Conclusions

We extracted all the data from the previous experiments and prepared four graphs, each for OD absorbance/fluorescence and LM8523 / UVM4.

From this initial data we can determine that for samples containing 280 and 385mM nitrate concentration in their medium, there was barely any growth, to the point of resembling white sample levels, in both UVM4 and LM8523 strains. For nitrate concentrations; 1mM, 15mM, 45mM and control (0mM), seem to perform the best and present out of all the cultures. In some of the cases, even 105mM presents good performance.

After completing growth analysis of the Chlamydomonas culture mediums, we used the remaining of the mediums to follow the protocol titled “Measuring nitrate levels from a liquid medium”.
The first step was the preparation of the nitrate calibration curve, from which we obtained the following graph, from were we extracted the formula of the tendency line so we could calculate the concentrations of the algae growth mediums:

However, upon using the value of the calibration curve to obtain the final nitrate concentrations of our algae we soon realised that there must have been a mistake during the procedure, as the results are the ones from the table below:

The data is clearly inconsistent, especially because of the presence of negative value concentrations. After revising our data and calculi, we have found an error in the calculi for the preparation of the sample dilutions. It seems as though we didn’t dilute the highest concentrated growth mediums enough, and also like we didn’t get enough data points to calculate the nitrate calibration with enough accuracy.
We are improving the protocol, re-calculating and re-writing it, so that when we eventually repeat this experiment we get usable data.
Therefore, we haven’t been able to accurately get data on nitrate absorption performance of Chlamydomonas reinhardtii strains LM8523 and UVM4, but we have been able to determine the ideal nitrate concentration range for their growth; [1, 45] mM.

The intent of this experiment, in the beginning, was to determine the nitrate uptake from the culture medium when samples of Chlamydomonas reinhardtii were cultivated in a medium that recreates wastewater (artificial wastewater). The protocol used for the preparation of this experiment is listed under the title “Determining the efficiency of nitrate uptake from the culture medium in two Chlamydomonas reinhardtii strains”.

Initial picture of the culture mediums immediately after preparation of the plates (time = 0h). On the left, UVM4 strains, and on the right, LM8523 strains. At this time, there are no signs of algal activity.
Over the course of the days, we noted no significant difference.
On the final day of the experiment, we took one last progress photo that demonstrates no evolution in the algae cultures, as the cultures at t=120h are the same as in t=0h.

Image of the wastewater nitrate assimilation experiments at t=120h. We observe no difference from the beginning of the experiment, and all the culture mediums behave the same as the white wells.

Conclusions

Given the results in this experiment (lack of any sign of algal growth), we decided not to measure the nitrates in the medium, as they would yield us no significant data. Our objective for the experiment was determining whether Chlamydomonas reinhardtii strains UVM4 and LM8523 ability to absorb nitrates from the medium was, to some degree, hindered by the wastewater conditions.
Instead, we were able to determine that at least one of the components from artificial waste water inhibited the development of algae. We were also able to rule out that it was caused by the nitrates, as the simultaneous experiment we were conducting (Nitrate assimilation in ideal conditions) had the same nitrate concentrations and did present growth.

From the results of this experiment, we decided it was necessary to determine which of the artificial wastewater components was inhibiting the algae proliferation, and instead of measuring the nitrate concentration of the medium, we proceeded to design a follow-up experiment: Wastewater inhibiting component.

After demonstrating that algae did not grow in artificial waste water conditions in our previous experiment (Titled Nitrate assimilation in artificial wastewater, that can be followed with protocol ‘Determining the efficiency of nitrate uptake from artificial wastewater culture medium in two Chlamydomonas reinhardtii strains), we started a new experiment to determine which of the wastewater components presents inhibition towards Chlamydomonas reinhardtii’s growth.
The protocol for this experiment is titled ‘Determining the effect of wastewater components on two Chlamydomonas Reinhardtii strains' growth’.
This protocol is modified to test mediums with one sole component from the artificial wastewaters. It is also designed to really control algal growth, as this is what we are most interested in discovering with this experiment, finding the inhibiting component of algae proliferation.

96-well plate prepared at t=0 to measure initial activity of algae in the mediums. No activity can be observed. UVM4 strain is in the top rows, whilst LM8523 is in the bottom rows.

