through too quickly for the bacteria to convert the nitrates. Powers

Auberle-Jacobs 6
pumps had an inflow rate of 100 mL/min, which was done to maintain a hydraulic retention time
of 6 hours. The water used was tested for temperature, pH, and conductivity, then filtered and
refrigerated for a predetermined amount of time.
Powers found the strictly woodchip bioreactors had an average nitrate removal of 32.1%
and the biochar-woodchip mix bioreactors had an average nitrate removal of 42.7%. Overall, the
biochar bioreactors outperformed the woodchips bioreactors, which was a trend throughout the
entire experiment. However, there was no big difference between the bioreactors in removal rates
for the phosphorus.
This experiment was useful because it detailed the procedure of an experiment with a lab
bioreactor run with constant variables, which is the basis of the experiment conducted.
In a similar experiment, a student by the name of Natasha Hoover from Iowa State
University conducted an experiment using bioreactors as well. Her experiment was based on
finding the nitrate removal rate based on hydraulic retention time and temperature. The water
being pumped through the bioreactors was brought to different temperatures for each trial, and
changed the pump speed to test hydraulic retention rate. Although her concluding results showed
that both the bioreactors succeeded in lowering the nitrate levels, it is not quite clear which
treatment - if either - worked better (Hoover).

Auberle-Jacobs 7

Figure 4. Bioreactor
Figure 4 above shows the containment for the woodchips in Hoovers trial. The vertical
aspect of the set-up allows the water to run cleanly through the woodchips and drain without
outside interaction, which may have had an effect on her results as compared to Siobhan Powers
horizontal layout.
Woodchip
Containment

Drainage
Containers

Figure 5. Hoovers Experimental Setup

Nitrate
Supplement

Auberle-Jacobs 8
The pictures in Figure 5 show Hoovers setup for her experiment. The pumps were
temperature controlled, as shown in (b) above, and each bioreactor was drained vertically as
opposed to Powers horizontal design. Note that the bioreactors on this experiment are much
more compact than that of Powers experiment.
While the experiment is being influenced by these other experiments, there are
differences that set the experiments apart. For example, while the two others experiments were
determining nitrate removal rates based on different variables, such as temperature or the type of
carbon media used, this experiment will be solely focusing on the amount of nitrates removed
from the water. In comparison, this experimental setup was based more closely off of Hoovers
than Powers, in size and shape. Powers was far too big to maintain in the space and time during
which this experiment was run, and the horizontal setup would have been much more mercurial
in terms of time for draining. The vertical setup was much more reliable, and the size was much
closer to that which we required.

Auberle-Jacobs 9
Problem Statement and Hypothesis
Problem Statement:
The purpose of this experiment was to determine the percent efficiency of woodchip
denitrifying bioreactors in removing nitrates from water. The higher the percent efficiency, the
better the bioreactor is at cleansing the water, which makes for a healthier ecosystem.
Hypothesis:
The woodchip denitrifying bioreactor will be between 40% and 60% efficient in
removing nitrates from the water
Data Measured:
The nitrate run-off from the denitrifying bioreactor will be measured in parts per million
(ppm) using a liquid nitrate test solution. The water passing through the bioreactor was a mixture
of fertilizer and the nitrate levels were raised to 10 ppm throughout the experiment. The nitrate
test solution was added to samples of the water before and after passing through the bioreactor,
and the difference was recorded. A matched-pairs t test was used to compare the means of the
original and the final nitrate levels in ppm to test for significance. Then, the percent difference
was calculated for the means to determine the effectiveness of the bioreactor.

Auberle-Jacobs 10
Experimental Design
Materials:
Miracle Gro Sphagnum Peat Moss Fertilizer
(4) 2cf bags woodchips
(2) 2 gallon plastic buckets (see Appendix
B)
Peristaltic Pump (See Appendix C)
API Freshwater Master Test Kit Nitrate
testing kit

5 gallon plastic bucket

Thermometer
Hot Plate
100mL Beaker
Tongs
ExTech DC Regulated Power Supply

Procedure:
1. Empty the bags of woodchips onto a large flat surface, spreading the woodchips out so that all of
the pieces have access to the air (Figure 7). Allow to dry for at least 24 hours. Repeat until all the
woodchips are dry (Figure 8).
2. While woodchips are drying, set up the buckets for the trials (Appendix B).
3. Fill one of the 2 gallon plastic buckets with 2 gallons of woodchips, making sure there is little to

no air left once the lid is closed.

