Writers: Şiyar Özmen, Muhammed Emir Akat, Dicle Dilan Yardımcı, Dicle Ezgi Ekinci, Diren Kayışkıran, Eren Arda Aktaş, Hasan Mert Altaç
Abstract:
Radiation is the emission of excess energy in an atom to be more stable. It can have immense effects on living tissue or DNA, which may lead to upcoming health problems. Astaxanthin is a secondary carotenoid restored inside the vacuole in Chlorella vulgaris. It is a red pigment that draws its colour due to two additional oxygen atoms on each benzene ring. Astaxanthin is applied to protect an organism from the internal or external effects of radiation. That occurs by the absorption or assimilation of astaxanthin. Taking these into consideration, it can be used to harness its ability to restore radiation damage.
The design of the study was initiated by creating the growth culture in the laboratory. The growth culture demonstrated a rapid growth rate, and the amount of C. vulgaris needed was provided two weeks after cultivation. For the harvest of the algae, the culture was centrifuged and filtered. The filtered algae were kept in a warm environment to be purified by evaporation.
The next part of the design is the preparation of quince seed gel. The quince seed gel was prepared to enable algae usage on the skin. The quince seeds were placed in the boiling water and stayed there until they left their essence. Then the mixture was filtered to dispose of the quince seeds. Finally, the two components-purified algae and quince seed gel-were mixed in a beaker. The odour of the mixture was closer to that of the algae. The colour of the mixture was pale yellow.
This mixture is produced for skin use in humans for protection from radiation and healing of the damage caused by radiation. Quince seed gel is known for its beneficial effects and has been used for the application of C. vulgaris. Astaxanthin contains conjugated double bonds, hydroxyl and keto groups. It has both lipophilic and hydrophilic properties. Previous studies prove that astaxanthin can be absorbed through skin, which implies that the mixture is effective since the whole process of production does not include any step in which astaxanthin molecules can be damaged. The reason why we studied a product which is applied topically is that it not only heals radiation damage, just like the oral intake of astaxanthin, but it also provides protection against radiation. This paper explains the problem of radiation and the solution of usage of C. vulgaris for this problem.
Introduction:
All matter in our universe is composed of atoms which are made of the nucleus (protons and neutrons) and the outer shell (electrons). Therefore, the nucleus carries a positive electrical charge, while the outer shell carries a negative electrical charge. These electrical forces in the atom are intended to reach a strong, stable balance by getting freed of abundant atomic energy, which is radioactivity. While this occurs, unstable nuclei can radiate a certain amount of energy, and this random emission is called radiation. Radiation is the energy released by matter within the frame of high-speed particles or rays.
There are two different radiations: ionizing and non-ionizing radiation. The former has enough energy for ionization, which is the process of knocking electrons out of atoms. Ionizing radiation threatens living organisms because it can damage tissue and genes in the DNA. Examples of sources of ionizing radiation can be cosmic particles from outer space, x-ray machines, and radioactive elements. Radioactive decay is the way radioactive elements emit ionizing radiation. This emitted ionizing radiation can include alpha/beta particles and/or gamma rays.
Non-ionizing radiation is the term given to radiation in the part of the electromagnetic spectrum where there is insufficient energy to cause ionization. It includes electric and magnetic fields, radio waves, microwaves, infrared, ultraviolet, and visible radiation. It can be created by unstable atoms or machinery. Therefore, it is possible to be exposed to any part of the globe. While small amounts of radiation do very little damage, excessive exposure to radiation can be fatal. Occupational groups whose workplaces or interests have a relationship with advanced machinery or radioactive matter are exposed to higher amounts of radiation. This mostly affects human health negatively by influencing DNA or living cells.
If the radiation damage in a cell is enough to kill it, the effect may not be noticeable before hours or days. The death of the cell can happen in two different ways. The first one is caused by internal ionization, in which the cell can no longer perform its function. This damage can be done by a radiation dose of 100 grays. The second one is mitotic inhibition or reproductive death, which may happen when a cell loses the ability to reproduce, but can still perform its other vital functions. This situation can be done with 2 grays of radiation.