Wastewater component culture update at t=24 hours. We still cannot observe indicators of algae growth. On the left, LM8523 cultures, and on the right, UVM4 cultures.

48 hour update on the cultures. UVM4 strain on the left and LM8523 strain on the right. There are clear signs of algae growth in all wells, except for the white wells and compound J wells (Na2EDTA·2H2O). It seems we have already found the growth inhibitor, but we will still study the development of the experiment.

We took a second measurement at t=48h, and this is the 98-well plate structure that we used in the SpectaMax. UVM4 is in the upper rows and LM8523 in the lower rows.

Update picture on the cultures at time 120h. On the left, UVM4 cultures, and on the right, LM8523 cultures. We observe further growth, but some of the wells have lost moisture and have started drying out, the most clear examples is F compound of LM8523, J compound of UVM4 and B compound of UVM4.

This is the 96-well plate we used for the 120h measure of algal growth. For the wells that were starting to dry, we decided to use half the volume of the sample and then multiply the data of those wells times two during the analysis. To prevent further drying, we will seal the well plates with plastic wrap moving forward.

Update picture on the cultures at time 144h. On the left, UVM4 cultures, and on the right, LM8523 cultures, same disposition as the update picture for 120h. We observe stable growth, and it also seems as though compound H well of UVM4 has started drying out.

Update picture on the cultures at time 168h. On the left, UVM4 cultures, and on the right, LM8523 cultures, same disposition as the update picture for 120h. We observe stable growth, in all wells except for the white wells and the J-compound wells.
At this stage, it is safe to assume that compound J (Na2EDTA·2H2O) is what was inhibiting the algae growth.

98-well plate preparation for the algal growth preparation at t=168h. This is the last measurement of the experiment. UVM4 cultures on top, and LM8523 cultures on the bottom. Again, the only wells where we don’t observe any algae growth are the whites and J-compounds.

Conclusions

From the very first stages of algal growth we determined that Na2EDTA·2H2O was most likely the inhibitor of the algal growth, and the fluorescence evolution values demonstrate it clearly, to the degree that the sample that contained Na2EDTA·2H2O in its medium has values that resemble those of the white sample.
We were therefore successful in detecting the main lone-acting inhibitor of Chlamydomonas reinhardtii growth from the components of artificial wastewaters.
We observed some unexpected behaviours in certain mediums that were able to surpass the growth rate of the control sample, especially at the peak of the algal exponential growth (120h).

ߦFor UVM4, the components were: CaCO3, NaCl, KH2PO4, MnCl2·4H2O, CoCl2·6H2O, FeCl3·6H2O and CaCl2·2H2O. All the components are ordered from highest growth boost to lowest growth boost above the control sample.
ߦFor LM8523, the components were: CaCO3, MnCl2, NaCl, CoCl2·6H2O, KH2PO4, FeCl3·6H2O, MgSO4·7H2O, ZnCl2. All the components are ordered from highest growth boost to lowest growth boost above the control sample.
- It is important to note that whilst components CaCO3, MnCl2, NaCl, CoCl2·6H2O and KH2PO4, reached algae growth peak at 120h, they proceeded to diminish in value at the next measurement (168h)
- In contrast, components FeCl3·6H2O, MgSO4·7H2O, ZnCl2 had not yet reached the growth peak even at 168h.
We found the results of this experiment very intriguing, as the prospect of creating an enriching growth boosting culture medium for algae with fewer or cheaper components is very promising. We have therefore decided to study this phenomenon further by designing a new set of experiments, Culture medium enricheners, which can be followed in the protocols titled “Determining the effect of salt supplements on two Chlamydomonas reinhardtii strains’ growth”.

1. Experiment 1

With the observed results from the ‘Wastewater inhibiting component’ experiment, we designed a new test concerning those salts that showed a growth boost of our microalgae:
- CaCO3
- FeCl3
- MnCl2
- NaCl
- ZnCl2
We designed 7 sets of mediums:
- 5 excluding one of those components in each
- Medium with every enrichener salt
- Negative control (TAP medium).
The procedure is described in the following protocol ““Determining the effect of salt supplements on two Chlamydomonas reinhardtii strains’ growth (1)”.
We wanted to discover if the mixture of the growth-boost components was favourable for algae growth and to sse which one of the enricheners was more determinant (both in UVM4 and LM8523 strains).

