The problem is that conventional ways to treat oily wastewater aren't enough effective and reuse isn't suitable for most reuse applications. In India 77 million human doesn't have access for clean water and 1600 people die in every day due to the diarrhea. Viggor, et al. (2018) workgroup had previously characterized used strains thus predicted result was that ICTN13 is dominant. McGenity, et al. (2012) research paper states that for complete degradation is required consortium so other predicted outcomes were that mixture is better on crude oil and on phenol.
I chose this topic, because microbiology interests me, it's important and in the future fresh water is even more rare. Technologies based on microbes are diverse and they can be used as multifunctional tools.
There has been other studies that observe other bacteria biodegradation ability in different substrates. Knowing different bacteria species' biodegradation efficiencies allows for the development of more effective bioremediation techniques. A fluorescent marker was added to Pseudomonas pseudoalcaligenes ICTN13 to distinguish from Acinetobacter venetianus ICP1. Next step was to create a graph where is shown optical density dependency on amount of bacteria. Then the microcosm studies allowed to create bacterial growth graphs. The results show that the most efficient way to remove phenol is to use only ICTN13, otherwise to educated guess. The best way to biodegrade crude oil and phenol is to use mix of both bacteria like it was predicted. To study it more precisely, chemical tests should be conducted. Obtained knowledge can be used for futher studies or to make comparisons with different strains.
I'll submit the study to the student research journal, participate in National (Estonian) Student Research Contest and work in the future as microbiologist. It would be great, if chemical tests will be also made.
There has been several studies regarding biodegradation, but in science every scientifically conducted research is small, yet vital part in a bigger system. To create better biofilter, the more available data regarding different substrates, bacteria, mobility etc is better. I had ICTN13 and ICP1 cells and there weren't any data regarding how well they biodegrade so I used the opportunity. As I wanted to contribute to find answer, I posed following questions.
After I read Viggor, et al. (2018) working group characteristics these strains, I expected that Pseudomonas pseudoalcaligenes ICTN13 will be dominant.
From McGenity, et al. (2012) research paper, where is written that mixed (two strain) consortium outperforms single species, I concluded that the same should happen with my crude oil microcosm tests.
The same prognosis was made for phenol as for crude oil consortium based on educated guess.
The results should be showing approximate biodegradation ability, since the analytical methods which were used are not precise thus additional microcosm tests, so the results will be statistically more correct, or chemical experiment should be conducted to know qualitative results will be available.
Danger of crude oil pollution has risen due to the increased marine traffic, oil transportation and extraction. Leakage of oil products in the marine environment affects it's ecosystem - from bacteria to mammals.
Crude oil is major threat on Baltic Sea due to the increased maritime activity. There is in every moment approximately 2000 ships and 20% of them are oil tanker, with estimated 166 million tons of crude oil products (HELCOM, 2010). It is vital to act operatively so influence on sensitive shoreline is reduced.
Crude oil one of the most complex mixture of organic compounds which contains alkanes, cycloalkanes and aromatic compounds. Aromatic compounds are made of one or more cyclical benzene core(s) as known as aromatic core. Arenes may form during incomplete burning, formation of crude oil or industrial producing (Polycyclic Aromatic Hydrocarbons, 2017). A lot of bacteria cand degrade arenes due to the aromatic core, which makes them good pollutants, make the most toxic part from crude oil and may have cancerogenic effect (Adbel-Shafy, Mansour, 2016) and due to that I used phenol one model substrate. Phenol do not have cancerogenic effect.
Bioremediation is a technology, which is used for cleaning up oil spills since 1970-s and with that method the environment is being cleaned with microorganism life activity, restoring previous situation. That technology is considered as the best way to restore marine ecological environment because of cheap price, environment friendliness and absence of secondary pollution (Chen et al., 2017). Bioremediation can be boosted including efficient oil degrading bacteria that are from crude oil degrading consortiums. That why it is important to find and characterize bacteria, who would be capable to degrade quickly and effectively crude oil.
Biostimulation and bioaugumentation are one form of bioremediation. During the biostimulation additional nutritients are added so microorganism life activity is supported. Bioaugumentation means adding specific bacteria with certain biodegradation ability. (Microbial Biodegradation, 2008). During the Deepwater Horizon oil spill bioaugumentation was used.
Used strains has already been characterized by workgroup (Viggor, et al., 2018. manuscript). Knowing characteristics made my project more reasonable, because it would have been futile to try to guess biomass production on different substrates and allowed me pick aerobic environment.
NB! The location, glossary, full list of materials and tools used is under bibliography, references, and acknowledgements. The safety precautions are under health & safety.
Following method has already been used in similar research, so I adapted it.