To this day, we still don't have adequate data to select between the distinctive models that are proposed to clarify cell death by the events at the level of atoms and molecules interior to a cell. On the off chance that sufficient significant cells inside the body totally terminate to operate, the impact is deadly. Death may result if cell generation ceases in parts of the body where cells are ceaselessly being supplanted at a high rate (such as the blood cell-forming tissues and the lining of the intestinal tract). If 100 gray radiation is sent to the entire body, the death-inside will occur in 1-2 days. If 2.5 to 5 gray (250 to 500 rad) radiation is sent to the body, this may result in death inside in a few weeks. At lower or more localized dosages, the impact will not be death, but particular indications due to the loss of a huge number of cells. These impacts were once called non-stochastic; they are presently called deterministic. A beta burn is an illustration of a deterministic impact.
The effects of radiation are not always fatal, but they can also cause damage that will affect the continuing life of the person. Radiation may alter the DNA code in various ways which can result in an error in the DNA blueprint (it may also be totally harmless depending on the location of damage). The damage of radiation is a kind of mutation whose effects are based on the nature of the error and the transcription of the damaged location. These effects are called stochastic because the process is random. For example, cancer and mutation in germ cells which can be inherited are stochastic results of radiation.
Due to the lack of evidence of human genome alteration caused by radiation, the genetic risk estimates are compared with the data obtained from laboratory animals. However, these estimates vary greatly depending on the animal. For instance, fruit flies’ considerable chromosomes are susceptible to radiation.
Aim:
We know that severe radiation in space gives serious damage to people. Our body is very vulnerable to radiation and deadly diseases. However, this does not apply to every living thing. Some living things may not be affected by radiation and these creatures can survive in a radioactive environment. For example, a single cell, green, freshwater algae called Chlorella can survive against radiation. In fact, it is proven in reliable scientific sources that it has not evolved for about 2.5 million years. In other words, despite the radiation exposure, its DNA has not changed. Since it is not affected by radiation, it does not need to change itself and, therefore, remains unchanged for millions of years without being mutated. Chlorella alga has the ability to repulse radiation, which has been proven by scientific studies. In the experiments performed by Herbert B. Posner and Arnold H. Sparrow, the most powerful radiation type, Gamma rays were directed to Chlorella alga, but it was able to survive. As we understand from this study showing that its resistance to radiation is more than many living things, Chlorella alga is a species that can survive for many years in space. More strikingly, having the ability to help us overcome perhaps the most challenging obstacle to live on another planet, Chlorella algae can be found in many parts of our country.
Chlorella Vulgaris:
Chlorella vulgaris is a species of green alga described by Dr. Martinus Willem Beijerinck in 1890 from the first pure culture of a eukaryotic microalga. This species is largely found in many habitats, such as freshwater, marine, and terrestrial environments. It is also capable of photosynthesis and rapid growth under different conditions. All of these characteristics have made it one of the first microalgae considered for large-scale cultivation and commercial production. For 2.5 million years, Chlorella-like organisms have been living on this planet.
C. vulgaris has spherical, subspherical, or ellipsoid cells without flagella with 2 to 10 µm in diameter. It can be found as single cells or colonies of up to 64 individuals. Chlorella has a cup-shaped chloroplast with or without pyrenoids (storing starch grains). C. vulgaris is a non-motile microalga. It reproduces through asexual autospores. The mother cell divides into 2 to 32 autospores. Auto Sporulation occurs within the species.
Scientific studies were conducted to estimate a way to cultivate C. vulgaris with the highest efficiency. Mohsenpour and Willoughby exposed different wavelengths of light to a culture of C. vulgaris. The results showed an increase in the population by 20.3 percent on the red light and 14.4 percent on the green light.
C. vulgaris has not evolved in 2.5 million years, which is the reason why it is used for radiation protection. This is due to its thick cell wall and the pigments within. Pigments are compounds responsible for the absorption and reflection of certain wavelengths for the photosynthesis process. Microalgae are known for their high production of pigments and compounds such as carotenoids, chlorophylls, and phycobiliproteins. Chlorophyll makes up to 1-2 percent of C. vulgaris’ dry weight when carotenoids like astaxanthin, lutein, lycopene et cetera make up about 0.4 percent of its dry weight.
Astaxanthin is believed to provide the highest protection from radiation and now we will take a deeper look at this unbelievable pigment.
