Practical 4:Microalgae growth measurement

Introduction:
There are several methods that can be used to measure the microalgal growth. The examples of the mthod are estimation individual dry weight, optical density reading, and chlorophyl analysis. During this practical, we have done this 3 methods.

Material and method:
1. Estimation of individual dry weight:
    Dry wight of algal cells can be determined by filtering and drying algae from aliqquots of culture of known concentraation:

• 3 duplicate counts was accurately determine their concentration of algal cutured to be sampled for dry weight analysis 

• An exact volume [V] was filtered on pretared glass microfibre filters [W] using a Buchner set up connected to a vacuum pump (in triploicate). The filter was washed with a solution of ammonium formate (0.5 M) to remove salts.

•   The filters were dried at 100◦C for 4h to volatilize the ammonium formate..It was then weighed on an analytical balance [W*-B*].

•  The dry weight per algal cell was calculated according to the formula:DW (g/cell)=DWwc-DWbc/N × V 

• The same procedure was followed with control filters on which an equal volume of 0.22 μm filtered seawater is filtered (in triplicate) [B].

2. Optical density reading:

     The optical density measurement was done with a spectrophotometer at 670nm. The spectrophotometer is fitted with a 10cm long cell. In this cell,we bring the sample to be measured. As a blank measurement we take the culture medium=seawater.

    When a bundle of light (I◦) is sent through an absorbing material,the bundle of light will have a lower intensity (I) when leaving the material. The absorption is dependent on the absorbing material in the solution (the algal cells) but also on the concentration and thickness of the layer (cell length).

This relation is described by the Lambert-Beer law : OD or A (absorption) = log10 I◦/I

Practically,we measured the absoprtion A of the seawater one time. We assume thisvalue to be I◦ since we are comparing it against the absorption of the algal cells in solution.

Then we set up a series of algal dilutions at 100%,80%,60%,40%,20%,0. The absorption of this series of dilutions is measured with the spectrophotometer and values are calculated according to Lambert-Beer law. The dilutions are also counted for number of algal cells with the haemacytometer counter chamber under the microscope.Using MS Excel we then put the values in a graph; Haemacytometer counts on the X-axis and OD values on the Y-axis. The a suggestion line is fitted and this can be used in the future to calculate cell concentrations for certain OD value.

3. Chlorophyl analysis:

• 50ml sample was filtered through GF/F filtered.
•    3 to 5 drops of MgCOᴈ was added to the sample as it is being filtered.
• The edges of filter which are not coated with residue were trimed away.
•  The filter was homogenized with 5ml acetone for 1 minute. 5ml of acetone was added more and grinded for 30 seconds.
•   The sample extract was sampled in refrigerator in the dark for 1 hr.
• The sample was centrifuged at 3000 rpm for 10 minutes.
•  The absobance of sample extract was measured at 750nm,664nm,647nm, and 630nm.

Results

1)Estimation of Dry weight

Weight(g)	Filter with seawater	Filtre with microalgae
Before	0.12	0.11
After	0.13	0.13

2)Optical density analysis

%	Cell/ml	OD
0%	0	0
20%	9 000	0.1889
40%	150 000	0.3919
60%	260 000	0.5827
80%	360 000	0.7547
100%	1 000 000	0.9438

3) Chlorophyll analysis

Wavelength(wm)	630	647	664	750
Absorbance	0.0432	0.0567	0.0431	0.0204