Download Environmental Chemistry I Lab. 5. The impact of acid precipitation

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 ringshaped 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.
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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.
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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 byproduct 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.
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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.
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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
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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:
, (
−
)∙
V1 - volume of extract sample use for examination, dm3
V2 - volume of sample use for filtration, m3
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.
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