Astaxanthin:
When consumed, astaxanthin (C40H52O4) is a pigment, provides radiation protection and damage repair. It is a product of the chloroplast. It is a member of xanthophylls and also has additional oxygen atoms on each benzene ring, which makes it a secondary carotenoid. This addition gives astaxanthin a deep red color and massive antioxidant properties. Unlike beta-carotene, it is not a vitamin A precursor. Secondary carotenoids are in the oil droplets of the cell and their main function is to protect the cell.
Methodology:
We carried out many of our projects in the laboratory, in the Biochemistry Laboratory of the Department of Chemistry in the Faculty of Science at Dicle University.
The operations we performed are, respectively,
• Preparation of Algae in Nutrient Media
• Cultivation of the Algae
• Observations on the Cultivated Algae
• Purifying the Algae
• Drying the Algae
• Preparation of the Quince Seed Gel
• Preparation of the Lotion
Preparation of the Algae in Nutrient Media:
In order to reproduce our algae, we gathered samples of our algae through Professor Semra Mirici at Gazi University. We then created 3 different media: tap water + mineral water, pure water + mineral water and BG11 medium media. We first started with a tap water and mineral water mixture. Equal amounts of tap water and mineral water were mixed with the help of magnetic stirrers in the beaker. To equalize the pH of the mixture to 7.5, we used hydrochloric acid (HCl) and sodium hydroxide (NaOH) where needed. After obtaining our mixture of pH 7.5, we used an autoclave, which created a high-temperature and high-pressure medium to remove living organisms. After the autoclave process, our first nutrient medium was ready.
Then we started to prepare a mixture of pure water + mineral water. We mixed an equal amount of pure water and mineral water with a magnetic stirrer in a beaker. When the mixture was homogeneous, we adjusted the pH of the mixture to 7.5 and then applied it to the autoclave to purify the living organisms. We have prepared the 2nd nutrient medium from cooling. In the BG11 medium, the processes were slightly different because this was the most commonly used medium in scientific studies. We have weighed the necessary minerals with the help of precision scales to create a nutrient medium.
Then, we dissolved these substances that were in 5 different tubes filled with pure water. By mixing the dissolved substances in an appropriate manner, we obtained a new solution. We used acidic (HCl) and basic (NaOH) substances to equalize the pH of this solution to 7.5. When we reached the appropriate pH, we cleaned the living organisms with the help of the autoclave machine. Our final nutrient medium was ready. This solution was the most suitable medium for algae because all the minerals that algae needed were present in this environment and our estimates were that this would be the environment in which the adaptation period would be the shortest. After the BG11 medium was cooled down and we noted our predictions, we started cultivation.
Cultivation of the Algae:
We have completely sterilized the environment to obtain the appropriate environment (free from living organisms) where we can plant algae. After preparing the environment, we started processing for cultivation. We created 4 media of 125 mL from each nutrient medium. We completed the cultivation process by fitting 1000 µL of Chlorella alga in every 25 mL of nutrient medium. We then placed these nutrient media in an ample light environment.
Observations on Cultivated Algae:
After planting, we left the algae where the light was ample, allowing them to proliferate. On plantation day, we could not observe a difference among nutrient media. They were all very similar and the ambient conditions were the same in order to get accurate results after algae reproduction. Therefore, it was difficult to comment on the first day, but our predictions were that the growth in the BG11 medium would be faster.8 days after planting, we made new observations on algae. BG11 medium was green. We were expecting a turning to green in this nutrient medium, but we were surprised that it was faster than we had expected.
There was also a little greening in the tap water and mineral water mixture. In the mixture of pure water and mineral water, there was almost no greening. Algae are very fast-growing creatures, but they need a little more time to reproduce as they adapt to the environment. 15 days after the plantation, we visited the algae again. The BG11 medium was totally green, and algae proliferated very quickly. The mixture of fountain water and mineral water was also slightly greener. Algae in the mixture of pure water and mineral water should still be in the process of adaptation as there was almost no difference from its first process. Literally 18 days after planting, the algae in the BG11 medium were ready to be harvested. Because the rate of greening in other nutrient media was low, they needed some time to be harvested. Our next operation was to purify BG11 medium by harvesting it.