4. Add water to a 5 gallon bucket until the bucket is almost completely full and place one end of the
hose from the pump into the bucket.
5. Start the pump and allow it to run until the water passing through the woodchips is clear, then
turn off the pump.
6. Fill the input tote with water until the bucket is almost completely full. Empty and rinse out the
drainage tote.
7. Refill the 2 gallon bucket with woodchips until full.
8. Plug in the hot plate. Fill the 100 mL beaker with water and place on the hot plate.
9. Once the water begins to boil, place the test tubes needed for the nitrate testing kit into the water
to sterilize. Allow to stay in for one minute.
10. After one minute, use the tongs to take the test tubes out of the beaker and place on a piece of
paper towel to dry.
11. The thermometer should also go in the water to sterilize after each use.

Auberle-Jacobs 11
12. Use the thermometer to measure the temperature of the water and test the nitrate levels of
the water using the API Freshwater Master Test Kit (See Appendix D for how to use).
13. Add Miracle Gro fertilizer slowly into the water. Stir and test the nitrate levels often until
the concentration reads 10 ppm.
14. Measure and record the temperature again.
15. Turn the pump back on and allow one hour to pass. After one hour, turn the pump off.
16. Test the nitrate levels of the water in the drainage bucket and measure the temperature.
Record.
17. Rinse out and dry both buckets.
18. Repeat steps 6 - 17 until 30 trials have been completed.
Diagrams:

Beaker

Hot plate
Tongs

Nitrate
Testing Kit
Thermometer
Syringe

Figure 6. Materials

Auberle-Jacobs 12
Pictured in Figure 6 are the materials used for data collection for the experiment.
Not pictured are the materials used during the duration of the experiment for pumping the
nitrates through the woodchips, and can be found in Figure 9 below, Setup.

Figure 7. Woodchips Drying

Figure 7 shows the woodchips drying on a tarp in the lab space. The woodchips
needed to be dried before each experiment so that they would not be waterlogged and to
allow for the maximum absorption of nitrates by the bacteria.

Figure 8. Wet Woodchips vs. Dry Woodchips

As shown in the figure above, drying the woodchips had an outstanding effect on
the appearance of the woodchips. The woodchips were much lighter once dried, and
much easier to manipulate.

Auberle-Jacobs 13

Nitrate fertilizer

Woodchip bucket

Peristaltic pump

Nitrate mixture
bucket
Drainage bucket

Figure 9. Setup
Figure 9 shows the setup of the experiment. Not pictured is the Extech DC Regulated
Power Supply, since it was placed behind the peristaltic pump. A diagram of the pump and power
source can be found in Appendix C.

Auberle-Jacobs 14
Data and Observations
Data:
Table 1
Data Table
Trial

Temp Before

Temp

Temp After

Nitrate

Nitrate

Nitrate

Percent

Fertilizer(C)

Before

Trial (C)

Level

Level

Level

Difference

Start of

Before

Before

After

(%)

Trial(C)

Fertilizer

(PPM)

(PPM)

(PPM)
0

21.3

21.3

19.6

10

-50

19.9

20.1

20

10

-50

20.1

20.4

19.9

10

-50

20.4

20.5

20.2

10

-50

19.6

19.7

19.8

10

-50

20.2

20.2

20.2

10

-50

20.8

20.9

20.8

10

-50

19.9

20

19.9

10

-50

19.8

19.8

19.4

10

-50

20.1

20.1

20.1

10

-50

10

18.9

18.7

20.3

10

-50

11

20.9

20.8

20.1

10

-50

12

19.6

19.5

18.5

10

-50

13

22.1

22

21.1

10

-50

14

22

22.2

19.5

10

-50

15

22

21.8

21.5

10

-50

Table 1 shows the values received in the duration of the experiment. For each trial, the
temperature was measured before the fertilizer was mixed into the water, right before the trial
started, and at the end of the trial. The temperature was measured in Celsius, and was measured

Auberle-Jacobs 15
to ensure it did not have an effect on the results of the experiment. Since all the nitrate levels
were measured to be the same for every trial, it was determined that any change in temperature
from the beginning of the trial until the end had no effect on the data. The nitrate levels were also
measured for each trial in the same intervals as the temperature, and this was measured in parts
per million (PPM). Trials 2, 7, 8, 9, and 10 were redone because of errors in nitrate readings.
Observations:
Table 2
Observation Table
Trial
0

Observations
Control trial run without fertilizer.
Started with 10 ppm of nitrate, the standard level in ground water for the US

Environmental Protection Agency.

Above standard nitrate levels, redone.

