Environmental Chemistry I Lab. 5. the Impact of Acid Precipitation On

Environmental Chemistry I

Lab. 5. The impact of acid precipitation on vegetation

EPM sem.

PHOTOSYNTHETIC PIGMENTS

Pigments are chemical compounds which reflect only certain wavelengths of visible light. This makes them appear colorful. Flowers, corals, and even animal skin contain pigments which give them their colors. More important than their reflection of light is the ability of pigments to absorb certain wavelengths.

Because they interact with light to absorb only certain wavelengths, pigments are useful to plants and other autotrophs – organisms which make their own food using photosynthesis. In plants, algae, and cyanobacteria, pigments are the means by which the energy of sunlight is captured for photosynthesis. However, since each pigment reacts with only a narrow range of the spectrum, there is usually a need to produce several kinds of pigments, each of a different color, to capture more of the sun's energy.

There are three basic classes of pigments:

 Chlorophylls are greenish pigments which contain a porphyrin ring. This is a stable ring- shaped molecule around which electrons are free to migrate. Because the electrons move freely, the ring has the potential to gain or lose electrons easily, and thus the potential to provide energized electrons to other molecules. This is the fundamental process by which chlorophyll "captures" the energy of sunlight.  Carotenoids are usually red, orange, or yellow pigments, and include the familiar compound carotene, which gives carrots their color. These compounds are composed of two small six-carbon rings connected by a "chain" of carbon atoms. As a result, they do not dissolve in water, and must be attached to membranes within the cell. Carotenoids cannot transfer sunlight energy directly to the photosynthetic pathway, but must pass their absorbed energy to chlorophyll. For this reason, they are called accessory pigments. One very visible accessory pigment is fucoxanthin the brown pigment which colors kelps and other brown algae as well as the diatoms.  Phycobilins are water-soluble pigments, and are therefore found in the cytoplasm, or in the stroma of the chloroplast. They occur only in Cyanobacteria and Rhodophyta.

The picture at the right shows the two classes of phycobilins which may be extracted from these "algae". The vial on the left contains the bluish pigment phycocyanin, which gives the Cyanobacteria their name. The vial on the right contains the reddish pigment phycoerythrin, which gives the red algae their common name.

Phycobilins are not only useful to the organisms which use them for soaking up light energy; they have also found use as research tools. Both phycocyanin and phycoerythrin fluoresce at a particular wavelength. That is, when they are exposed to strong light, they absorb the light energy, and release it by emitting light of a very narrow range of wavelengths. The light produced by this fluorescence is so distinctive and reliable, that phycobilins may be used as chemical "tags". The pigments are chemically bonded to antibodies, which are then put into a solution of cells. When the solution is sprayed as a stream of fine droplets past a laser and computer sensor, a machine can identify whether the cells in the droplets have been "tagged" by the antibodies. This has found extensive use in cancer research, for "tagging" tumor cells.

CHLOROPHYLL

Chlorophyll is a term used for several green assimilation pigments of plants, algae and cyanobacteria. These pigments belong to a group of porphyrins. Together with carotenoids and phycobilins they take part in absorption and conversion of light energy into chemical energy used in photosynthesis. Chlorophyll is hydrophobic, hardly dissolves in water but easily dissolves in oils and organic solvents. A chlorophyll molecule has a hydrophobic "tail" that embeds the molecule into the thylakoid membrane. The "head" of a chlorophyll molecule is a ring called a porphyrin. The porphyrin ring of chlorophyll, which has a magnesium atom at its center, is the part of a chlorophyll molecule that absorbs light energy.

We can differentiate few kinds of chlorophyll marked with a, b, c, d and f symbols. The basic, most important chlorophyll kind present in plants is chlorophyll a

(C55H72O5N4Mg). This is the molecule which makes photosynthesis possible, by passing its energized electrons on to molecules which will manufacture sugars. All plants, algae, and cyanobacteria which photosynthesize contain chlorophyll a. A second kind of chlorophyll is chlorophyll b (C55H70O6N4Mg), which occurs only in green algae and in the plants. The main difference between them is the presence of aldehyde group in chlorophyll b instead of methyl group (chlorophyll a) (Fig.1.). A third common form of chlorophyll is chlorophyll c, and is found only in the photosynthetic members of the Chromista as well as the dinoflagellates. The differences between the chlorophylls of these major groups was one of the first clues that they were not as closely related as previously thought.

Fig.1. The molecular structure of chlorophylls

PHOTOSYNTHESIS

Living systems cannot directly utilize light energy, but can convert it into C-C bond energy through a complicated series of reactions called photosynthesis. Photosynthesis is a two stage process. The first process is light dependent (called light reactions). It requires the direct energy of light to strike chlorophyll a in such a way as to excite electrons to a higher energy state. The light energy is converted into ATP and NADPH (energy carrier molecules) along an electron transport process. Water is split in the process, releasing oxygen as a by- product of the reaction. The light reactions occur in the grana of the chloroplasts.