1.1. Results analysis

C → Control. Tap medium.
1 → TAP, CaCO3, FeCl3, MnCl2, ZnCl2
2 → TAP, CaCO3, FeCl3, NaCl, ZnCl2
3 → TAP, CaCO3, NaCl, MnCl2, ZnCl2
4 → TAP, NaCl, FeCl3, MnCl2, ZnCl2
5 → TAP, CaCO3, FeCl3, MnCl2, NaCl
6 → TAP, CaCO3, FeCl3, NaCl, MnCl2, ZnCl2

As observed in the graphs, mediums 2 and 3 showed a lower microalgae growth than the control for both strains.

When considering the LM8523 strain, medium 1 was the only one that showed a clear growth boost in comparison with the control. Mediums 4, 5, 6 present a similar growth pattern than the control.

For UVM4 strain, mediums 1 and 6 seem to have a better growth than the control, showing a surprisingly high boost at 192h.
Mediums 4 and 5 have a similar growth pattern as the control.

1.2. Conclusion

As medium 2 and 3 didn’t contain either MnCl2 nor FeCl3, we need to conclude that these salts are indispensable for microalgae growth when mixed in a medium with other salts. Perhaps their presence contributed to damaging the cells in a determined way.

Medium 4, lacking CaCO3, showed us how this component might be also important in the salt synergy for microalgae growth.

The 2 strains don’t present an important difference in their reaction to the enricheners. In addition, it is likely that the observed boost of growth for mediums 1 and 6 was caused by contamination.

Nevertheless, the measures weren’t taken evenly and we should repeat the experiment in order to obtain valid results.

2. Experiment 2

With the results from experiment 1, we have developed a new experiment selecting the best medium and salts combinations (mediums 1,5,6) for both strains, repeating their culture and creating 5 new medium combinations (all of them slight variations of mediums 1,5,6).

The procedure describing the medium preparations is included in the protocol “Determining the effect of salt supplements on two Chlamydomonas Reinhardtii strains' growth (2)”.

2.1. Results analysis

Mediums (if there is an added *, it signifies an increase of the component concentration):
1. TAP (control)
2. TAP + MnCl2 + FeCl3 + CaCO3
3. TAP + MnCl2 + FeCl3+ CaCO3 + NaCl + ZnCl2
4. TAP + MnCl2 + FeCl3 + CaCO3 + NaCl
5. TAP + MnCl2 + FeCl3+ CaCO3 + ZnCl2
6. TAP + MnCl2* + FeCl3*+ CaCO3*
7. TAP + MnCl2* + FeCl3+ CaCO3
8. TAP + MnCl2 + FeCl3*+ CaCO3
9. TAP + MnCl2 + FeCl3+ CaCO3

We observe how the controls present better growth for both strains. The peak is observed at 96h, and from that point, OD measures values decrease.
All mediums with increased concentrations show a bad growth performance, not even reaching half the growth of the control at the last measure.

Mediums 2, 5 and 5 in LM8523 strain don’t achieve their peak and growth decline. It would be possible that because of these supplements they decrease their growth speed but they still achieve the control maximum OD.
The UVM4 strain reaches growth peak at 168h, and decreases in OD during the several following measures.

2.2. Conclusion

Results seem to show that Experiment 1 results are not reliable, as they do not present lasting continuity. We are therefore able to determine that the combination of these enricheners doesn’t seem to favour the growth of Chlamydomonas reinhardtii, for either of its strains.

Hence, it is better to culture this microalgae species in TAP medium rather than combine it with many other components. This addition will only decrease the growth speed of the culture.

Introduction

As a part of their university studies, two of our team members are taking a course on Biochemistry and Plant Physiology, where they had wet lab class related with the use of a biostimulant in Solanum lycopersicum, a tomato variety.
We decided that using the data of this experiment would be a great opportunity for us, as we would be able to gather information on the effect of cytokinins on the plant.
The experiment is based on the use of kinetin, a type of cytokinin. Literature reading let us determine they are associated with salinity-response stress mechanisms, which is of important interest to us, as those can often be a side-effect of drought.
Our goal for this experiment was using moderately salt water (result of mixing fresh water and sea water), and test whether it would be possible to irrigate crops in areas of high water stress with it.