Pump had to be reset multiple times due to electrical problems.

Trial ran as planned.

Pump reset due to electrical problems.

Bucket leaked water near the nozzle.

Fertilizer sat out in the water for too long, redone.

No readable change in nitrate levels due to closeness in color of nitrate testing

values. Redone.

Above standard ppm of nitrates. Redone.

No readable change in nitrate levels due to closeness in color of nitrate testing

10

values. Redone.

11

Trial ran as planned.

12

Trial ran as planned.

13

Trial ran as planned.

Auberle-Jacobs 16
14

Large temperature difference.

Temperature recorded while fertilizer was in the water, possibly reading wrong

15

temperature.

Table 2 shows the observations taken during the trials. Again, trials 2, 7, 8, 9, and 10 had to be
redone, which was recorded in the table, and trial 9 had above standard nitrate levels, but was
kept because the percent difference was in line with the other trials. A consistent difficulty during
experimentation was the pump having problems with both construction and the power source,
which would often lead to the pump having to be restarted. However, this was all done in a
timely manner and it did not seem to have an effect on the final results.

Figure 10. Before and After of Control Trial 0

Shown in Figure 10 is the before and after nitrate levels for the control trial without any
nitrates added. The first and second picture both show a level of 0 ppm, which is consistent with
the assumption for the control.

Figure 11. Before and After of Trial 1

Auberle-Jacobs 17
Figure 11 shows the before and after nitrate levels of the first trial. The first picture shows
the before levels, which are measured in ppms, and it was closest to 10 ppm. The second picture
is the nitrate levels at the end of the trial. These levels were measured to be closer to 5 ppm.

Figure 12. Before and After of Trial 2

Figure 12 shows the results from the experiment for trial 2. In the before picture, it is
shown the nitrate level is closest to 10 ppm and in the after picture, the nitrate level is closest to 5
ppm.

Figure 13. Before and After of Trial 3

Shown in the figure above, the nitrate levels before the third trial were closest to 10 ppm,
and the levels after the trial were closest to 5 ppm.

Figure 14. Before and After Trial 4

Auberle-Jacobs 18

In Trial 14, the before and after nitrate levels of the water were close; however, the nitrate
levels before were closer to 10 ppm and the nitrate levels after were closer to 5 ppm.

Figure 15. Before and After Trial 5

In the figure above, the nitrate levels are shown for before and after the run of the trial. In
the before picture, it shows that the nitrate levels were closest to 10 ppm. In the after picture, it
shows that the nitrate levels were closest to 5 ppm.

Figure 16. Before and After Trial 6

The nitrate levels before experimentation were closest to 10 ppm and the nitrate levels
after experimentation were closest to 5 ppm.

Figure 17. Before and After Trial 7

Auberle-Jacobs 19
The figure above shows that the nitrate levels before the trial were found to be closest to
10 ppm and the nitrate levels after were found to be 5 ppm.

Figure 18. Before and After Trial 8

Before experimentation, the nitrate levels were 10 ppm, and after, the nitrate levels were
5 ppm.

Figure 19. Before and After Trial 9

As seen in the figure above, the nitrate levels before were found to be 10 ppm and the
nitrate levels after the experiment were found to be 5 ppm.

Figure 20. Before and After Trial 10

Figure 20 above shows the nitrate levels before the trial, which were closer to 10 ppm,
and the nitrate levels after, which were closer to 5 ppm.

Auberle-Jacobs 20

Figure 21. Before and After Trial 11

The picture above shows the nitrate levels for trial 11. The levels before the trial were
closest to 10 ppm and the levels after were closest to 5 ppm.

Figure 22. Before and After Trial 12

Figure 22 above shows pictures of the before and after nitrate levels for trial 12. The
levels before were closer to 10 ppm and the levels after were closer to 5 ppm.

Auberle-Jacobs 21
Data Analysis and Interpretation
Once all the data was collected, the data was analyzed to determine whether the
hypothesis would be accepted or rejected. To analyze the data, a matched-pairs t test was
used. This statistical test was chosen because the nitrate levels before were compared to
the nitrate levels after, both measured in parts per million (ppm). Sample calculations for
each equation used in the matched-pairs t test can be found in Appendix A.
Before the matched-pairs t test was used, the data had to be determined to be
normal. This was done by graphing the data and examining the graphs, looking for any
outliers in the data when it is plotted.
12
10
8
Nitrate Levels (PPM)

6
PPM Before

PPM After

2
0
0 2 4 6 8 10 12 14 16
Trial

Figure 23. Graph of the Data

As seen above, the graph of the data above contains the points for the nitrate levels both
before and after the water went through the bioreactor for all 15 trials. There are no abnormalities
in the data; in fact, all the data was the same for every trial.

