PROJECT N°53
Study into the potential use of triglycerides
for the creation of biofuel/biodiesel.
Francisco Javier Ramos Sánchez, Iacopo Esposito, Javier Rafael Sánchez Villalba
European School Strasbourg
2 Rue Peter Schwarber, 67000 Strasbourg, France
S5
Abstract:
In this experiment, two different strains of algae were grown in two different media. The growth of
euglena gracilis in commercially-purchased media was the most successful, in both media with the
botryococcus braunii failing to grow significantly during the course of the experiment. Various
separation methods were investigated, with the addition of a dichloromethane / methanol mix being
the most successful. Extraction and isolation of material soluble in dichloromethane was completed
and the product tested as a potential fuel.
Microscope image taken of euglena gracilis algae
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1. Introduction
In this experiment the potential to use algae as a method to create fuels was investigated. When algae
grow, lipids are formed within them, with different quantities and types of lipids being formed,
depending on the strain of algae grown. We hoped that these lipids, once harvested, could then be
converted into a usable biofuel.
Algae, being plants, photosynthesise, taking carbon dioxide from the air and converting it into glucose.
This glucose is then converted into lipids through a range of complex processes in the algae. If these
lipids could be isolated and converted into fuel, the carbon dioxide that would be released upon
burning this fuel would be the same carbon dioxide absorbed by the algae in photosynthesis, meaning
that the fuel would be carbon neutral, hence having a null carbon footprint.
In this experiment, as well as investigating if such a process can be done relatively easily, we
investigated the effect of different growing media, different strains of algae and different extraction
techniques, to see which was the most effective at obtaining lipids.
We believe that algae offer a potentially viable substitute for fossil fuels in the future, reducing global
warming and preserving crude oil stocks. The relatively rapid growth rate of algae makes them an
interesting
study
organism
for this
to
purpose.
During our research into the
project, we also learned that
some countries, such as
Sweden
have
already
realized
this
and
started
working
have
for
a
cleaner planet through the
use of algae.
2. Materials and Methods
This experiment put together several scientific laboratory experiments, thus requiring a large amount
of materials for each step of the process. In order to be able to start the experiment, the first material
required is a significant amount of algae. We opted to use two strains of algae. The first was euglena
gracilis. We chose this strain because it is known that it can be easily grown in a school laboratory and
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the conditions for growth of this algae (23-25 Celsius) were close to ambient temperature in our school.
This was important, as we wanted to ensure that we could first of all grow the algae, then harvest the
lipids produced. The second strain of algae that we chose was one that could potentially contain a very
high percentage of lipids. This was botryococcus braunii. Whilst we waited for the algae to arrive we
prepared the media for them to grow in. Medium one was a ‘home made’ medium made from
chemicals in the laboratory, whereas we opted to purchase medium two. The details of the medium
that we made are given in Table one.
Medium 1
Concentration in
Concentration in
stock solution
medium
K2HPO4
40 g/L
2.3 x 10-4 M
MgSO4
30 g/L
1.22 x 10-4 M
CaNO3
30 g/L
1.27 x 10-4 M
FeCl3
0.5 g/L
1.85 x 10-4 M
KNO3
200 g/L
2 mM
Soil extract
-
-
Component
Table one: Composition of media used to grow the algae
In order to prepare the soil extract for medium one, 500g of ordinary soil was taken from the garden.
1 litre of distilled water was added and the mixture was filtered, first under gravity, and then through
a membrane with a vacuum pump. The filtrate was then collected and added as needed to the
medium. This process presumably collected soluble materials present in soil that would help the algae
grow.
To prepare the stock solutions necessary to create the media, masses of each component were
measured, using a balance and taking into account the relative formula mass of each component in
order to create solutions of precise concentrations. Then, precise amounts of each component stock
solution were added to distilled water to create the final medium, which was finally autoclaved to
sterilize.
When the euglena gracilis algae arrived, we decided to split it up, and add it to both media. To do this,
we created a sterile environment using two heaters and alcohol to clean the equipment, such as
scissors and work space.
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The pipette and cotton, which was then used to close the medium back up, were pre-sterilized. When
all the apparatus was ready, we began the procedure. First the air above the workspace was heated in
order to preserve a sterile environment.
Then the algae was opened and removed from its sterile
bottle, using the pipette, and added to the medium. Finally,
a piece of sterile cotton was cut and placed in the bottle
top. This was to ensure that there is a permanent flow of
air and CO2 nonetheless no bacteria can enter. The cotton
lets through the gases but traps the bacteria at the top.
Once the algae were in the medium, they were exposed to
a significant amount of light and a temperature of 25-30 degrees Celsius for optimal growth. The same
process was done for the botryococcus braunni algae, however as the sample that we received of this
was small, we decided to just add it to medium one, as this was the one that we had researched as
being ideal for this strain to grow in.
