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Issue 7 Jan-Feb 2009


YOUNG SCIENTISTS Young Scientists Journal

Issue 7 路

January-February 2009 路

Pages 1-51 路

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Published by MEDKNOW PUBLICATIONS & MEDIA PVT. LTD. B5-12, Kanara Business Center, Off Link Rd, Ghatkopar (E), Mumbai - 400075, INDIA. Phone: 91-22-6649 1818/1816; Fax: 91-22-6649 1817; E-mail:, Web:

Issue 7 | Jan - Feb 2009

Contents... From the Chief Editor - Jonathan Rogers .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 1

Original Articles Effect of Aflatoxin B1 from Aspergillus flavu on MDA-MB-231 Human Breast Cancer Cells - Ana Victoria Colón .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 3 Inhibiting Biofilm Formation of Pseudomonas Aeruginosa: A Two-Pronged Attack - Katherine Cheng, Daniel Anderson, Andrei Dan .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 8 Feasibility of a Low-cost MFC for Electricity Generation and Wastewater Treatment - Kartik Sameer Madiraju. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .14 Pulmonary Effects of Ozone -generating Air Purifiers - Otana Jakpor .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .20

Research Articles Fullerenes - Chris Slaney .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .32 What are Tilings and Tessellations and how are they used in Architecture? - Jaspreet Khaira . .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .35

Book Review João Magueijo’s “Faster than the Speed of Light”: A review - Christopher Barry . .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .47

Opinion Darwin’s Theory of Evolution - Nelson Bridgford .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .48

In Brief Formulae of Squares and Square Roots - Rittik Gautam .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .51

Young Scientists Journal Editors Chief Editor

Editorial Board (articles)

Jonathan Rogers (UK) Technical Malcolm Morgan (UK) Jeffrey Chan (UK) David Sales (UK) Courtney Williams (UK) Publicity Team Kartik Madiraju (Canada) Jolyon Martin (UK) David Sales (UK) Donnchadh O’Sullivan (Ireland) Commissioning Elisabeth Muller (UK) Kim Dunn (UK)

Jonathan Rogers (UK) Christopher Barry (UK) Kim Dunn (UK) Pamela Barraza Flores (Mexico) Natalie Hackman (UK) Aaron Hakim (Canada) Otana Jakpor (USA) Muna Oli (USA) Kimi Tur (UK) Courtney Williams (UK) Kartik Madiraju(Canada) Courtney Williams (UK) George Harvey (UK) Timey Moju (UK) Issy Wingrad (UK) Samuel Gearing (UK)

International Advisory Editors Christina Astin (UK) (Chair) Alexander Willey (UK) Anna Grigoryan (USA) Linda Crouch (UK) Peter Mose Larsen (Denmark) Ghazwan Butrous (UK) Janne Manaster (USA) Andréia Azevedo Soares (UK/Brazil) Thijs-Kouwenhoven (China) Steven Chambers (UK)

This magazine web-based Young Scientists Journal is online journal open access journal ( It has been in existence since June 06 and contains articles written by young scientists for young scientists. It is where young scientists get their research and review articles published. Published by MEDKNOW PUBLICATIONS AND MEDIA PVT. LTD. B5-12, Kanara Business Center, Off Link Rd, Ghatkopar (E), Mumbai - 400075, INDIA. Phone: 91-22-6649 1818/1816; Fax: 91-22-6649 1817; E-mail:, Web:

From the Chief Editor Science has revolutionised our lives, transforming civilisation in the Industrial Revolution and now renovating it once more in a digital revolution that promises to change our lifestyles seemingly beyond recognition from those of our grandparents. In parallel with this, medical advances continue to extend life and improve its quality, while fundamental physics explores realms that would have been unthinkable a century ago. In the backdrop of this scientific age, one of the most exciting phenomena is young people engaging in these developments and considering how we can shape the future.