Auberle-Jacobs 22
12
10
8
Percent Difference (%)

6
4
2
0
0 2 4 6 8 1012
Trial

Figure 24. Percent Difference for Each Trial

The figure above shows the percent differences for each trial. Because the nitrate levels
were the same for every trial, this means the percent difference for each was the same. The
nitrate levels for every trial decreased by 50%.
Since the data was found to be normal, the match-pairs t test can be performed. First,
however, the assumptions have to be met. For a matched-pairs t test, the assumptions include
that the data is normal and the experiment was a simple random sample. The first assumption
was met; the data was found normal in the figures above. The second assumption, that the
experiment was a simple random sample, was met simply because since each trial was done
exactly the same way, there was no need for any randomization.
Hypothesis:
Ho: = 0
Ha: > 0
The hypothesis above shows the hypothesis for the matched pairs test, where represents the
difference between the two data sets.

Auberle-Jacobs 23

Table 3.
Matched-Pairs Data Table
Pair

PPM Before

PPM After

Difference (di)

Mean
Difference (di
D)2

10

10

10

10

10

10

10

10

10

10

10

11

10

12

10

13

10

14

10

15

10

The table above shows the data used in the matched pairs test. The difference, di, between the
data sets in the pair were consistent throughout the trials with a difference of 5 ppm. The mean
difference, (di - D)2, adds up to zero.

Auberle-Jacobs 24
d iD

=
For the matched-pairs t test, the standard deviation, , needs to be found. To find the
standard deviation, the square root of the sum of the mean differences is divided by 1 less than
the number of paired values, n.
When the calculation was worked out, the standard deviation for the data was calculated
to just be 0. This makes sense since there was no difference in the results of each trials.

1
(nN)
n
SE=
n1

For the statistical test to work, the standard error, SE, needs to be found. To find the
standard error, the standard deviation, , is multiplied by the square root of the product of the
inverse of the number of pairs, n, and the difference of the number of pairs and number of
populations in the sample, N, divided by one less than the number of pairs.
The result came to be zero because each data point was the same, so there was no
deviation, and therefore no error.
The degrees of freedom, DoF, need to be found in order to figure out the p-value after the
t value has been calculated. To find the DoF, it is simply one less than the number of pairs, n.
For this experiment, the degrees of freedom were found to be 14.
Finally, the t value was calculated. To find the t value, the hypothesized difference
between population means, dh, was taken from the difference of the means for each pair, (1

Auberle-Jacobs 25
2). This value was then divided by the standard error, SE, which was shown how to be found
earlier.
The calculations for the degrees of freedom and the t-value follow the earlier trend in
which the end result is zero. Since the standard deviation and the standard error are both zero, the
probability that the results were achieved by chance alone is non-existent.
Ho was rejected because the p-value of 0 is below the alpha level 0.05. There is strong evidence
that bioreactor was successful in lowering the nitrate levels in the water. The chance of receiving
these results by chance alone is zero.

Auberle-Jacobs 26
Conclusion
Denitrifying woodchip bioreactors have been designed as a solution to the excessive
nitrate levels in water systems such as rivers and lakes. It is important to remove these excess
nitrates because the nitrates deprive the aquatic life, both plant and animal, from oxygen. The
purpose of this experiment was to determine the efficiency of woodchip bioreactors. For this
experiment, the hypothesis, that the woodchip denitrifying bioreactor will be between 40% and
60% efficient in removing nitrates from the water, was accepted. For every trial in the
experiment, there was a decrease in the amount of nitrates (measured in ppm) of 50%. This
percent difference is between the predicted 40 - 60%; therefore, the hypothesis was accepted.
In a bioreactor, nitrate saturated water is pumped through a container of woodchips in order to
lower the nitrate levels. There are anaerobic bacteria in the woodchips that, when deprived of
oxygen, consume nitrates and converts the compound into nitrogen gas (Woodchip Bioreactors).
While the nitrates in the water and soil are harmful, nitrogen gas being released into the system
had very little negative environmental effects, since 78% of the earths atmosphere contains
nitrogen gas already (Composition of the Atmosphere). In a typical farm bioreactor, the
woodchips are buried underground and deprived of oxygen naturally. In order to simulate this, he
woodchips used in the experiment were placed in a container with an airtight lid for the duration
of the trial, and once the water began to pump through, the oxygen in the system was quickly
consumed and could not be replaced. Therefore, the bacteria in the system reverted to nitrogen
consumption, releasing the nitrogen gas along with the water and lowering the nitrate levels in
the reactor.
The conversion of nitrates into nitrogen gas is shown in the equation below. The
anaerobic bacteria uses carbon, C, nitrates, NO3, and water, H2O, to produce nitrogen gas,