The growth conditions of each strain were fairly balanced and similar. The botryococcus braunni, once
placed in its own medium, was then placed at constant light and temperatures ranging between 27-30
degrees Celsius. This, however, was interrupted due to a need of transportation, which was potentially
problematic for the algae’s healthy growth. Notwithstanding, once this displacement was made, they
returned to a fixed temperature, with however, significantly more light. This transportation and
sudden change in temperature was perhaps a little troubling for the algae. After a few weeks of very
little to no change at all in their growth status, we took the decision of adding CO2 tablets to see if this
helped their growth. The euglena algae was kept at a stable temperature and stable amount of light
throughout the experiment.
Various methods were researched in order to flocculate the algae. Small samples of each algae were
taken in order to research the best to use. Initially, centrifugation was attempted as a method to
concentrate the algae. An alternative method was the use of an acidic pH, so differing amounts of nitric
acid was added to each of 5 small euglena samples. A third method involved adding warm isopropanol
to the algae. Finally, a method involving the addition of a 50:50 mix of dichloromethane and methanol
was investigated. It was envisaged that once the algae had been separated from their medium the
lipids contained within them could be dissolved in an organic solvent, which would then be removed
under distillation, to yield the lipids in crude form.
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3. Results
When discussing the results, we can split the experiment up into four distinct sections, these being:
growth of the algae; effect of different media; flocculation methods and purification methods.
In terms of the algae growth, the growth of the euglena was vastly superior to that of the botryococcus
braunni. The effect of the different media was that the euglena (the strain exposed to both media)
grew best in the commercially purchased media. As for the separation methods, whilst centrifugation
showed some potential, there was not a distinct separation between the algae and the medium,
although some concentration of the organic
material was noted. Addition of various amounts of
warm isopropanol did not seem to isolate the algae
from the medium, contrary to the literature that
we read. Similarly, addition of nitric acid to lower
the pH did not seem to have a big effect on the
dispersion of algae in the medium. The image
shows the experiment investigating the effect of
adding hot isopropanol.
The extraction involving the mix of dichloromethane and methanol was the most successful method.
Upon addition of this to a small test sample, the methanol mixed with the water of the medium
whereas the dichloromethane, being immiscible in this water methanol mix, separated and sank to the
bottom of the tube. After a few moments and agitation, the bottom layer became green and looked
to have dissolved significant material from the algae. Given that this method showed that the algae
cells had been disrupted, resulting in the dissolution of plant material in the solvent, we decided that
we would pursue this method of separation with the main samples of algae. To this end, the solvent
mix was added to the algae samples and the bottom green
dichloromethane layer was separated using a separating funnel.
The solvent was then removed by distillation. This left us with a
small amount of a thick black/green oil, as shown in the picture
on the previous page. Attempts to light the oil with a match did
not work, indicating that further processing would be necessary,
if, this organic material could be used as a fuel.
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4. Discussion
One of the disappointing aspects of our experiment is the poor growth rate of botryococcus braunni,
although this has been noted by other researchers. This could have been to do with the transportation
problems discussed earlier. It is unlikely that temperature or the amount of light affected it, as the
euglena grew well in similar conditions. In addition, it is unlikely that the prepared medium was
contaminated in some way, as the euglena grew successfully in this medium. We would have liked to
have more time to investigate the flocculation and separation methods as many are reported and we
did not find some successful. Whilst we did appear to isolate some organic material from the euglena
algae, further work is necessary to investigate the exact nature of this material. It could perhaps be
studied by chromatography to see if it is pure or a mixture of various compounds. Furthermore, it could
be treated in a similar way to the synthesis of biodiesel to see if this would yield a more flammable
product.
5. Conclusion
Whilst it cannot be said that we set out to achieve all our goals in this investigation, we have at least
shown that it is possible to obtain organic material that could potentially be used as a fuel from algae.
Indeed, the large amount of euglena that we grew would have absorbed significant carbon dioxide
from the atmosphere. When we return from the holidays, we intend to continue the study, to see if
further results can be obtained.
6. Acknowledgements
We would like to express our deepest thanks to Mr. David Griffiths and Mr. Fred Brubach for their
continuous invaluable help and support all throughout this project; our project would not have been
possible without their inputs and commitment. Also we would like to thank Algobank without which,
quite literally, this project would not have even started and last but not least, we would like to also like
to thank the (school), which provided us with quite a large part of the apparatus required for our
research and experiments.
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7. References
Here is a list of websites and places which we looked to for additional research needed for our
project:
University of Caen Algobank. http://www.unicaen.fr/algobank/depot/milieu_11.pdf. Accessed
04/11/2016.
DW.com http://www.dw.com/en/sweden-algae-as-energy/av-6655016. Accessed 15/02/2017.
Botryococcus braunii. Wikipedia. Wikimedia Foundation.
https://en.wikipedia.org/wiki/Botryococcus_brauni. Accessed 13/02/2017
Cyberlipid. http://www.cyberlipid.org/extract/extr0006.htm. Accessed 04/11/2016
Pubmed. https://www.ncbi.nlm.nih.gov/pubmed/23612166. Accessed 15/02/2017
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