The light independent process (called dark reactions) occurs when the products of the light reaction are used to form C-C covalent bonds of carbohydrates. The incorporation of carbon dioxide into organic compounds is known as carbon fixation. Carbon dioxide enters single-celled and aquatic autotrophs through no specialized structures, diffusing into the cells. Land plants must guard against drying out (desiccation) and so have evolved specialized structures known as stomata to allow gas to enter and leave the leaf. In the Light Independent Process, carbon is captured and modified by the addition of hydrogen to form carbohydrates. The energy for this comes from the first phase of the photosynthetic process (from ATP and NASDPH). The dark reactions take place in the stroma of the chloroplasts. The C-C bond energy can be stored and finally released by glycolysis and other metabolic processes.

The dark reactions can usually occur in the dark, if the energy carriers from the light process are present. Recent evidence suggests that a major enzyme of the dark reaction is indirectly stimulated by light, thus the term dark reaction is somewhat of a misnomer.

Fig. 2. Overview of the two steps in the photosynthesis process

ACID RAIN

Acidic precipitation causes a number of negative consequences in forest ecosystems. It not only acidifies soil but also interferes with plants growth by directly affecting their green parts and root system. Falling on leaves or needles acid rain drops cause sorts of "burns" (stains). Destroying the leaf cuticle (epidermis) they penetrate inside, causing damage to cell membranes, enzyme system and alteration in organelles (the plant cell structure). In a further step it leads to an imbalance in the functioning of cells, reduce the chlorophyll content necessary to carry out photosynthesis, induce the chlorosis (eg. yellowing leaves) and necrosis (death). Depending on the content of acidic components in atmospheric precipitation one can differentiate the following three types of changes in the photosynthetic activity of plants:  an increase in biological activity – a defensive reaction observed at low levels of acids in atmospheric precipitation or at the initial stages of precipitation with higher levels of acids;  reversible decrease in biological activity – observed for plants subjected to periodic impact of highly acidic precipitation; the speed and rate of reversibility are driven by plant sensitivity, time of exposure and pH of precipitation;  irreversible atrophy of biological activity – caused by precipitation containing acids in a concentration leading to plant necrosis. Carbon dioxide assimilation rate decreases at necrosis of more than 5-20% of leaves area. It leads to stunting, reduction of wood weight gain, decreased resistance to environmental factors e.g. extreme weather events (droughts, floods, frosts, gales etc.), insects, microorganisms etc. Moreover, acid rains leach calcium, magnesium and potassium from soil (limiting the nutrients available to plants) and increase the migration of aluminum and heavy metals (exposing plants to toxic substances slowly released from the soil).

It was found that the largest share in causing the acidity of summer rainfall was sulfuric acid (H2SO4) (73%). In winter nitric acid (HNO3) dominates in rainfall, due to emissions of nitrogen oxides (NOx) from increased combustion of fuels in that period.

Reference

Kubiak J., Tchórz A., Nędzarek A., Analityczne podstawy hydrochemii, Wydawnictwo Akademii Rolniczej, Szczecin 1999. Wachowski L., Kirszensztejn P., Ćwiczenia z podstaw chemii środowiska, Wydawnictwo Naukowe UAM, Poznań 1999. Purves W.K., Orians G.H., Heller H. C.R, Life: The Science of Biology 4th Edition, Sinauer Associates Inc, 1994. http://www3.epa.gov/acidrain/effects/forests.html http://www.ucmp.berkeley.edu/glossary/gloss3/pigments.html

LABORATORY PROCEDURE

Preparation of vegetation samples in simulated acid precipitation conditions

Weigh about 2 g samples of green leaves or needless in triplicate, jot down their masses. Place the samples in the crystallizers and mark them with the name of the group and the following conditions: H2O (reference), 0,1% H2SO4 or 1% H2SO4. Place the crystallizers in the proper exsiccator: H2O, 0.1% H2SO4 or 1% H2SO4, respectively.

Preparation of the samples for chlorophyll a content assessment

After one week of keeping the samples in the acid precipitation conditions or reference (H2O) prepare them for chlorophyll content assessment. Take the samples out of exsiccators, dry with tissue-paper if necessary and place in porcelain mortar. Add 10 ml of distilled water and mash until uniform paste is obtained. Transfer the paste quantitatively to the phial and add

15 mL of extraction solution (MgCO3 in acetone) (rinse the mortar with the solution 3 x 5 mL). Shake the phial for 5 minutes, decant the extract, filter it and collect filtrate to conical flask (100 mL), close the flask with a cork.

Chlorophyll a content assessment

Pour 3 mL of the extract to the cuvette and measure the absorbance at the wavelength 664 nm (zero the spectrophotometer with acetone). Then add 0,1 ml of HCl to the cuvette and measure the absorbance at the wavelength 665 nm (zero the spectrophotometer with acetone).

Calculate the concentration of chlorophyll [µg/L]:

, ( − )∙ = where: 3 V1 - volume of extract sample use for examination, dm 3 V2 - volume of sample use for filtration, m L – width of the cuvette, cm 664a - absorbance of the extract at the wavelength 664 nm 665b - absorbance of the acidified extract at the wavelength 665 nm

Report Compare the appearance of all samples. Recalculate the chlorophyll content for µg of chlorophyll per g of plant. Calculate the loss of chlorophyll in the samples kept under acidic conditions and compare with the reference. Draw the conclusions.