Auberle-Jacobs 27
N2, along with bicarbonate, HCO3 -, and carbon dioxide gas, CO2, through respiration
(Powers).
+CO 2
+ H 2 O 2 N 2 +4 HCO3
5 C+ 4 NO 3
While the experiment did run smoothly overall, there were a few design flaws which
could have affected the data. One such flaw was that the way the pump was built prevented it
from going at a slower speed than it did, which may have prevented more accurate results from
occurring.. If the pump had gone at a slower speed, the water would have gone through the
bioreactor more slowly. This would, in turn, give the bacteria in the woodchips more time to
absorb the nitrates in the water and convert more of it into nitrogen gas. This would have shown
a greater percent difference in the data. Another flaw was the nitrate level testing kit. Although it
did work throughout the experiment, a more accurate test, such as one with a direct numerical
reading of the nitrate levels, would have led to more accurate results because there would have
been less guessing on what the nitrate level was exactly. There was the problem of colors on the
key being extremely close together, which made it difficult to tell the colors, and thus the levels,
apart.

Auberle-Jacobs 28

Figure 25. Nitrate Color

As can be seen in Figure 25, the colors which represent 5.0 ppm, 10 ppm, and 20 ppm are
all very close in color. This could lead to a mix-up in colors, which would then lead to
inaccuracy in the data. Even though this was a problem in the experiment, it was still obvious
throughout the experiment that the nitrate levels were decreasing because the color of the water
became lighter between the beginning of the experiment and the end.

Figure 26. Before and After Nitrate Levels of Trial 7

Figure 26 above shows the results from trial 7. It is very obvious the coloring of the
before picture, the picture on the left, is darker than the after picture, the picture on the right. On
the color chart, it shows that the lighter the color, the lower the nitrate levels. So, it can be

Auberle-Jacobs 29
assumed that the lighter color of the water in the picture on the right means there is a lower level
of nitrates.
The data for the experiment was analyzed using a matched-pairs t test, which compared
the mean values of two groups, usually a final and initial group. The test concluded the average
percent difference of 50% had no chance of being achieved by chance alone if there was no
difference in the groups, which there was not in this experiment.
This experiment did not forge any new ground in the field of research or the field of
denitrification. However, this experiment did have more consistent results. Instead of the
difference in nitrate levels being anywhere from 30 - 70% as in other lab experiments, this
experiment had a consistent 50% decrease in nitrate levels. These results show that it is possible
to get a smaller range in the percent difference, which could help researchers who are working
with bioreactors in the real world work more toward getting more consistent results.
Denitrifying bioreactors are of great interest in research, especially in rural areas with a
lot of farming. Scientists are always looking for new ways to improve these systems in order to
further decrease the amount of nitrates in water. Some further research that could be beneficial to
bioreactors is testing different types of mediums, such as a mixture of different woodchips or
woodchips mixed with some other substance to see if there is a difference in nitrate removal
based on the carbon medium itself. Temperature and volume of the bioreactor system could also
be manipulated to see if those values have any outstanding effects.
The hypothesis for this experiment, that the percent difference between the final and
initial nitrate levels would be between 40-60%, was accepted. The anaerobic bacteria in the
woodchips succeeded in lowering the nitrate value by 50% by converting the nitrates in the water

Auberle-Jacobs 30
into nitrogen gas. Although there were some complications, the experiment ran as planned and
produced the expected outcomes, resulting in a successful experiment overall.

Auberle-Jacobs 31
Acknowledgements
Thank you to Mark Supal for help with the paper and for use of the workshop, Greg
McMillian for use of his DC Power Supply, and for consultations with the electronics portion of
the experiment. Jamie Hilliard, who helped us refine our experiment idea and pushed us to
question everything, and Scot Acre and Christine Tallman who helped with the statistics, were
also a great help in the process. We would also like to thank Patricia Jacobs-Soulard for
providing the nitrate testing kit, R. Daniel Auberle for help in building the peristaltic pump, and
finally Jacob Stanczyck for his aid in working with the motor and associated electronics parts.

