Effects of Acid on Chlorophyll Production of Common Duckweed (Lemna minor L.)

Corinne Breymeier and Cosima Wiese, Ph.D. Department of

Biology

Introduction

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

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.

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).

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.

Introduction

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

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.

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.

Hypothesis

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

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)

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.

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.

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.

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

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.

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.

Results

Figure 1

Results

Figure 2

Results

Figure 3

Results

Figure 4

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).

Results-Biomass

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.

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).

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.

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.

Further Research

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

Acknowledgements Special thanks and appreciation to Misericordia

University’s summer research fellowship program, the student research grant program, and the Biology

and Chemistry departments.

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.

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