Problem

With almost no foreknowledge about microalgae cultivation and growth, we talked to several experts on this topic (Prof. Dr. Arnd G. Heyer, Vitor Verdelho, Luft and PD Dr. Michael Schweikert) helping us to implement the cultivation of Chlorella vulgaris and Chlorella sorokiniana in our lab. Although cultivation of the microalgae was with a constant setup (16:8 hours of illumination, constant gassing and mixing) the growth curve (measurement of optical density (OD)) of the microalgae exhibited several discrepancies compared to our expected growth curve. As an example, the growth of the algae was different for each day, sometimes exhibiting an exponential growth, sometimes a linear or stagnating growth. With our fear that constantly varying growth behavior might cause different compositions of our algae extract we were keen on fixing the growth behavior of the microalgae in order to obtain a constant, non-changing growth rate.

The role of MicroBiolytics

We reached out to MicroBiolytics, a local company offering quality control services for the food and pharmaceutical industry. The CEO of the company (Andreas Wolf) was very interested in our project, especially the idea to develop an eco-friendlier process. We set up a meeting with Andreas in which we told him about our iGEM project, whereas he explained us what MicroBiolytics offers. In short, MicroBiolytics analyzes fluids using infrared-spectroscopy, being able to analyze the changes of compounds in the fluid. With the inconsistent algae growth in mind we came to the conclusion that we could detect and measure the changes in the growth medium helping us understand the algae growth. Therefore, we collected daily samples of the airlift bioreactor in which we cultivated our algae and noted the corresponding OD at 750 nm. With the captured OD we can deduce on which day the algae exhibit stagnating growth and link that to the liquid in the reactor by analyzing the collected sample. Furthermore, we were offered to analyze our algae extraction batches for purity and differences in compounds.

After the meeting, we collected the airlift samples over the next 2 weeks and brought them to MicroBiolytics. We were then explained the analyzation method and were able to use their device for measurements. Afterwards, we were explained how they deduce the relevant data from the huge data sets which accumulate for infrared spectroscopy and how to interpret the results.

Results

Samples were taken according to the following scheme.

On each date four samples were taken from the reactor. Two for the measurement and two for pH determination. Two additional samples C1 DSNA1 and C2 DSNA2 were taken as control for the analysis of composition. We also calculated the concentrations of each supplement of the DSN media which was used for the Chlorella sorokiniana cultivation as additional information for Micro Biolytic. The concentrations are shown in the following table.

In a presentation results were presented and explained. Each sample was measured 3 times. They did a quantitative analysis of known spectra from a database on oure samples.

In the following figure the concentrations of sulfate are shown in each sample.

It is notable that the sulfate concentration is steady increasing over the time in the reactor except for sample A01 and A02. I may have something to do with the evaporation of the media during the cultivation. Sulfate may accumulate.

The next figure shows the nitrate concentration in each sample.

In figure 2 the real nitrate concentration (C1 and C2) seam to perfectly match the calculated concentrations of nitrate. The nitrate concentration in A01 and A02 seem to be divergent to the other from samples A03 to A08. Concentrations in A03 to A08 seem not to change much. From sample A09 on there is a significant jump in concertation from near 0.30 g/L to almost 58 g/l. This can be explained by the fact, that we inoculated 3-4 L of DSN media on this date (10.07.2019) because the corresponding volume evaporated.

The concentration of sodium chlorin in our samples are shown in the next figure.

It is noteworthy that sodium chloride concentrations in all samples are much lower than they should be according to the recipe for the DSN media. The values are slightly increasing from sample A09. This can be again an effect of the addition of DSN media.

Phosphate was not detected in any samples.

Draw-back

With the measurement of the composition of the media in our reactor, we were able to draw back at least one important part for future algae cultivation. As can be seen, after addition of new media in the reactor (A09). After this timepoint we were able to detect growth of the microalgae. Since the only significant effect was able to be detected for nitrate and the other components not exhibiting a significant effect we wanted to test if weekly addition of supplemental nitrate would increase the algae growth and remove the shaky growth of the algae.
With this in mind, we exemplary tested the nitrate supplementation for the next two weeks. The additional nitrate supplementation was able to stabilize algae growth with the daily OD measurement not exhibiting any stagnating growth (Figure 4).

Thanks to the weekly nitrate supplementation we were able to stabilize the growth of the microalgae, thereby increasing our biomass yield. Furthermore, due to the detected evaporation effect we decided to weekly refill with new media. This was only possible with the help of MicroBiolytics which kindly supported us along our project.