Purifying Algae:
In order to be able to use algae, we separated them from all minerals in the nutrient medium. We used the centrifuge device for this process. The centrifugal device density rotates the mixtures of different substances at high speed to allow the heavy materials to settle to the bottom. In order to centrifuge the algae in the nutrient media, we have placed them in appropriate tubes. Then we repeated these operations with the help of the centrifuge device 6 times. After each treatment, we rinsed with pure water to ensure that no particles remained. Then we collected all the Chlorella in a single tube and repeated these processes one last time. To avoid any imbalance in the device, we placed tubes filled with pure water equal to the other 3 compartments of the 4-chamber device. After the last treatment, we had pure Chlorella.
Drying Algae:
We needed powder form to use the alga lotion. The alga that we collected in a single tube by purifying waited for 3 days in an environment of 45 degrees centigrade, because even if we purified it, it was still alive and there was still a high percentage of water in the cell structure. We kept it in the lab for 3 days at 45 degrees Celsius and our dry alga was ready. The dry alga’s weight was 0.728 g. In order to reach the powder form, we mashed it in a pot. Chlorella alga, which became a powder, was ready for use in lotion.
Preparation of the Quince Seed Gel:
The other substance of the lotion was quince seed gel. Quince seed gel is a widely used substance in the community and is frequently recommended by experts. In order to obtain this gel, we reached a gel form by boiling 8 quince seeds in 100 ml of pure water. By boiling quince seeds in the laboratory, we obtained quince seed gel, the second substance of our lotion. The next process was to produce lotion.
Preparation of Lotion:
After making quince seed gel and Chlorella algae ready for use, we started to produce lotion. For the production of lotion, we slowly added the powdered Chlorella algae that were in the beaker by means of the magnetic stirrer by mixing constantly. We continued mixing for about 1 hour to make the mixture homogeneous. After this procedure, our radioprotective and highly applicable lotion that appeals to past, present, and future lotion was ready.
Findings:
We have obtained some findings while carrying out our project work. We observed that the Chlorella algae we obtained from Gazi University were not homogeneously distributed in their environment; instead, if kept there, they would settle to the bottom. The algae at the bottom were a dark green color, and when shaken, they could spread throughout their environment without losing their vitality. We have seen that the adaptation time for the alga in BG11 nutrient medium during reproduction processes is about 7 days, which is shorter than the tap water + mineral water and pure water + mineral water nutrient media.
We also found that the Chlorella alga, after adapting to the environment, reproduced very rapidly and gave a green color to the environment. As the density of Chlorella alga increases in the environment, the darkness of the green color increases. If kept so long before being harvested, metabolic wastes that are left in the nutrient medium can damage alga. Chlorella undergoes some changes depending on the color of the light directed to it. In our experiments, we observed that the amount of astaxanthin contained in the Chlorella alga reached the maximum value when reproduced under blue light. The chlorophyll ratio was higher in our algae under green light. Ones growing in the red light had higher nutrition values than the others. In other words, we have tested the environment in which Chlorella should be reproduced in order to get the highest efficiency according to the purpose of use. In centrifugation, we observed that algae survived after being treated 7 times for 10 minutes in centrifuge devices operating at 5,000 rpm (Revolutions per minute).
In addition, we observed that after each process, the washing treatments with pure water were effective in purifying the algae. It is important that this event occurs slowly when drying algae. In the case of rapid drying, the possibility of degradation of substances is high; especially protein-based substances, they may be denatured. The point to consider when preparing the quince seed gel is the proportion. The consistency of the gel prepared with 8 quince seeds in 100 mL water is optimum. If the quince seed proportion is low, its consistency is rare, and if it is too high, its consistency may be intense.
Since such situations will make the use of lotion difficult, the seed-water proportion is important at this point. While preparing our lotion, we should add the algae that is powdered (mashed) to quince seed gel slowly by stirring continuously. Thus, the alga distributes the lotion homogeneously and the radioprotectivity of the lotion reaches the optimum level. Another consideration we should make when preparing the lotion is that the environment is sterile. The lotion can be more effective if the medium is sterilized. We have found that there is no drawback in applying lotion to human skin. If the lotion is applied to the human skin, it continues to have its effect and the quince seed is absorbed by the body under the influence of the gel.
Innovation:
Quince is a small tree from the family Rosaceae grown and cultivated for its edible fruit. It is native to Iran, Turkey, Greece, and the Crimean Peninsula. It is also the only member of the genus Cydonia.
Quince seed releases specific supplements when boiled, thickens the water without any taste or smell. This gel is beneficial in a variety of aspects, and is therefore used in different industries like culinary (food decoration), traditional medicine (open wounds), the beauty industry (natural hair sprays), and pharmacy (dermatologic diseases).