Auberle-Jacobs 32
Appendix A: Sample Calculations
To analyze the data for this experiment, a matched-pairs t test was used. Here, sample
calculations will be performed in order to show how everything was calculated in the data
analysis.
Table 4.
Experiment Data
Pair
PPM Before
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15

PPM After
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10

Difference (di)
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5

5
5
5
5
5
5
5
5
5
5
5
5
5
5
5

Mean
Difference (D)
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0

The table above shows the data used in the matched pairs test. The difference
between the data sets in the pair were consistent throughout the trials, so the final
difference calculation, (d-D)2, adds up to zero.
For the matched-pairs t test, the standard deviation, , needs to be found. To find
the standard deviation, the square root of the sum of the mean differences is divided by 1
less than the number of paired values, n.
(d iD)2
=
n1

Auberle-Jacobs 33
To show how to use this formula, a sample calculation using the data from pair 15 will be
used to replace the corresponding variables, as shown below.
2

( d D )
=
i

( 55 )

n1

151

0
14

Figure 27. Standard Deviation Sample Calculation

The sample calculation above shows how the standard deviation was found. For this
calculation, the final result came to be 0, which fits the data because there is no deviation
between the data sets.
For the statistical test to work, the standard error, SE, needs to be found. To find
the standard error, the standard deviation, , is multiplied by the square root of the
product of the inverse of the number of pairs, n, and the difference of the number of pairs
and number of populations in the sample, N, divided by one less than the number of
pairs.

1
(nN )
n
SE=
n1

1
( nN )
n
SE=
n1

1
( 1530 )
15
0
=0
151

Figure 28. Standard Error Sample Calculation

The calculation above shows the standard error of the data sets. The result came to be
zero because each data point was the same, so there was no deviation, and therefore no error.
DoF=n1

Auberle-Jacobs 34
DoF=n1
151=14
Figure 29. Degrees of Freedom Sample Calculations
The degrees of freedom, DoF, need to be found in order to figure out the p-value
after the t value has been calculated. To find the DoF, it is simply one less than the
number of pairs, n.

t=

t=

( 12 )d h d i d h
=

SE

( 12 )d h
SE

SE

d id h 50
=
=0
SE
0

Figure 30. Degrees of Freedom and T-value Sample Calculations

The calculations for the degrees of freedom and the t-value follow the earlier
trend in which the end result is zero. Since the standard deviation and the standard error
are both zero, the probability that the results were achieved by chance alone is nonexistent.

Auberle-Jacobs 35
Appendix B: Bucket Set-up
Materials:
(2) 2 gallon buckets with lids

Drill bit

(4) 1/2" x 3/4 Nylon hose barb adapters

Compass

Drill

Peristaltic pump construction

Procedure:
1. Take the lids off the buckets and mark the exact center on
each of the lids using the compass (Figure 30).
2. Using a drill bit, drill a hole into the center of each lid.
3. Thread a nylon hose barb adapter into each hole (Figure
31).
4. Mark a spot near the very bottom of each bucket to put the
remaining adapters for drainage.
5. Use the drill bit to drill the holes, then thread in the
adapters (Figure 32)
6. Replace the lids, making sure the seal is airtight. One
bucket will be used during the trial while the other will be filled with woodchips, then
replaced in order to quicken the pace of trials.
7. Attach one end of the tubing from the peristaltic pump onto
the adapter in the lid of one of the buckets
8. Ensure that the motor is connected to the ExTech DC
Power Supply so that the pump runs from one end of the tubing into the tubing connected
to the bucket. If not, switch the positive and negative wires.
Diagrams:

Auberle-Jacobs 35

Figure 31. Marking the Bucket Lid

Figure 31 above shows the correct set-up for drilling the hole in the lid, along with the
proper markings in order to determine the exact center of the lid.

Figure 32. Nylon Hose Adapter Connection

Figure 32 shows how the nylon hose adapter should be threaded into the lid of the bucket.
Notice the adapter portion of the piece is facing outwards from the bucket.

Auberle-Jacobs 36

Figure 33. Complete Bucket Set-up

Figure 33 shows the completed set-up of one bucket. Notice how the hose from the pump
is attached to the top of the bucket. If the hose is not cut long enough, a connector can be bought
and used to connect two pieces of hose together.