The idea for this use of cytokinins and salt water stems from our team visit to Comunitat de Regants del Sindicat Agrícola de l’Ebre, who are an irrigation community really struggling with the effect of high salinity in their crops.

Therefore, we are using Mediterranean sea water as a reference for the concentration of the saline water, which has a concentration of 38 g/l.
We also discovered that 100mM of NaCl is considered “severe stress” for S. lycopersicum, whilst our saline water concentration reference is of 651mM.
To prevent conducting the experiment in a very harmful environment for the plan, we decided to dilute the sea water with fresh water, from which we can discover the adequate concentrations of cytokinins to use that can mitigate the effects of the high salinity water on the plant.

For example, in the Ebre region, sea water often infiltrates into the soil, thereby increasing its salinity. This is why we thought that implementing our project for a situation such as theirs, would be a beneficial situation for both of the members of the deal.

Action mechanism of Kinetin

The action mechanism of kinetin, like that of any other cytokinin, is not yet fully characterised. Despite this, a dual-component signalling model, similar to the bacterial system, has been established as the preferred model to explain the functioning of CKs (Shi et al., 2011).
In this system, CK acts by binding to histidine-kinase sensors that initiate a signalling cascade. This receptor, when autophosphorylated, transfers a phosphoryl group to a protein phosphotransferase (HPt), which at the same time transfers it to specific response regulators (RR) located in the nucleus.
There are two types of RR: those of type B, which are transcription factors that activate certain genes involved in plant development; and those of type A, which allow a negative feedback to be carried out to regulate this whole process.
Cytokinin response factors (CRFs) have also been found to act in parallel on certain downstream targets of cytokinins, either independently or in conjunction with B-type RRs.
Up to 11 CRFs have been characterised in Solanum lycopersicum, although its mechanism is not known to the same level as Arabidopsis thaliana. However, SlCRFs (Solanum lycopersicum CRFs) are expressed differently in a variety of plant tissues, and some are more sensitive to cytokinins than others.
In addition, abiotic factors such as salinity can also induce certain SlCRFs, suggesting that CRFs are directly involved in the response to salinity stress and that they may act more generally on other types of stress (Shi et al., 2011) (Ahanger et al., 2018).

Experimental design

For this experiment, we have set two experimental groups, with 5 plants (n=5) in each one. Each group will be treated with 5 mg of kinetin twice a week, in spray, and will be irrigated with a different salinity concentration: 108 mM or 217 mM of NaCl.
According to the consulted literature, 100 NaCl mM can be considered a severe salinity for plant development in Solanum lycopersicum, and 200 mM is practically lethal (Ahanger et al., 2020). From now on, we will be addressing our groups as “Severe” for the one with 108 NaCl mM, and “Extreme” for the one with 217 NaCl mM treatments.
The duration of the treatments was 3 weeks, when we also took the fruits and treated them to measure their biochemical parameters.

Results analysis

Firstly, we wanted to see the physiological effects of the sea-water treatment on our plants, so we measured the relative growth of the stem and the amount of chlorophyll in the leaves. The following graphs show how there are no significant differences between the «Severe» and the «Extreme» groups, which means that the amount of CK we applied was enough to neutralize the effects of high salinity.

However, and still in the physiological department, the stomatal conductance did show significant differences between the two groups, which means that there were differences in water uptake.

On the other hand, we also wanted to measure the quality of the fruits, so as to understand if plant survival was to the expense of yield and quality. In the fruits, we measured lycopene content, the amount of antioxidants and the sugar/acid ratio. The following graphs show that none of these parameters were significantly different between the two groups, so it is safe to say that the quality of the fruits was maintained.

Lastly, we want to stress that the absence of a proper control is a clear issue that must be addressed when repeating this experiment. The main question one may ask is whether or not the differences in salinity really affected the development of the tomatoes, because there are no significant differences in practically any parameter. However, the stomatal conductance did show a significant difference between the two groups, which was expected as the plants with a higher salinity will have more difficulty to absorb water, so they’ll close their stomata.

Therefore, salinity did have an effect on the plants, but kinetin was able to neutralize its effect on the development of our tomatoes. However, a control for the kinetin treatment (no salinity stress but kinetin application), and a control of the salt concentration (tomatoes under salt stress but without kinetin treatment), are very much needed to fully ensure the preliminary conclusions of this experiment.

References