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Exposing Lemna minor to Nitrogen Nutrients Increased Population Growth Rate

Elizabeth Kim
Lab Partners: Neda Acheampong, Doee Yang, Giselle Galeana
TA: Cody DePew
Section 005

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Introduction
As the human population has grown, the demand for produce and livestock has
exponentially increased as well. With continuously improving technology, society has been able
to develop an efficient mode of production to meet these demands in regards to time and money,
but the issues often left neglected are one of the most important problems that the world faces
everyday. The most prevalent consequence seen universally is the byproducts created by large
agricultural industries such as pollution via fertilizers, deposited into nearby waters. Fertilizers
are abundant of nutrients and minerals such as phosphorus and nitrogen, both of which are
essential in development of plants (Matson et al, 2005). Agricultural runoff has been an issue for
years, and evidence of the severity of this problem can be seen in large algal blooms that occur
shortly after irrigation of crops (Matson et al, 2005). These algal blooms are detrimental to
ecosystems such that phytoplanktons thrive on high nitrogen and phosphorus concentrations
from fertilizers, depleting the environment of nutrients from other organisms. Competition for
oxygen and sunlight among other marine species as well as exposure to toxins that algal blooms
sometimes create are a few of many complications these ecosystems encounter (Matson et al,
2005).
In order to combat the endemic of eutrophication, researchers have developed the idea of
using aquatic plants such as Lemna minor and Salvinia minima to affected waters to absorb
excess nutrients (Burpee et al, 2016). Using these plants as a method of phytoremediation could
potentially save our watersheds without causing more harm (Burpee et al, 2016). To determine
whether Lemna or Salvinia are able to perform the task of phytoremediation, we tested the two
species in various conditions by administering nitrogen or phosphorus to either plant in four
different combinations, as well as competition between both species to observe the affects of

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distinct conditions on the plants growth (Burpee et al, 2016). I predict that the population
growth rate of both aquatic plant species will increase with addition of nitrogen. Conversely, it is
possible that the data collected may not reflect what was originally hypothesized, but addition of
nutrients may limit population growth rate and have a negative affect. To test this hypothesis,
each group was assigned an aquatic plant and specific condition, in this report the affects of
excess nitrogen on Lemna minor. As a reference condition, a control group was made where no
nutrients were added. All plant populations were grown in a greenhouse and weekly observations
were collected. In order to analyze data, population growth was graphed and growth rates and
carrying capacity were extrapolated from them.
Materials and Methods
The following experiments were followed by procedures in the lab manual (Burpee et al,
2016). All plant populations were cultured in the Buckhout Greenhouse with constant a
temperature of 85F.
Experiment 1
A group of 4 students were responsible for the care of their assigned aquatic plant, Lemna
minor. The control group was set up with populations of 12 and 24 plants in respective cups
filled entirely of pond water. This provided the experiment with a basal growth rate of plants
without added nutrients.
Experiment 2
The same group of students was also assigned a variable to test the affect of Lemna
growth rate. This group was responsible for addition of nitrogen to 3 different Lemna
populations with starting population of 24 plants each. Half of the group watered and added 2

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mL of Ca(NO3)2 as the nitrogen source, and the other half of the group counted the thalli of all
populations. Both jobs were to be done at a consistent time every week.
Data Analysis
The populations of Lemna minor were graphed for population size with respect to time to
observe general growth patterns. Population growth rate values were then linearized using the
natural log to obtain the max per capita growth rate. Finally, the geometric rates of increase were
calculated via the slopes of the linearized graph to estimate the carrying capacities of all Lemna
cultures.
Results
Figure 1. Population Growth of Lemna minor

Thalli Number

500
400
300
200

Nitrogen

100

Control

0
0

10

20

30

Days

Figure 1 graphs the population growth of average number of thalli counted among nitrogen
treated group and control group populations of Lemna minor over the course of days. The
average control population growth was also plotted to compare to the nitrogen group. This data
collection was discarded because of the death of all plant populations, responsible for the rapid
decrease in growth.

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Figure 2. Population Growth of Lemna minor
900
800
Thalli Number

700
600
500
400

Nitrogen

300

Control

200
100
0
0

10

15

20

25

30

Days

Figure 2 shows the average growth of Lemna minor populations (thalli count) over the course of
28 days. This information was obtained from the previous class data and was used for the rest of
the data analysis exhibited in figures and tables following this graph.
Figure 3. Population Growth Rate [ln(N)] of Lemna minor vs. Time
8
7
6
Nitrogen
ln(N)

5
Control
4

Nitrogen Linear
Portion
Control Linear
Portion
Linear (Nitrogen
Linear Portion)
Linear (Control
Linear Portion)

3
2

y = 0.1639x + 3.1705

y = 0.1397x + 3.2445

0
0

10

15
Days

20

25

30

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Figure 3 is a comparison of nitrogen treated population and control group population growth, N=
thalli count, linearized with the natural log from values of Figure 2 in order to obtain the slope or
max per capita rate of increase, rmax, as represented in Figure 4 (Burpee et al, 2016).
LambdaNitrogen= 1.178 and lambdaControl= 1.145, the exponential growth rate observed in Figure 3.
Figure 4. Geometric Rate of Increase vs. Lemna minor Population Sizes in Nitrogen (2 mL)
Treated and Control Group
1.4
1.2

