Chlorophyll a　measurement

Chlorophyll a (Chla) is a light-absorbing pigment used by plants for photosynthesis. We will measure and use the amount of Chla as an estimate of the algal biomass. Since epilithic algae are tiny, we will filter them on a filter paper.

1. Collect some cobbles from riverbeds and remove algae from 6cm x 6cm quadrat using a toothbrush, wash well in a white tray

2. Bring back the sample in a 100mL plastic bottle.

3. In lab, measure absorbance using a spectrophotometer. Prepare vacuum filtration system (suction bottles, filtering apparatus, hand pump, 47mm GF filter).

4. Record the volume of re-suspension and amount filtered. Adjust the volume depending on the concentration (stop before the filter clogs).　Pour algae re-suspended water from the top of the filter apparatus and vacuum using the pump.

5. Place the filter on a KimWipe to dry.

6. Cut the filter into small pieces (3-4mm width) and place in an Erlenmeyer flask. Add 15ml acetone.

7. Cover the flask with a piece of parafilm and leave in cool & dark place (coolers) overnight.

8. Place a paper filter (ADVANTEC No.5, 90mm) on a funnel and filter (7) into a test tube.

9. Unesco method: Measure the absorbance of (8) at 750, 663, 645, 630nm by aspectrophotometer. Use 90% acetone as a control. Since absorbance at 750nm is the turbidity measure, subtract this value from all other measurements. Once measured, place the solution back into the test tube. Calculate total [Chla] using the formula 1.

10. Lorenzen method: Measure absorbance at 750 and 665nm then add 2 drops of 1N HCl to the solution and leave for a few mins before re-measuring. Calculate active [Chla] and [Phephythin a] from formula 2 &3.

Formula 1-3 show the amounts of Chlorophyll a and Pheaphythin a in 1mL of acetone extract.

Formula 1^A: Total [Chl a] (μg/ml) = 11.64 * E₆₆₃ – 2.16 * E₆₄₅ + 0.10 * E₆₃₀

Formula 2^B: Active [Chl a] (μg/ml) = 26.7*(E₆₆₅ – E₆₆₅a)

Formula 3^C: [Pheophythin a] (μg/ml) = 26.7*(1.7 * E_665a – E₆₆₅)

^A The values E₆₆₃ E₆₄₅ and E₆₃₀ are each absorbance measured at 663, 645, 630nm after subtraction by that at 750nm. i.e. absorbance measurement without turbidity measurement.

^B E₆₆₅ is the absorbance measured at 665nm minus that at 750nm. E_665a is the absorbance measured at 665nm after HCl addition minus that at 750nm.

^C Pheophythin a is a chlorophyll molecule broken down to lose 2 Mg²⁺. High [pheophythin a] indicates low algal activity i.e. many dead/weak cells.

11. From the area scraped for algae and amount of water filtered, convert the [Chl a] into mg/m².

Algal biomass (mg Chl a/m²) = 1/1000 * [Chl a] * amount of extract * (re-suspension/filtering water) * 1/scraped area