This study examined the effects of different acidity levels on the growth and chlorophyll content of common duckweed (Lemna minor L.). Duckweed was exposed to pH levels of 4.1, 5.4, and 6.5 (control) for 10-12 days. The results showed that more acidic conditions reduced biomass in some experiments, but did not significantly affect chlorophyll content. While the hypothesis that acid would reduce chlorophyll and inhibit growth was only partially supported, the study provides insight into duckweed's tolerance of acidic water pollution from abandoned mines.

1. Effects of Acid on Chlorophyll
Production of Common Duckweed
(Lemna minor L.)
Corinne Breymeier and Cosima
Wiese, Ph.D. Department of
Biology

2. Introduction
• Lemna minor L. (common duckweed) is a
vascular, light green, freshwater, aquatic plant
that floats freely on the surface of water.

3. Introduction
Usually 3 fronds are
present in each separate
duckweed plant.
The roots are long and
usually when the
duckweed grows in dense
colonies, the roots link up
together and form a
carpet like cover over the
surface of the water.

4. Introduction
• Lemna minor has high water purification
capabilities and therefore is used in water quality
testing to monitor heavy metals and other aquatic
pollutants (acid mine drainage AMD).

5. Introduction
• Acid mine drainage (AMD) from abandoned coal
mines is Pennsylvania’s largest single source of
pollution.
• Approximately 2,500 miles of stream are impaired
due to AMD and it will cost the state an estimated
$15 billion to fix all of the pollution issues caused by
the AMD
• At this rate, it will take centuries to restore all of
Pennsylvania’s AMD-impacted watersheds
• Duckweed can be a possible solution to this problem
due to its affinity for some pollutants in aquatic
ecosystems if it can tolerate exposure to the toxic
AMD.

6. Introduction
AMD
overflowing into
Shamokin Creek
from an
abandoned mine
shaft. Notice the
red-orange color
of the water.

7. Introduction
• Research has been done on duckweed and
its sensitivity to the mine drainage.
• The duckweed proved to be a good indicator
of the AMD but also susceptible to growth
inhibition which leads to a decline in
biomass.
• We want to know why the declines in
biomass are happening.

8. Introduction
• During light reactions, chlorophyll molecules
and other pigments capture light, which is
converted to chemical energy in form of ATP.
• ATP is the source of energy that drives the
production of carbohydrates.
• If chlorophyll molecules are depleted,
inhibited, or damaged by acidic pollutants, this
could disrupt the functioning of the light
dependent reactions of photosynthesis.

9. Hypothesis
• What effects will different levels of acid have
on the reproduction of the Lemna minor
plants?

10. Materials and Methods
• Experimental Set Up
– Approximately twenty fronds of duckweed were
placed in sterilized Erlenmeyer flasks containing a
modified Hoagland nutrient solution.
– Each flask was aerated and covered with a foam-
stopper to minimize contamination.
– The pH of the Hoagland’s solution was modified
with 0.25 M sodium hydroxide (NaOH) or 0.1 M
sulfuric acid (H2SO4) to get pH values of 4.1, 5.4,
and 6.5 (control)

11. Materials and Methods
• Experimental set up continued..
– Four replicate set-ups per pH treatment were
placed in a growth chamber at a constant
temperature of 25° C and a 16 hour light period
with a light intensity of 400 µmol m-2s-1.
– Solutions were changed every other day to
ensure consistency in duckweed exposure to
acidic conditions.

12. Here you can see the
experimental set up in
the growth chamber in
Dr. Wiese’s lab area.
Each flask is labeled,
topped off with a foam
stopper, and has a
glass tube inside
connected to air
supply so that the
duckweed was not
deprived of oxygen.

13. Materials and Methods
• Experimental set up continued..
– At the end of the exposure period, the
duckweed was removed from the flasks using
vacuum filtration, and a final biomass was
determined for each flask.
– Duckweed tissue samples were then flash
frozen in liquid N2 and stored at -80oC until
further use.

14. Pictures of Vacuum
filtration mechanism
used to remove
duckweed from flasks.

15. Materials and Methods
• Chlorophyll Extraction from Duckweed
– The duckweed tissue was ground in a frozen
mortar and pestle and combined with 2 mL of
80% acetone.
– The mixture was further homogenized and then
incubated at 4oC for 2 hours.
– Samples were centrifuged for 2 minutes at
12,000 rpm and absorbance of the supernatant
was measured at 663 and 645 nm.

