Monitoring of algae growth rate by measuring transmittance

We intend to grow microalgae Clorella in the laboratory scale bioreactor. The algae we choose could be used as protein enrichment for human food and animal feed. Clorella algae are also used as fertilizer.

Clorella are green algae (Chlorophyta) that range in form from unicells. These algae are autotrophic organisms and they use an inorganic form of carbon, such as carbon dioxide, to make up complex organic compounds.

Every bioreactor must be designed to obtain optimum conditions for growing organisms. For a bioreactor used for growing algae, it means the design supports algae's access to carbon dioxide and light. It also has to ensure that the bioreactor contents are thoroughly mixed and homogenized during the bioprocess.

During bioprocess the number of algae unicells is supposed to grow. We will be monitoring the growth of cell number with measuring the transmittance (the intensity of light passing through the sample) of the samples. The sample will be the mixture of algae unicells and liquid culture media in the bioreactor. Samples will be taken in defined time intervals.

The unicells of Clorella algae are green and contain chlorophyll. Their growing number increases the intensity of the sample's green colour. The unicells also absorb certain amounts of light, which affect intensity of light passing trough the sample.

The quantity (volume) of liquid culture media stays unchanged during the bioprocess; the number of algae cells is growing. This is the reason why the sample's turbidity differs. The turbidity of the sample increases over the period of the bioprocess.

With increasing turbidity the transmittance measured in the samples should decrease.

Because the samples of algae and culture media mixtures are coloured green, the LED light used for measuring transmittance will be red.


Transmittance depends on the number of algae cells in the sample

Hypothesis

An increasing number of algae cells is supposed to increase the turbidity of the samples taken during monitoring the bioprocess. An increase in the turbidity of samples should decrease their transmittance. This is why we believe it is possible to monitor the growth of algae Clorella by measuring transmittance in the samples.

In order to evaluate this hypothesis, different suspensions of algae unicells in culture media will be prepared, the unicells will be counted and the transmittance will be measured. The evaluation of the results will show the effect of the unicells number on measured transmittance.

Preparing samples for measuring transmittance

The sample has to be retrieved from the bioreactor with sterile equipment. This will be the initial sample.

Set five labelled sterile tubes and 50 mL of sterilized culture media (the same as used in the bioreactor). It is Important that there are no cells (dead or alive) or any other fragmets in the sterilized culture media, which is going to be used to dilute samples. Cells or any other fragments in the diluting solution would affect the measured transmittance. This is why all used tubes and pipettes must be clean and sterilized.

Transfer 10 mL of well stirred initial sample transfer into the first sterile tube using a sterile pipette.

Using a pipette transfer 1 mL of well stirred initial sample into the second tube and add 9 mL of the sterilized culture media. Stir well. This sample is 10-1 diluted.

Using a pipette transfer 1 mL of the sample from the second into the third tube and add 9 mL sterilized culture media. Stir well. This sample is 10-2 diluted.

Repeat the action two more times until five samples have been prepared:

  • initial sample
  • 10-1 diluted sample
  • 10-2 diluted sample
  • 10-3 diluted sample
  • 10-4 diluted sample

All samples must be well shaken before any solutions are transferred.


Measuring the transmittance

Fill five holes in the blister with the well stirred samples. Put each sample in one hole. Fill the sixth hole with sterilized culture media. The blister should be filled as shown below:

Blister hole

1

2

3

4

5

6

Content

initial sample

10-1 diluted sample

10-2 diluted sample

10-3 diluted sample

10-4 diluted sample

sterilized culture media

Sample volume

250 µL

250 µL

250 µL

250 µL

250 µL

250 µL

Put the filled blister on shaker to prevent cell gathering on the bottom of the hole. Set the shaker on 320 RPM (rotations per minute). Blister should be left on the shaker, until the spectrometer is ready to use.

Get the spectrometer ready and turn on the red LED light at the highest intensity.

Put the filled blister on the spectrometer. Set the transmittance measured at the sterilized culture media on 100.0 %. After that measure the transmittance of the other samples. If the measuring process lasts too long, the blister should be set on the shaker again, because the cells will begin to gather on the bottom of the blister hole which will affect the measurements.


Counting cells in the sample

The number of cells in 1 mL of sample could be defined using a hemocitometer and a light microscope. A hemocitometer is used to count cells in a small amount of sample. The number of cells in 1 mL of sample is calculated using the counting result. In order to get accurate results the cell number in the sample must not be too big or too small. It is impossible to count cells, if their number is too big. Such a sample should be diluted before counting.

The equation used to calculate the number of cells in 1 mL of sample:

Cell number/1 mL sample = average cell number · 4 · 106 · R

Average cell number is calculated by dividing the number of cells in all the hemocitometer squares with the number of squares.

R = 1 Sample's dilution (102, 103,…) is used just to calculate the number of cells in the initial sample from the cells counted in the diluted sample. When calculating the number of cells in a given sample dilution is 1 (R = 1).


Hypothesis evaluation

All the results obtained from counting cells and measuring transmittance in samples is written in the table shown. Results in the table are used to evaluate the affect of cell number on measured transmittance. With this evaluation we can prove the hypothesis right or wrong.

Example

Table 1: T measured in samples with defined cell number

sample

cell nr./mL

ln cell nr./mL

T

A

initial

1.3 · 108

18.62

94.0

0.0268

10-1

5.5 · 107

17.82

95.6

0.0195

10-2

1.1 · 107

16.21

97.7

0.0101

10-3

6.5 · 106

15.69

98.4

0.0070

10-4

3.0 · 106

14.91

99.1

0.0044

Transmittance is related to absorbance as described by the equation:

A = –log T

Evaluation: Measured transmittance decreases with increasing cell number/ mL .

Calculated absorbance decreases with decreasing number of cells.


Conclusion

The results show the effect of cell number on transmittance of the sample. Transmittance (measured with spectrometer) decreases with increasing number of cells, whilst the absorbance decreases with decreasing number of cells.

These results show that measuring a sample's transmittance could be used for monitoring the growth of algae cell numbers during the bioprocess.



Prepared by: Alma Kapun Dolinar