In accordance with our firm commitment to supporting all these young scientists, over the last few months we have been radically altering our structure to make the whole organisation more efficient and hopefully more helpful to young scientists. As such, the students who work on the journal are now divided up into four teams – Technical, Commissioning, Editorial and Publicity – which coordinate their respective areas under the management of team leaders. The Technical Team primarily administers the website and has recently been working hard to update and refine it. The site now has a new look, featuring multimedia, blogs and latest news, while our article submissions process has been vastly simplified with additional support for new authors. If you are thinking of submitting an article, you may wish to contact a member of the Commissioning Team, whose job it is to encourage and support prospective authors, while the Editorial Team takes over after the submission of an article to check and enhance it, ensuring that it undergoes a rigorous peer-review process. All these activities work hand-in-hand with those of the Publicity Team, which aims to highlight the work we are doing and encourage readership and partnerships with other organisations. If you are interested in joining the team, either as a student or as a member of the International Advisory Board, visit and get in touch.


In terms of the content that we publish, we have been considering how best to present some of the most exciting developments in complex science while ensuring that we remain accessible and engaging. Starting one’s articles at a scientific level considerably above that of the majority of readers is a perennial problem in many scientific publications, severely impairing the reader’s benefit and enjoyment. It is, however, by no means an insuperable barrier with publications such as New Scientist and the many scientific articles and sections in the mass media designed for lay people succeeding in educating readers with little or no specialist knowledge in a particular field. Primarily this is done by condensing the subject matter through leaving out complex details if they add little to the article’s main thrust. Moreover, it is possible to explain complicated facets of an argument by analogy to a process that a reader already understands, as with David Miller’s now famous explanation of the Higgs Boson in terms that even politicians can understand (see higgs3.htm). Also, it may be necessary to explain some of the basic science behind the research for the new material to be intelligible to most readers. (Note here that we are not advocating that we become a journal that merely publishes pithy résumés of current science with little scope for original research, but rather that we further our commitment to helping young scientists develop by providing them with interesting new advances in terms that they can readily understand.) Young Scientists Journal | 2009 | Issue 7


Moreover, too often scientific articles can be sterile and dull, written in monotonous prose devoid of varied constructions or interesting literary techniques. Young people of today’s generation are used to and deserve a more attractive way of writing, so this pitfall must surely be avoided. It is definitely possible to produce a much more engaging style of writing without compromising rigorous scientific accuracy, merely by using more diverse conjunctions, adjectives and turns of phrase.

This Issue

In this issue, we are particularly pleased to be able to introduce a very varied selection of articles, continuing our commitment to publishing original research while including other formats such as a book review, opinion and reviews. It is also encouraging to see articles covering such a large range of disciplines from fascinating research into human breast cancer cells by Ana Victoria Colón to Jaspreet Khaira’s eye-opening discussion of tiling and tessellation and their use in architecture. Environmental concerns are as ever pressing with Kartik Madiraju’s article insightfully investigating the possibilities of microbial fuel cells, while Otana Jakpor’s research poignantly demonstrates the dangers of ozoneemitting air purifiers for asthmatics. Other important areas of current concern have been addressed with an article covering a significant aspect of bacterial antibiotic resistance by Katherine Cheng, Daniel Anderson and Andrei Dan, and Nelson Bridgford’s article gives an interesting stance on the ever-present controversies surrounding creation and evolution. If reading all these isn’t enough for the aspiring scientist, Chris Barry gives an excellent review of João Magueijo’s Faster than the Speed of Light. Finally, we now have a new category of submissions for brief articles demonstrating engaging scientific phenomena, beginning with an interesting delve into square numbers by Rittik Gautam.

The Future

We sincerely hope you enjoy reading the articles in this edition of Young Scientists Journal. If you like it, why not consider getting involved by writing an article or posting on the online forums? It has been a great privilege to work on this issue over the past few months and I would like to thank all students on the team for their commitment and dedication and the International Advisory Board for their tireless energy and invaluable advice. Special thanks must go to Aaron Hakim, who did a superlative job as Acting Chief Editor for a few weeks in my absence over the summer. It is with great pleasure and anticipation that I now hand over the role of Chief Editor to Malcolm Morgan, one of our most experienced and accomplished members, whose tenure shall run to April 2010.