I used strains, Pseudomonas pseudoalcaligenes ICTN13 and Acinetobacter venetianus ICP1, which were isolated by workers of Institute of Molecular and Cell Biology of the University of Tartu from URAN FTP (located in India) crude oil plant cleaning station. Strains are also cataloged in microbe database CLEMS. Obtained cells were preserved in 34% glycerol at -84 °C or as agar culture on R2A broth.
Tagging was done with using OFP (Orange Fuorescence Protein) tag transmittion plasmid (circular DNA in bacteria) pminiTn7-lacItac-OFP (obtained from Rita Hõrak), which includes gentamicin (Gm) resistance gene and OFP coding gene (Koch et al., 2001; Lambersten et al., 2004). Plasmid was transmitted into Escherichia coli CC118λpir cells via electroporator.
For inserting plasmid (circular DNA) pminiTn7-lacItac-OFP into cells, the cells must be processed, so membrane will let plasmid go more easily through. For doing that a new seeding from E. Coli CC18λpir culture was made to LB liquid full broth and was grown for 2 hours. Then cells were separated from broth with centrifuge (12000 x g, 1 minute). Afterward cells were washed twice with 500 μl cold distilled water and for next step was suspensioned 2 times with 200 μl cold 10%-se glycerol and at last suspesioned in 50 μl 10%.
Competent cells were pipetted into electroporator cuvette and 0,8 μl pminiTn7-lacItac-OFP plasmid was added. Process was made at 2500 volts. After electroporating 1,5 ml was placed on LB broth vial and was grown for 1 hour at 37 °C
Strains ICTN13 and ICP1 and their mixture was grown in 150 ml Erlenmeyer flasks, which included 45 ml distilled water, 5 ml 10xM9 , 125 μl 400x micronutrient solution and as substrate 1,3 mM (millimolar) phenol. For inoculation was used on same substrate grown cells (30 °C, 180 rpm) with end optical density OD580=0,1. The test was made 3 times. Growth was observed with spectrofotometer at 580 nm and growth and generation speed was found with formula.
2 strain consortia was grown on 1,3 mM phenol and then samples was seeded on plates, which included Gm and isopropyl β-D-1-thiogalactopyranoside (IPTG) 0,5 mM for inducing glow. New samples were taken in 2, 5, 7, 9 hours and in different concentrations. ICTN13 colonies turned into orange, which allowed to differ it form ICP1. From plates were found multiplicity and relative proportions in mixture.
Biodegradation ability of crude oil was evaluated indirectly - visually.observing how crude oil changed and bacteria culture growth resulted haziness. It's presumed that if bacteria can grow on substrate then they can also biodegrade it. Consortiums were grown in 150 ml Erlenmeyer flasks, which included 45 ml distilled water, 5 ml 10xM9, 125 μl 400x micronutrient solution and as substrate crude oil (1%). Growth was observed for 5 days and every day the pictures were taken. For control was growth medium without cells. Tests were repeated 3 times, so validity can be ensured.
Figure 1. Growth rates on phenol
Microcosms (simulated ecosystems) were inoculated with cells evenly as possible. Initial seeding was OD580=0,1 and samples were taken each hour for 12 hours. Growth curves On figure 1 it is visible that all strains and have rather long lag phase before they start to actively grow. ICP1 adapted with the new environment in 7 hours and then growth accelerates followed by peak that is reached in 10 hours and then multiplicity suddenly drops. Strain ICP1 generation time is 1,78 hours and growth speed is 0,561-1 h whereas ICTN13 has shorter adaption period and actively grows from the 4th hour. In tenth hour the stationary phase begins. Mixture is adapting till 6th hour and then biomass production begins, which is not as big when compared to individual strains. The next objective was to learn which strain is more dominant in the mixture and for that ICTN13 was marked with fluorescent marker (figure 2).
Figure 2. Pseudomonas pseudoalcaligenes ICTN13-OFP under fluorescent microscope.
The biodegradation is mostly done in microbial consortium and that is why it is important to know how mixtures are biodegrading different substrates thus ICTN13 was marked. IPTG was added to induce the glow and cells were observed through fluorescent (figure 2) microscope or plate colour were checked (orange colour will be visible).
Figure 3. ICTN13-OFP and ICP1 relative proportion during the phenol tests.
From phenol microcosms mixed culture the samples were taken on 2th, 5th, 7th, and 9th hour and seeded in differently concentrated solutions to borth plates, which also included IPTG and gentamicin. Marked colonies became orange which allowed to differentiate from ICP1. It was devious method to determine multiplicity and does not give precise result, but gives clue which strain is dominant.
On figure 3 it is visible that ICTN13 was dominant during the test. On 2th hour ICTN13 had highest percentage and fell till 7th hour. It is because meta (ICTN13) and ortho (ICP1) pathway having culture population is changing in opposite phases. Same result was also observed by Heinaru et al. (2005) workgroup, who demonstrated similar fluctuation in opposite phases. Described circumstance shows that mixture can be observed as unified population, where every strain has only one induced biodegradation pathway so non-productive catabolic pathways will not be activated (Heinaru et al., 2005).