Auberle-Jacobs 37
Appendix C: Peristaltic Pump
Materials:
(1) 12 diameter Springform cake pan
(2)
(3)
(4)
(5)

(4) 2 casters
5 ft silicone hose
A block of wood
1 washers

(6) (2) washers

(7) Various screws and bolts
(8) #12 wood screws
(9) Compass
(10)
Drill
(11)
Drill bits
(24)
Procedure:

(12)
(13)
(14)
(15)
(16)
(17)
(18)
(19)
(20)
(21)
(22)
(23)

Dremel tool
7/16 rachet
7/16 wrench
Screwdriver
Framing square
Circular saw
(2) -20 t-nuts
Allen wrenches
Motor and matching key
ExTech DC Power Supply
Wires
Miscellaneous wood scrap

1. Lay the block of wood in the cake

pan with the casters and determine the size the wood needs to be in order to
accommodate the casters.
(25)
2. Use the framing square to mark the
cuts, then use the circular saw to cut the wood to size.
(26)
3. Use the framing square to mark the
exact center of your square and drill a hole at that point. Drill another hole large enough
to seat the t-nuts so that the top of the square is level.
(27)
4. Insert the t-nuts.
(28)
5. Take the wood block and lay it face
up on a horizontal surface and line the casters up against each side. Mark the center of the
holes on each caster.
(29)
6. Drill the holes for the screws using a
5/16 drill bit at an angle so the screws will not hit each other.
(30)
7. Line up the casters and screw the
#12 wood screws into the wood
(31)
8. Take out the bottom of the springform cake pan and find
the center using the compass.
(32)
9. Drill out the center of the cake pan, then smooth out the
edges using the dremel tool.

Auberle-Jacobs 38
(33)
10. Insert the bolt through the hole in the bottom of the cake
pan using washers as a buffer, adding washers to the opposite side as well.
(34)
11. Place the wooden block with casters onto the bolt,
tightening on with nuts buffered by washers.
(35)
12. Take the top portion of the cake pan and mark where the
holes for the tubing should go through the sides.
(36)
13. Draw in shapes near the bottom of the pan wall at the same
level as where the casters will be, then cut using the dremel tool. Grind away the sharp
edges, again using the dremel tool.
(37)
14. Insert the tubing through the holes just cut.
(38)
15. Place the cake pan wall and tubing over the bottom portion
with the wooden block and casters, making sure the tubing is in between the pan wall and
the caster wheels (Figure 4).
(39)
16. Attach the motor key to the top of the wooden block by
drilling holes into the block through the edge of the key. Secure using wood screws
(Figure 35).
(40)
17. Cut out two small block of wood the same size and line
them up around the outside edge of the cake pan, level with the top of the key.
(41)
18. Screw the two blocks into the pan walls, making sure not to obstruct the movement of the
casters or tubing (Figure 36).
(42)
19. Cut a thin strip of sheet wood long enough to fit over the diameter of the cake pan. Find
the exact center and drill a hole.
(43)
20. Place the end of the motor that attaches to the key through the hole. Secure the motor
with the bolts included in the motor set, or any bolts that fit.
(44)
21. Insert the end of the motor into the key and tighten with an allen wrench. Attach the long
strip of wood to the two blocks on the outside of the cake pan (Figure 37).
(45)
(46)
22. Connect the motor to the ExTech DC Power Supply using the wires.
(47)

Auberle-Jacobs 39

Springform
cake pan

Tubing
Allen
Wrench
Casters

Blocks
of wood
Motor

Fasteners
(48)

Motor Key

Figure 34. Materials

Figure 34 above shows the main materials used in the creation of the peristaltic
pump.
(49)

(50)

Figure 35. Pump Base Construction

Auberle-Jacobs 40
(51)

Figure 35 shows the completion of the first portion of the pump

construction. Notice how the casters are flush against each side of the wood block with
some space in between the wheels and the cake pan walls for the tubing.

(52)
(53)

Figure 36. Pump Base with Motor Key

The figure above shows the correct placement of the motor key onto the pump

base. It is imperative that the key be placed in the exact center of the block and the pan,
otherwise the pump will not work.

(54)
(55)

Figure 37. Support Block Placement

Figure 37 above shows the correct placement of the blocks on the outside of the

cake pan used to support the motor.