Jonathan Rogers Chief Editor (July – October 2009)


Young Scientists Journal | 2009 | Issue 7

Original Article

Effect of Aflatoxin B1 from Aspergillus flavu on MDA-MB-231 Human Breast Cancer Cells Ana Victoria Colón Academia del Perpetuo Socorro, San Juan, Puerto Rico. Email:


Aflatoxin is a known carcinogen, expelled from naturally occurring fungi such as Aspergillus flavus. In this experiment, different concentrations of aflatoxin were placed in samples of MDA-MB-231 human breast cancer cells and observed for five days. Four samples of MDA-MB-231 human breast cancer cells were prepared: a Control (no aflatoxin), 10 µmol/g aflatoxin B1, 50 µmol/g aflatoxin B1, and 100 µmol/g aflatoxin B1. The change in cell number was determined by cell counting. The results showed that aflatoxin B1 in a 100 µmol/g concentration terminated the breast cancer cells; while the other concentrations decreased the number of breast cancer cells in comparison to the Control. In addition, the morphology of all samples was examined, with pronounced differences between the treated and untreated samples.

Introduction Aflatoxin is a carcinogen from naturally occurring fungi such as Aspergillus flavus. The fungus growth is more prevalent in tropical climates where it tends to grow on food such as corn, nuts and rice. This poses a danger to humans as much of the developing world depends on such food. The mass consumption of carcinogen-tainted food can result in widespread cancer epidemics. This contamination has been linked to liver cancer epidemics in the said third world countries. Indeed, this is what led scientists to classify aflatoxin as a carcinogen. Cancer is defined as an uncontrolled proliferation of cells that are no longer responsive to growth regulating signals, and tend to spread to other parts of the body through direct contiguous extension, blood stream, or the lymphatics. Breast cancer is the second most common cancer world wide, and ranks fifth (after lung, stomach, liver, and colorectal) as a cause of death Young Scientists Journal | 2009 | Issue 7

(Kamangar et al. 2006). Breast cancer is a malignant tumour on the breast, affecting its tissue, and eventually spreading to the lymph nodes which grant the tumour access to the rest of the body. The molecular triggers of breast cancer are unclear, but abnormal oestrogen levels are said to be related. The Susan G. Komen Foundation estimated that 182,460 cases of breast cancer in women would be reported in 2008 alone. Methods to halt cancer growth and eliminate tumours include radiotherapy, chemotherapy, and surgery; all are, however, tied to extremely risky side effects. Scientists have been working for a long time to find less invasive and dangerous methods to attack this increasingly prevalent disease and in this experiment, the effects of Aflatoxin on breast cancer cells will be examined.

Materials and Methods Risk Assessment All procedures are to be handled in a biochemistry 3

laboratory at the University of Puerto Rico Medical Campus. Advanced and hazardous laboratory procedures are to be handled by trained laboratory aides and investigators are to serve as observers. A qualified scientist will be present at all times. Appropriate laboratory attire will be worn at all times, and proper disposal will be made, following the regulations of the University of Puerto Rico Medical Campus. All procedures involving the MDA-MB-231 human breast cancer cells will be performed under the chemical hood to avoid contamination. Preparing the MDA-MB-231 Human Breast Cancer Cell Samples The cells used for this experiment were harvested in 2007 and preserved in a sub-zero refrigerator at a temperature of -143º C. They were removed and placed in room-temperature water to unfreeze and placed in a 100-µL plastic flask with 10 µL of medium to prepare a stock solution of cells. The old medium was discarded after the cells were obtained. A tripsine solution was prepared to clean the cell plates, with 2mL of 25% of tripsine and 3mL of PBS. The flasks were washed with 1 mL of PBS 10% buffer each. 2mL of the tripsine solution was added to one flask, and then the flask’s contents were transferred to a second flask. 2.5mL of the second flask’s contents were then transferred to the first one. Both flasks were incubated. A microtube was prepared with 450 µL of PBS to later dilute the cells for counting. After incubating for 10 minutes, the cell flasks were removed. About 10mL of 2% medium was put in each flask and mixed. The 10mL of medium was transferred from the first flask to the second, now with 20mL of medium in the second flask. 1mL of cells was placed into a new microtube. Then, from that microtube, 50 µL of cells was placed in the PBS solution of the first microtube prepared for counting. Counting the Cells About 20 µL from the cell microtube was placed in the counting chamber. One quadrant of the counting chamber was counted. According to the number of cells counted, the amount of cells and medium needed per well was determined for the experiment. To determine this, the total number of cells was divided by (the number of sections counted in a quadrant), then multiplied by 10 (the mL of PBS 10% buffer used for dilution), then multiplied by 104 (a standard number used when determining cell number). The result was 2.75 x 105 / mL; this is the number of cells per well, round the number off to 2.6 4