Figure 4. Crude oil microcosm trial. A - abiotic (without cells) test; 1 - strain ICP1; 13 - strain ICTN13; S - strain mixture; I - trial at the beginning; II - trial after 24 hours; V - trial after 5 days.
Crude oil is vital in modern world, but with using it the pollution follows so the next task was to evaluate bacteria ability to biodegrade oil. Strains were grown for 5 days in 150 ml Erlenmeyer flasks and evaluated indirectly via observation. Photos were taken. Control was crude oil growth environment without cells (figure 4). After 5 days of orbital shaker, control stayed same. Other flasks had small visible oil drops (figure 5).
Figure 5. Two strain consortia from crude oil microcosm
Crude oil pollution is one of the most serious type of pollution, which affects both humans and ecosystems. Bioremediation is important technology for cleaning up pollution using microbe life activity. Especially good results are achieved when local, which means strains that were isolated from nature is being used. Crude oil biodegradation speed is influenced from pollution extent and composition (Atlas, Hazen, 2011).
So the aim was to use strains, which were isolated from nature, to study crude oil and phenol disintegration. Investigated strains, Pseudomonas pseudoalcaligenes ICTN13 and Acinetobacter venetianus ICP1 can degrade several aromatic and aliphatic compounds. I consider experiment success, because microcosm trials proved visually good biodegradation ability, especially ICTN 13 had good capability, and in mixture they supported each others growth. Only one predicted outcome was wrong. I don't know why exactly, but one plausible explanation might be that ICTN13 is more specialized for degrading phenol than ICP1, thus likely ICP1 hindered degradation in mixture and underperformed in single strain consortium. As previously I mentioned, the chemical tests should be also conducted, so more precise results will be available. The next things that can be studied is how studied strains perform when they are immobilized, how are they performing in different consortum and on different substrates. When constructing new water purifying system, then those strains can be used included. I'll write laboratory diary more systematically next time, because it will make life more easier.
I'm 18 year old enthusiastic, curious and courageous Estonian. When I was younger, I read my older brother textbooks because they were interesting. In 3rd grade my grandfather showed me how to make my own webpage in HTML5 and CSS which was my first coding experience. I've been each year several times in local science center since it was opened in 2011. I'm sure that it has big influence on me.
I've participated in hackathon and two times in a prototyping event for high and middle school students. First time when I participated there I built prototype to check how it would work for student company. I got chance to see how bog is being restored and what kind of environmental monitoring scientists did. This year I built discovery learning kits, inspired from bog visiting, and the minimum viable product that we finished was a (self assemblable) device that should be able to measure if juice has natural or artificial food coloring..
I even discussed with Minister of the Environment on that prototype where we had 2nd prototype.
In hackathon I joined with team that was going to build interactive model of a local museum that got in the end first place.
In one youth exchange project I conducted workshop about bogs during the Researches' Night Festival.
In October, I conducted workshop where other students learned more about modular laptops.
All these events have given me valuable experience how to be a well functioning team member and how to realize ideas. I want to work as science communicator, scientist and later as entrepreneur. I'm inspired by various people who are making the world a better place to live on.
Winning would mean a lot for me, because I'm not from the wealthiest family and a lot of resources is used for my younger brother and sister, both with unique disorder, thus it would allow me to obtain education abroad and focus on studying instead of working.
Name: Merike Jõesaar
NB! I wrote my paper originaly in estonian and which was bit longer than I could write here, but I feel that I still have to credit authors, whose materials I used, hence here is full list of articles that I read and used in final edition, but did not made to my project description here in Google Science Fair due to the shortening.
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Ayala, Marcela, Eduardo Torres, 2004. Enzymatic activation of alkanes: constraints and prospective. Applied Catalysis A: General, nr 272(1-2), pages 1-13.
Bao, Ying, Douglas P. Lies, Haian Fu, Gary. P. Roberts. 1991. An improved Tn7-based system for the single-copy insertion of cloned genes into chromosomes of gram-negative bacteria. Gene, nr 109(1), pages 167–168.
Boyer, Herbert W., Daisy Roulland-Dussoix, 1969. A complementation analysis of the restriction and modification of DNA in Escherichia coli. Journal of Molecular Biology, nr 41(3), pages 459–472.
Chakraborty, Romy, John D. Coates, 2005. Hydroxylation and Carboxylation—Two Crucial Steps of Anaerobic Benzene Degradation by Dechloromonas Strain RCB. Applied environmental microbiology, nr 71(9), pages 5427-5432.
Chandra, Subhash, Richa Sharma, Kriti Singh, Anima Sharma, 2013. Application of bioremediation technology in the environment contaminated with petroleum hydrocarbon. Annals of microbiology, nr 63(2), pages 417-431.