Auberle-Jacobs 41
(56)

Figure 38. Completed Peristaltic Pump

The figure above shows the completed peristaltic pump, complete with motor. The
pump, when connected to a DC Power Supply, should be able to slow down and speed up
in respect to the current.
(57)

Auberle-Jacobs 42
(58)
(59)
(60)
(61)
(62)
(63)

Materials:

(64)

Procedure:

Appendix D: Nitrate Testing

20 mL Syringe
API Freshwater Master Nitrate Testing Kit
5 mL test tube

1. Fill the syringe with water.

2. Use the syringe to fill a test tube with 5 mL of water, which is indicated by the white line
(65) on the test tube.
3. Shake Bottle #1 and then put 10 drops of the solution into the test tube.
4. Shake Bottle #2 and then put 10 drops of the solution into the test tube.
5. Shake the test tube and allow to sit for 5 minutes. The color of the test tube should start
(66) out as a yellowish color and may change depending on the nitrate level of the
water.
6. After 5 minutes, compare the color the water in the test tube is now to the different colors
(67) on the chart. These different colors indicate different nitrate levels and are notes
underneath the color and measured in parts per million (ppm).
(68)
(69)
(70)

(71)
(72)
(73)
(74)

Diagrams:

Figure 39. Filling the Syringe

The syringe does not need to be filled with a certain amount of water. It

just needs to have enough in order to fill the test tube with 5 mL of water.

Auberle-Jacobs 43

(75)
(76)
(77)
(78)

Figure 40. Test Tube

The figure above shows the test tube once filled with water. As can be

seen, there is a white line which wraps around the test tube to show where the 5 mL mark
is. This is as full as the test tube should be.

(79)
(80)
(81)

Figure 41. Nitrate Testing Solution Bottle #1

(82)
(83)
(84)

Figure 42. Nitrate Testing Solution Bottle #2

(85)
(86)

Figure 43. Test Tube with Testing Solution

Auberle-Jacobs 44
Figure 43 shows about what color the water in the test tube should be after

(87)

the testing solution has been added. The color of the water may change after the five
minute waiting period if the nitrate levels are above 0 ppm.

(88)
(89)
(90)

Figure 44. Comparing the Solution to the Color Chart

(91)

The figure above shows how to compare the color of the water after five

minutes has passed to the color chart. For this test, the color of the water is yellow, which
is 0 ppm.
(92)

Auberle-Jacobs 45
(93)

Works Cited

(94)

1. Buckner, John. "Bioreactors Take On 'The Dead Zone'" AgWeb. Farm Journal, 7 Jan.

(96)

2012. Web. 1 Dec. 2015. <http://www.agweb.com/article/bioreactors_take_on_the

(95) _dead_zone/>.
2. Composition of the Atmosphere | Climate Education Modules for K-12. Composition
of the Atmosphere | Climate Education Modules for K-12. NC State University. 9 Aug
2013. Web. 19 Nov, 2015. <https://climate.ncsu.edu/edu/k12/.AtmComposition>.

(97)

3. Costello, Christine, W. Michael Griffin, Amy E Landis, and H. Scott Matthews.

"Impact of Biofuel Crop Production on the Formation of Hypoxia in the Gulf of Mexico."
Environ. Sci. Techno 43.20 (2009): 7985-991. Environ. Sci. Technol. American Chemical
Society. Web. 25 Sept. 2015. <http://pubs.acs.org/doi/abs/10.1021/es9011433>.

(98)

4. "Denitrifying Woodchip Bioreactors." Denitrifying Woodchip Bioreactors. Iowa State

University, 2015. Web. 25 Sept. 2015. <http://agwatermgmt.ae.iastate.edu/content/
(99)

denitrifying-woodchip-bioreactors>.

(100) 5. Hoover, Natasha. "Denitrification Woodchip Bioreactor Two-phase Column Study:

Evaluation of Nitrate Removal at Various Hydraulic Retention times and Effect of
Temperature on Denitrification Rates." Iowa State University, 2012. Web. 25 Sept. 2015.
<http://lib.dr.iastate.edu/cgi/viewcontent.cgi?article=3355&context=etd>.
(101) 6. Joyce, S. The Dead Zones: Oxygen-Starved Coastal Waters. Environmental Health
Perspectives 108.3 (2000): A120A125. Print.
(102)
Print.
(103)

7. Knowles, R. Denitrification. Microbiological Reviews 46.1 (1982): 4370.

Auberle-Jacobs 46
(104) 8. Powers, Siobhan. "Nitrate Reduction and Phosphate Uptake in Lab-Scale Denitrifying
Bioreactors with Woodchip and Biochar Media." Cornell University, 3 May 2012. Web.
25 Sept. 2015.<https://ecommons.cornell.edu/bitstream/handle/1813/33243
(105) /finalpaper.pdf?sequence=2>.
(106)
(107) 9. Woodchip Bioreactors. Woodchip Bioreactors. N.p., n.d. Web. 25 Sept 2015.
<https://engineering.purdue.edu/watersheds/conservationdrainage/bioreactors.html>.
(108)
(109)