x 106. This number was then multiplied by 70 (number of wells to be prepared, although only 50 wells will be used). Result- 14 x 105. This number was divided by 2.75 x 105 (use the original number of cells to assure precision) to determine the amount of cells in mL per well. The result was 5.09mL which was to be subtracted from 70mL (the total volume/number of wells to be prepared) to determine the amount of medium. The result: 64.9 mL. The solution was prepared in a new flask using the amounts determined. The amounts were estimated using 70 wells, or 70 mL, because of the percentage error during the preparation of the cell solution. This error is due to the formation of air bubbles in the solution when preparing the wells. Using the new flask with the cell solution, the cell plates were prepared. About 1 mL of solution was placed in each well- 60 wells in total. 5.09 mL of 10% serum medium was placed in the second flask of cells, where the 5.09 mL of cells were taken from. About 10 mL from the second cell flask was taken, and placed in the first (empty) cell flaks so that there were two 10 mL cell flasks and one cell solution flask. The newly prepared cell plates and the three cell flasks were incubated. Preparation of the Aflatoxin B1 Concentrations About 0.936 mg of pure aflatoxin B1 was weighed. It was provided by Sigma Aldrich and placed in a 75 mm test tube. About 1mL of DMSO was added to the tube. The tube was placed in the Vortex diluter to dissolve the aflatoxin B1 in the DMSO; one more mL of DMSO was added and vortexing was repeated. The solution was 1,000 µmol/g of aflatoxin. The tube was placed in a heated bath of water for a few minutes until the water reached 37ºC. About 1 mL of DMSO was added and the tube was vortexed. Four 45mL tubes were obtained and labelled: one each for the Control, 10 µm/mg of aflatoxin B1, 50 µmol/g of aflatoxin B1, and 100 µmol/g aflatoxin B1. The amount of 2% serum medium and aflatoxin B1 needed in each concentration sample was determined to add up to a total 15,000 µL of solution. For the 10 µmol/g of aflatoxin B1, 14,850 µL of medium and 150 µL of aflatoxin B1 were needed; for the 50 µmol/g sample, 14,250 µL of medium and 750 µL of aflatoxin were needed; and for the 100 µmol/g, 13,500 µL of medium and 1,500 µL of aflatoxin were needed. Then 18mL of the 2% serum medium was added to the Control tube, and 14mL to the other three tubes. From the Control, 850 µL of medium was taken Young Scientists Journal | 2009 | Issue 7