Chen, Qingguo, Jingjing Li, Mei Liu, Huiling Sun, Mutai Bao, 2017. Study on the biodegradation of crude oil by free and immobilized bacterial consortium in marine environment. PLOS ONE 12(3): e0174445
Conney, Allan H, 1982. Induction of microsomal enzymes by foreign chemicals and carcinogenesis by polycyclic aromatic hydrocarbons: GHA Clowes Memorial Lecture. Cancer research, nr 42(12), pages 4875-4917.
Harayama, S., Monique Rekik 1989. Bacterial aromatic ring-cleavage enzymes are classified into two different gene families. Journal Biological Chemistry, nr 264(26), pages 15328-15333.
Head, Ian M., D. Martin Jones, Wilfred F.M. Röling, 2006. Marine microorganisms make a meal of oil. Nature Reviews Microbiology, nr 4(3), pages 173-182.
Heinaru, Eeva, Merike Merimaa, Signe Viggor, Merit Lehiste, Ivo Leito, Jaak Truu, Ain Heinaru, 2005. Biodegradation efficiency of functionally important populations selected for bioaugmentation in phenol- and oil-polluted area. FEMS Microbioly Ecology, nr 51(3), pages 363-73.
HELCOM, 2010 = Maritime Activities in the Baltic Sea – An integrated thematic assessment on maritime activities and response to pollution at sea in the Baltic Sea Region. Baltic Sea Environment Proceedings No. 123.
Herrero, Marta, Victor de Lorenzo, Kenneth N. Timmis, 1990. Transposon vectors containing non-antibiotic resistance selection markers for cloning and stable chromosomal insertion of foreign genes in gram-negative bacteria. Journal of Bacterioloy, nr 172(11), pages 6557–6567.
Hoffman, David J., Martha L. Gay, 1981. Embryotoxic effects of benzo [a] pyrene, chrysene, and 7, 12‐dimethylbenz [a] anthracene in petroleum hydrocarbon mixtures in mallard ducks. Journal of Toxicology and Environmental Health, Part A Current Issues, nr 7(5), pages 775-787.
Janbandhu, Anjali, M. H. Fulekar, 2011. Biodegradation of phenanthrene using adapted microbial consortium isolated from petrochemical contaminated environment. Journal of hazardous materials nr 187(1), pages 333-340.
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Juhanson, Jaanis, 2010. Impact of phytoremediation and bioaugmentation on the microbial community in oil shale chemical industry solid waste. (doctor dissertation). Tartu: University of Tartu Press
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Kriek, Erik, Margarita Rojas, Kroum Alexandrov, Helmut Bartsch, 1998. Polycyclic aromatic hydrocarbon - DNA adducts in humans: relevance as biomarkers for exposure and cancer risk. Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, nr 400(1), pages 215-231.
Lambertsen, Lotte, Claus Sternberg, Søren Molin, 2004. Mini‐Tn7 transposons for site‐specific tagging of bacteria with fluorescent proteins. Environmental microbiology, nr 6(7), pages 726-732.
Lenskaja, Anastassija, 2014. Alkaani hüdroksülaasi (AlkB) mitmekesisus Läänemere bakterites. (bachelor thesis). Tartu: University of Tartu
McGenity, Terry J., Benjamin D. Folwell, Boyd A. McKew, Gbemisola O. Sanni, 2012. Marine crude-oil biodegradation: a central role for interspecies interactions. Aquatic Biosystems, nr 8(1), page 10.
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van Schie, Paula M., Lily Y. Young 2000. Biodegradation of phenol: Mechanisms and Applications. Bioremediation Journal, nr 4(1), pages 1-18.
Viggor, Signe, Jaanis Juhanson, Merike Jõesaar, Mario Mitt, Jaak Truu, Eve Vedler, Ain Heinaru, 2013. Dynamic changes in the structure of microbial communities in Baltic Sea coastal seawater microcosms modified by crude oil, shale oil or diesel fuel. Microbiological Research, nr 168(7), pages 415-427.
Viggor, Signe, Merike Jõesaar, Paulo Santos, Pedro Soares-Castro, Atya Kapley, Maia Kivisaar, 2018. Ability of two bacterial strains isolated from crude oil refinery wastewater treatment plant degrade aromatic and aliphatic hydrocarbons. Manuscript.
Watanabe, Kazuya, Maki Teramoto, Hiroyuki Futamata, Shigeaki Harayama, 1998. Molecular Detection, Isolation, and Physiological Characterization of Functionally Dominant Phenol-Degrading Bacteria in Activated Sludge. Applied and Environmental Microbiology, nr 64(11), pages 4396-4402.
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Institute of Molecular and Cell Biology of the University of Tartu biosafety level 1 laboratory (location: Riia 23, Tartu, Tartu county, Estonia)