Lambda

1
0.8

Nitrogen

0.6

Control
Linear (Nitrogen)

0.4

Linear (Control)
y = -0.0002x + 1.2137

0.2
0
0

200

400

600

800

1000

y = -0.0002x + 1.1926

N (Population)

Figure 4 displays the geometric population growth rate calculated from slopes of average
nitrogen and control population size from Figure 3, representing the population growth rates of
Lemna minor in nitrogen treatment and control group at a specific population size.
Table 1. Carrying Capacity of Lemna minor
Population Group

Carrying Capacity (K= #

Thalli)
Nitrogen
1069
Control
963
Theoretical
1452
Table 1 utilized the linear equation calculated from Figure 4. When y=1 or lambda=1, growth
rate is 0, which was used to calculate the carrying capacity of each population.
Calculation: ynitrogen= -0.0002x + 1.2137
1= -0.0002x +1.2137
x= 1068.5 = 1069 thalli

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Table 2. Surface Area of Growth Container and Lemna Leaf (Burpee et al, 2016).
Item
Surface Area
Standard white container
5809 mm2
Lemna leaf
4 mm2
Table 2 was used for the calculation of theoretical carrying capacity displayed in Table 1.
Calculation: AreaContainer/AreaLeaf = K
Discussion
In this experiment, data was collected from the aquatic plant Lemna minor to test the
effects of administration of excess nitrogen would have on the growth of its population growth.
The original data as illustrated in Figure 1, was abandoned because all plant populations died,
resulting in the negative population growth seen in the graph. This could have been a
consequence of experimental errors such as mold or bacteria that might have grown in the
cultures, competing with the Lemna and creating an unsuitable environment in which they could
not continue to grow. Better results could have been obtained by perfectly isolating the plants
from any debris that may impair optimal growth, and checking into the greenhouse more often to
solve any issues that arise before it affects the plants. The individual who collects data should be
kept to a minimum in order to ensure consistency. Each person may be counting thalli or
administering the nutrient differently, which may be the result of variation in data. Data analysis
was done on a previous class data collection due to these complications. By the end of previous
data collection, we were able to conclude that our hypothesis was proven correct, that the effects
of adding excess nitrogen would increase the population growth rate of Lemna minor
populations. This was evident throughout the analysis of the observed population growth data
collected. Population growth values from Figure 2 were linearized in order to model the
exponential growth curve that was to be expected, and the max per capita rate of increase was
obtained from both the nitrogen treated group and control group slopes. It is evident that the

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geometric rate of increase was larger in value for the nitrogen group compared to the control
group, allowing us to conclude that the Lemna treated with nitrogen were positively affecting
population growth by increasing its geometric growth rate. This is observed in Figure 2, where
the nitrogen treated group and control group started out at the same initial population size, but
the nitrogen treated group ultimately outgrew the control group in same amount of time of 28
days.
In a previous study, similar results were obtained by the administration of 20 mM of NO3
or 4.7 mL to Lemna minor cultures and exhibited an exponential growth model similar to the
results seen in Figure 2 (Joy, 1969). In both this experiment and the previous study, the plant
population displayed an exponential growth initially but began to level out and exhibit a
logistical growth pattern. The slight decrease of Lemna observed after day 21 may have been due
to overcrowding of plants as they grew and no longer had the theoretical unlimited resource
condition that was taken advantage of in the first few weeks. With this being said, the reality of
population growth is that they will eventually slow down or even stop as they reach carrying
capacity because resources are eventually depleted, whether it is nutrients, sunlight, or space.
Figure 4 and Table 1 express the carrying capacities of nitrogen treated and control groups in
comparison to the theoretical values. The nitrogen and control groups were comparable to the
theoretical value but were slightly different, which may be the result of the cultures either not
grown in optimal growing conditions, thalli of each Lemna are not always produced in the same
size, affecting the actual area in which each plant takes up the container, or combination of both
of these experimental errors. The carrying capacity of all Lemna populations regardless of
treatment should be similar, independent of how quickly the population reaches capacity. This

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ensures that growth was not significantly affected by external variables that could have
manipulated the results also ensuring reliability of the data collected.
The increase in population growth rate of the nitrogen treated Lemna population proves
this species as a potential candidate for phytoremediation projects for clean up of agricultural
run-off. Future studies to be employed next could involve the efforts to discover the most
optimal plant mass-to-excess nutrient ratio without over harvesting nutrients from the ecosystem
and causing more damage. By doing so, researchers could ultimately determine the most
efficient solution to reversing eutrophication and contribute to a wide range of bioremediation
projects that continue to exist today.

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References
1. Matson, Pamela A., J. Michael Beman, and Kevin R. Arrigo. "Agricultural Runoff Fuels
Large Phytoplankton Blooms in Vulnerable Areas of the Ocean." Nature 434.7030 (2005):
211-4. Web.
2. Burpee, D., Hass, C., Ikis, D., and K. Richter, eds. A Laboratory Manual for Biology 220W:
Populations and Communities. 2016. Department of Biology, The Pennsylvania State
University, University Park, PA.
3. Joy, K. W. "Nitrogen Metabolism of Lemna minor. I. Growth, Nitrogen Sources and Amino
Acid Inhibition." Plant Physiology 44 (1969): 845-48. Print.