Based on thorough literature research and the results of his own work, Dr. Kurmayer suggested 11 strains of Cyanobacteria, which were non-toxic (a crucial criterion for their later use) and which had, according to him, the potential for fast growth and a good dry mass yield. We also preferred strains that were prone to fix a lot of atmospheric nitrogen.
In a first test series we aimed to substantiate our assumptions. We wanted to select the strains with the greatest potential.
1st test series - preselection
11.12.2008 - 15.01.2009 E,K
18.12.2008 - 15.01.2009 F,G
02.01.2008 - 08.01.2009 I
24.12.2008 - 15.01.2009 A,H
24.12.2008 - 08.01.2009 B
24.12.2008 - 19.02.2009 J,D,C
We used the capital letters to code the bottles for cell culture. In the table below you will find the names and codes of the strains according to "Pasteur Culture Collection of Cyanobacteria" (Paris) and to the Institute for Limnology of the Academy of Sciences (Mondsee) respectively.
We used a photometer to measure the growth of the cell cultures twice a week. We also assessed the dry mass once a week. At the end of the test series we counted the number of strains and their bacteria as well as the proportion of heterocytes with the Abbe-Zeiss cell counting chamber under the microscope. Each time we froze a 1ml sample of each strain in order to carry out the nitrogen analysis later. We initially assumed that the production of dry mass was directly related with nitrogen fixation. We could later put this assumption to the test.
Difficulties and their Solutions:
There were some problems with the lighting of the stacked racks, which led to varying light
The lighting was optimized. We kept the bacteria in two different lightings: First the cyanobacteria were grown under good proliferation conditions (40µmol m-2s-1). Once an extinction coefficient of 0.1 -0.2 hadbeen reached, we switched to a more intense lighting (up to 200µmol m-2s-1). The selection of five strains also reduced the amount of time we had to spend with laboratory work. We used an exact timer, to simulate 16h of day and 8h of night.
On cost grounds it was not possible to increase the CO2 content of the substrate which would have had a beneficial influence on the rate of growth. Since the tube reactor at the HTL Braunau lets oxygen pearl into the reactor, this environment is certain to contain a higher concentration of CO2 as CO2 dissolves better in water than nitrogen.
We also created one extra culture of every strain and called them "To the Bitter End" (indicated with double capitals). With these cultures we wanted to find out the extent of maximum yield that could be reached before the algae stop growing, enter a dormant stage, or die. They can either stop to grow due to a lack of light once the bacteria concentration grows so strong that only the outer layers get enough lighting. On the other hand side, there is only a limited supply of nutrients (phosphorus in particular) in the solution. With this part of the experiment we wanted to show which bacteria could reach the highest density in the nutrient solution. Once the bacteria had stopped growing, we measured the final dry-mass. We were hoping to find out the perfect harvesting time, when the concentration of Cyanobacteria was the greatest. The highest density of algae ensures maximum yield.
The table below shows the final results of our 2nd test series. The top five strains selected in the course of the first test series are sorted by extinction
intensities for the different cell culture bottles. We also made a mistake when setting the timer for the lighting which was supposed to simulate day and night. We discovered this mistake after the first testing series. Since light is a
decisive factor, it is likely that the results of this first series were affected by this mistake. However, it was the aim of this test series to select suitable strains of Cyanobacteria. As the conditions were the same for all strains, we were able to identify the fastest-growing blue-green algae.
In a real bio-reactor the bacteria are in constant movement, leading to a homogeneous distribution and better light supply. We tried to simulate these conditions through shaking the cell culture bottles once a day. We could not afford an agitator that could handle so many bottles.
Cyanobacteria lower their metabolism as soon as the environmental conditions (temperature, lighting, nutrition) worsen. This trait makes it easy to store blue-green algae in dark conditions. As soon as the living conditions improve, they start to grow and multiply at a regular rate. When we started the test series, all of the strains were in this kind of dormant state. Some strains accelerated their exponential growth quicker than others. This led to a time lag in the first test series.
The problem of measuring the pure nitrogen will be described in the discussion of the second test series.
The table below compares the 11 strains examined in the first test series, sorted by extinction coefficient in descending order.
coefficient in descending order. The levels of temperature and µEinstein are mean values. The extinction coefficient, concentration of dry mass and total nitrogen are from the final measurement. The bacteria were kept at a mean temperature of 26.5°C. The entire 2nd test series carried out at a light. intensity of about 200µmol m-2s-1 to reach maximum dry-mass yield. Please note that there is no correlation between high extinction coefficients and great dry-mass concentration. It was also interesting to observe that this extreme lighting situation led to a decrease in nitrogen production
Difficulties and their Solution:
We were full of expectation when we brought our 117 samples from the 1st and 2nd test series to the FH Wels. There we wanted to measure the absolute "Internal Nitrogen" using the "Dumatherm Nitrogen/Protein Analyser". As our samples mainly consisted of water, we first had to prepare our samples before using this device. However, due to the binder we had to use for that purpose, we also had to change the cup collecting the residue much more often than usual. During this maintenance someone unfortunately forgot to put the lid back onto the rotating table. Therefore, the results of 43 samples is faulty, since air, which consists of almost 80% of nitrogen got into the Dumatherm. We sent those strains of which we still had enough frozen material to The Austrian Agency for Health and Food Safety (AGES), which kindly enough was enthused by our idea and helped us out.