16. Materials and Methods
• Chlorophyll Extraction from Duckweed continued..
-Calculations of chlorophyll content were carried
out as described by Lichtenthaler and Buschmann
(2001).
-Means of biomass and chlorophyll data were
compared using Analysis of Variance (ANOVA)
and a Student’s T-test.

17. Results
Figure 1

18. Results
Figure 2

19. Results
Figure 3

20. Results
Figure 4

21. Results-Biomass
– Experiment 1, showed higher final biomass
results than experiment 2 did.
– In experiment 1, the final biomass increases as
pH value increases; however, there is no
significant difference in the biomass between
any combo of the pH treatments.
– In experiment 2, the biomass data did not show
the same trend from experiment 1. The final
biomass for the highest pH treatment (pH=6.5)
is not the highest final biomass, pH 5.4
treatment shows the highest biomass. In figure
2, there is a significant difference in the biomass
between pH 4.1 treatment and pH 5.4 treatment
as well as pH 4.1 treatment and pH 6.5
treatment (control).

22. Results-Biomass

23. Results-Chlorophyll Content
• For both experiments, trends in chlorophyll
content (chlorophyll A, chlorophyll B, and
total) are the same.
• Chlorophyll A content was higher than
chlorophyll B content for all pH treatments
in both experiments.
• Neither experiment showed a significant
difference in chlorophyll content between
any combination of two pH treatments.

24. Discussion and Conclusion
• In the analysis of Lemna minor’s (common
duckweed) biomass and chlorophyll content after
being exposed to acidic conditions, the results did
not fully support our hypothesis.
• The data suggests that acidic conditions did not
pose a threat to L. minor’s chlorophyll content
after an extended period of exposure (10-12 days).

25. Discussion and Conclusion
• We were more consistent in changing our solutions in
experiment 2.
• Changing the solutions more frequently benefitted in
two ways, pH values were consistent throughout the
entire duration of the experiment and algal growth was
very limited because of having new, fresh solution
added to the flasks and discarding the old solution.
• If left untouched, acidic pH values would become more
basic (increase) over time, which would create an ideal
pH for duckweed to grow, thus no growth inhibition
would occur.
• In experiment 1, many problems occurred during the
harvest due to a large amount of algal growth within
the replicate flasks. It is possible that algae contributed
to the weight of the harvested duckweed.

26. Discussion and Conclusion
• In conclusion, results of this study only partially
support our hypothesis.
• In conclusion, results of this study only partially
support our hypothesis. That is, the acidic
conditions are affecting the biomass; however, it is
not a decline in chlorophyll content that is the
cause of the declining biomass.

27. Further Research
• Future studies can be conducted using the
same protocol but:
– elongating the exposure period
– Adjusting the acidic pH values,
– other plant molecules

28. Acknowledgements
Special thanks and appreciation to Misericordia
University’s summer research fellowship program,
the student research grant program, and the Biology
and Chemistry departments.

29. References
• Ge X, Zhang N, Phillips GC, Xu J. 2012. Growing Lemna minor in agricultural
wastewater and converting the duckweed biomass to ethanol. Bioresource
Technology 124(0):485-8.
• Gerhardt A, Janssens de Bisthoven L, Guhr K, Soares A, Pereira M.J. 2008.
Phytoassessment of acid mine drainage: Lemna gibba bioassay and diatom
community structure. Ecotoxicology 17:47-58
• Lichtenthaler HK & Buschmann C. 2001. Chlorophylls and Carotenoids:
measurement and Characterization by UV-VIS Spectroscopy. In: Current
Protocols in Food Analytical Chemistry F4.3.1-F4.3.8.
• Smith MW and Skema VW. 2001. Evaluating the potential for acid mine
drainage remediation through remining in the tangascootack creek watershed,
clinton county, pennsylvania. Mining Eng 53(2):41-8.
• Taraldsen JE and Norberg-King TJ. 1990. New method for determining effluent
toxicity using duckweed (Lemna minor). Environmental Toxicology and
Chemistry 9(6):761-7.
• Wang W. 1986. Toxicity tests of aquatic pollutants by using common duckweed.
Environmental Pollution Series B, Chemical and Physical 11(1):1-14.

30. Thank you for listening!