Regarding its availability in the Middle East both economically and geographically, quince seed gel is the optimum base for astaxanthin to reside in for several reasons. First, it is cheap; therefore, the cost of the product will be inexpensive. Second, quince trees are self-pollinating and they are adaptive to the soil: their optimum soil is well-drained but they do fairly well in dry or wet soil. Third, the preparation of quince seed gel produces too little waste and is compostable, which makes the product no-waste compared to a possible chemically produced variant that is significantly better for the environment. The final reason is that it does not react to any compound originating from C. vulgaris and the most important of which is astaxanthin.
Acknowledgements:
The authors thank Assoc. Prof. Dr. Murat Yavuz (Dicle University) for his mentorship on the project and supervision in the Biochemistry Laboratory of Dicle University, and Prof. Dr. Zübeyde Baysal (Dicle University) for her assistance in the preparation of quince seed gel. The authors also thank Prof. Dr. Semra Mirici (Gazi University) and Gazi University for providing samples of Chlorella vulgaris samples for us to cultivate.
References:
1. Borowitzka, M.A., 2018. Biology of Microalgae.
2. Brennan, L., Owende, P., 2010. Biofuels from microalgae - A review of technologies for production, processing, and extractions of biofuels and co-products. Pages 217-232
3. Champenois, J., Marfaing, H., Pierre, R., 2015. Review of the taxonomic revision of Chlorella and consequences for its food uses in Europe.
4. Flores-McLaughlin, J., 2016. A Mechanistic Model of Environmental Oxygen Influence on the Deterministic Effects to Human Skin from Space Radiations.
5. Gouveia, L., Gomes, E., Empis, J., 1996. Potential use of a microalga (Chlorella vulgaris) in the pigmentation of rainbow trout (Oncorhynchus mykiss) muscle. Pages 75-79.
6. Kobayashi, M., Takashi, O., 2000. Protective role of astaxanthin against u.v.-B irradiation in the green alga Haematococcus pluvialis. Pages 177-181.
7. Krienitz, L., Huss, V.A.R., Bock, C., 2015. Chlorella: 125 years of the green survivalist. Pages 67-69.
8. Liang, Y., Sarkany, N., Cui, Y., 2009. Biomass and lipid productivities of Chlorella vulgaris under autotrophic, heterotrophic and mixotrophic growth conditions.
9. Panahi, Y., Darvishi, B., Jowzi, N., Beiraghdar, F., Sahebkar A., 2016. Chlorella vulgaris: A Multifunctional Dietary Supplement with Diverse Medicinal Properties.
10. Pfendler, S., Alaoui-Sossé, B., Alaoui-Sossé, L., Bousta, F., Aleya, L., 2018. Effects of UV-C radiation on Chlorella vulgaris, a biofilm-forming alga.
11. Přibyl, P., Cepák, V., Zachleder. V., 2012. Production of lipids in 10 strains of Chlorella and Parachlorella, and enhanced lipid productivity in Chlorella vulgaris. Pages 549-561
12. Queiroz, M.L.S., Rodrigues, A.P.O., Bincoletto, C., Figueirêdo, C.A.V., Malacrida, S., 2003. Protective effects of Chlorella vulgaris in lead-exposed mice infected with Listeria monocytogenes. Pages 889-900.
13. Safi, C., Merah, O., Pontalier, P.Y., Zebib, B., 2014. Morphology, composition, production, processing and applications of Chlorella vulgaris: A review. Pages 265-278.
14. Schaffer. S.A., 1985. The bioenergetic response of Chlorella vulgaris to alpha radiation. Pages 1-6.
15. Sorokin, C., Myers, J., 1957. The course of respiration during the life cycle of Chlorella cells.
16. Tomaselli, L., 2004. The Microalgal Cell. Pages 1-19.
17. Yamamoto, M., Fujishita, M., Hirata, A., Kawano S., 2004. Regeneration and maturation of daughter cell walls in the autospore-forming green alga Chlorella vulgaris (Chlorophyta, Trebouxiophyceae).
18. Yeh, K.L., Chang, J.S., 2012. Effects of cultivation conditions and media composition on cell growth and lipid productivity of indigenous microalga Chlorella vulgaris ESP-31. Pages 120-127