As the cell culture bottles were very close to the lighting there was hardly any space for the light sensor of the LI-250A. As soon as it was held at a different angle, it also measured different levels of brightness. We measured the light tube once without all the cell culture bottles in it and confirmed a measuring range from 170-230µmolm-²s-1.
look at the Table below
3rd test series - attempting sedimentation
We could observe that some of the Cyanobacteria
tended to settle. Therefore we had to shake the cultures once a day in order to keep up a homogenous solution. In an algae tube-reactor the Cyanobacteria would be in constant movement as well, preventing the algae from settling. Towards the end of the 2nd test
The first two columns, i.e. mean temperature and mean light intensity, give more information on the living conditions of the bacteria. The success of a strain was assessed by its extinction coefficient, concentration of dry mass and nitrogen. The values
series we found out that the dry-mass yield we were hoping to obtain could not be reached as the bacteria were not multiplying effectively enough. We had the idea to use the disadvantageous property of settling for our purposes. We decided to leave some of the
are from the last measuring. The extinction coefficients tagged with the asterisk * are too low, because the photometer was unable to correctly measure the concentration of bacteria due to the high degree of coagulation. One time
the beam of light hits an agglomeration of bacteria, and another time it does not. Therefore the results were not particularly significant.
The strains marked red were included into the second test series. It was our aim to greatly increase the production of dry mass by increasing the lighting. The strains E and F showed a higher concentration in nitrogen then in dry mass. One reason for this could be that dead and other bacteria digested by Cyanobacteria were not detected by our filter. Another reason might be that the strain also releases nitrogen into the substrate - a circumstance that would definitely need further looking into.
As D, C and J are indigenous strains and less problematic to be used for the purpose of fertilization, we observed them over a longer period of time as it took them longer to reach exponential growth. As they tend to flocculate and agglomerate it was not possible to make any significant measurements. However, this does not have any influence on the dry mass or nitrogen production.
2nd test series - increase of yield
22.01.2009 - 19.02.2009 G
15.01.2009 - 19.02.2009 E
19.01.2009 - 19.02.2009 F, H, K
15.12.2008 - 19.02.2009 EE, FF, KK
29.01.2009 - 19.02.2009 HH
We now focused on five favourite strains. We continued to measure lighting, extinction coefficient and dry mass, and also froze samples for later nitrogen measurements. We aimed at dramatically increasing the production of dry mass.
cultures for several hours without shaking, until the bacteria had settled completely. Then we took some ml of the sediment and measured the dry-mass concentration. We found out that a ten times higher dry-mass yield could be reached with the sediment rather than with the homgenous algae solution.
4th test series - Native Cyanobacteria.
From the results of the 1st series of measurements one can see that the sample of D Nostoc is one of the best nitrogen producers under consideration. Since it is a native species to Austria, it would be ideal to use on the fields. Microorganisms are being used by organic farmers to help stabilize the soil from erosion. Maybe, this specimen could as be used for such applications as well.
It seems that Nostoc has roughly the same potential as the winning sample from the first experiments. Nostoc was removed from these experiments because initially its tendency to clump interfered with the measurement devices we were initially using.
We can show now, that Nostoc has a dry weight of 1kg /m3 and an internal Nitrogen content of 300g/m>3.
5th test series - Air and CO2 Carbonation - bubble control
We assumed that the growth of Cyanobacteria in an alge tube reactor would be accelerated by the introduction of air, since CO2 is naturally water soluble. Our hypothesis was that the blue algae would have an increased growth rate as the CO2 content of the reactor was increased. Naturally their would be enough Nitrogen since injected air is about 78% Nitrogen. Additionally the bacteria would have a harder time attaching to the bottom or the walls since, the substrate would be kept in constant motion from the injected air. So we started an experiment to compare the growth of bacteria exposed to air and to CO2 fortified air. Air was injected with a conventional aquarium pump through a hose. At the pump a sterile filter was attached to prevent contamination of the reactor. CO2 was supplied with a CO2 fertilizer for aquariums. We started using the Nostoc samples because of the good showing it had in fourth row of measurements. After a short time it was very apparent that the samples with air fortified with CO2 grew at a much faster rate than the sample in normal air. The data below shows that the dry -mass of the sample proportionally increased while the Nitrogen production did not increase in the same proportions. We believe that the bacteria are simply conserving on nitrogen production if they detect that there is a plentiful supply in the substrate surrounding them.