Environmental Engineering Reference
In-Depth Information
should be carried out under light suction pressure (no
greater than 3 atm) to avoid damage to delicate algal
cells. Once filtration is complete, the filter should be
immediately removed and pigment extraction ideally
carried out straightway. If extraction is to be delayed,
filters should be folded and stored at −20
◦
C until
required.
absorption. In practice, chlorophyll-
a
is frequently
used as the sole measure of algal biomass, since it
is present in all taxonomic groups and is the most
abundant of all the algal pigments. Chlorophyll-
a
concentration (
C
) can be determined (Jespersen and
Christoffersen, 1987) by measuring UV absorption
at 665 nm (chlorophyll-
a
) and 750 nm (turbidity)
using the following equation:
Pigment extraction
Chlorophylls and caroten-
oidpigmentscontainedwithinalgalcellsareremoved
by physical break-up of the cells followed by extrac-
tion with organic solvents (acetone, hot or cold
methanol). To carry out an acetone extraction:
C
=
AV
∕
V
s
f
∕
L
(2.2)
where
C
is the chlorophyll-
a
concentration per unit
volume of lake water (mg l
−1
),
V
the volume of sol-
vent (ml),
V
s
the volume of sample (l),
L
the light
path (cm),
f
= (1/specific extraction coefficient) ×
1000 and
A
= absorbance
665
− absorbance
750
.
1. Place the filters into the base of a Teflon/glass
homogeniser, add 3-4 ml of 90% alkaline acetone
plus a small amount of glass powder and grind the
sample for a period of 45 s under a fume hood.
Compensation for degradation products
Although
chlorophyll concentrations are frequently estimated
as a direct spectrophotometric reading, their degra-
dation products should also be taken into account.
Chlorophylls degrade to phaeophytins, which are
structurally similar except that the magnesium atom
is lost from the ring structure. Because absorption of
light by these compounds is similar to chlorophylls
(but less strong) they can interfere with chlorophyll
analyses. They have to be determined separately and
the estimate then deducted from the total 'chloro-
phyll' value. To do this the total amount of pigment
(chlorophyll plus phaeophytin) is determined in alka-
line acetone. The sample is then acidified, converting
all chlorophyll to phaeophytin, the chlorophyll con-
centration determined by the change in absorbance
(Wetzel and Likens, 1990).
2. Decant the homogenate into a centrifuge tube,
then wash out the remains of the homogeniser by
adding a further 3 ml of the acetone solution and
grindforafurther15s.Addtherinsesolutiontothe
existing homogenate and maintain the stoppered
centrifuge tube in darkness until centrifugation.
3. Centrifuge at 300-4000 rpm (about 1000 g) for
5 min, using a refrigerated centrifuge (5-10
◦
C) if
available. Remove and tap the tubes to dislodge
any particulate matter that has stuck to the sides,
then recentrifuge for a further 2 min.
4. Carefully remove the clear supernatant (without
disturbing the sediment) and pipette the clear liq-
uid into a clean (non-acidic) spectrophotometer
cuvette.Toavoidpigmentdilution,theuseofsmall
(10 ml) cuvettes, with light paths of 5 or 10 cm, is
recommended.
Spectrophotometric analysis of chlorophylls-a,
-b,and-c
For a more detailed description, see Eaton
et al
.
(2005).
Although chlorophyll-
a
is routinely used to monitor
total algal biomass, analysis of other pigments can
also be used for this and can also provide information
onthetaxonomiccompositionofphytoplanktoncom-
munities. Spectrophotometric estimation of the range
of chlorophylls can be carried out by determining
extinction coefficients at 664, 647 and 630 nm, using
the trichromatic method of Jeffrey and Humphrey
Spectrophotometry
The concentrations of
major algal pigments - chlorophylls (
a
,
b
,
c
) and
carotenoids (carotenes and xanthophylls) - can be
individually determined by measuring light absorp-
tion at their respective wavelengths of maximum
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