and placed in the 10 µmol/g tube, and 250 µM for the 50 µmol/g tube; 500 µL of medium was taken from the 100 µmol/g tube. The amounts of aflatoxin determined for each concentration were placed in the corresponding tubes. Tubes were sealed and mixed. Adding Aflatoxin B1 to the MBA-MD-231 Human Breast Cancer Cells The cell well plates were removed from the incubator and cells were checked. The old medium was discarded, and 1 mL of PBS 10% buffer added to each well. The PBS was discarded and the process was repeated. The well plates were labelled to indicate the concentration to be added in each well. Counting was to take place over five days, and for each day there were three samples of each concentration counted, for a total of 12 wells per day (three Control wells, three 10 µmol/g of aflatoxin B1 wells, three 50 µmol/g of aflatoxin B1 wells, and three 100 µmol/g of aflatoxin B1 wells). 1 mL of the concentration solution corresponding to each well was added. Wells were incubated. Counting the MDA-MB-231 Human Breast Cancer Cells Twenty four hours after preparing the cell plates, one plate was removed from the incubator. Cells were checked, old medium was discarded from the wells to be counted and 200 µL of tripsine was added to each and discarded. After this, 200 µL of tripsine was readded to the wells, and the cell plate was reincubated for 10 to 15 minutes. The cells were removed from the incubator, and 300 µL of PBS 10% buffer was added to each well to be counted. Twelve microtubes were taken, and labelled according to the sample to be placed in it and the number of the sample: C1, C2, C3, 101, 102, 103, 501, 502, 503, 1001, 1002, and 1003. Each of the well’s contents was placed in the corresponding microtubule. The Control1 sample was placed in the counting chamber. One quadrant of the two-quadrant counting chamber was counted. The Control2 sample was placed in the other quadrant, count, and recorded. The chamber was cleaned with distilled water and dried between samples. This process was repeated with all 12 of the cell samples. After counting all the samples, the cells were destroyed according to laboratory protocols. To obtain the number of cells, an average was taken for each treatment. Cell Morphology Study Before counting the cells each day, the wells (with Young Scientists Journal | 2009 | Issue 7

samples) were were observed under a microscope. Each individual sample was photographed as evidence for the morphological study. The photos taken were meticulously examined and the changes in shape and size of the cells recorded. Cell pictures of the 10 µmol/g, 50 µmol/g, and 100 µmol/g of aflatoxin B1 samples were compared to the Control sample to take note of the changes due to carcinogen exposure. The most important cell changes to be seen were apoptosis (programmed cell death) and change in the nuclear structure of the cells. Statistics T-tests were used to determine statistical significance of the cell numbers between the Control and the samples and between the different samples. Observations Table 1 – Cell count: All samples Control 10 μmol/g 50 μmol/g 100 μmol/g

Day 1

Day 2

Day 3

Day 4

Day 5

8 11 8 0

9 6 4 1

35 11 3 0

53 19 2 0

64 13 5 0

Statistical Analysis Table 2 - T - test results Samples tested


Control and 10 μmol/g Control and 50 μmol/g Control and 100 μmol/g 10 μmol/g and 50 μmol/g 10 μmol/g and 100 μmol/g 50 μmol/g and 100 μmol/g

0.0949 0.0323 0.0179 0.0117 0.0050 0.0039

Null-Hypothesis (no difference) Fail to reject Reject Reject Reject Reject Reject

The T-test analysis between the Control and 10 µmol/g samples had a P value of 0.0949, which is not statistically significant. On the other hand, the Control with the 50 µmol/g of aflatoxin B1, the Control with the 100 µmol/g of aflatoxin B1, the 10 µmol/g of aflatoxin B1 with the 50 µmol/g of aflatoxin B1, the 10 µmol/g of aflatoxin B1 and the 100 µmol/g of aflatoxin B1, and the 50 µmol/g of aflatoxin B1 and the 100 µmol/g of aflatoxin B1 were all statistically significant.

Discussion of Results Current modalities of breast cancer treatment are surgical excision, chemotherapy, and radiotherapy. Carcinogens are not used as a way to halt cancer growth. This study was designed to test the effects of different concentrations of aflatoxin B1 from the Aspergillus flavu on MDA-MB-231 human breast 5

cancer cell stocks. All treatments were compared to a Control stock of MDA-MB-231 human breast cancer cells to determine the change in the numbers of cells. Also, pictures were taken of each sample on each day of counting to observe the morphological changes of the cells and the cell death process of the MDA-MB-231 human breast cancer cells. The Control sample exhibited normal growth, as shown in Figure 1. It began with a lag stage during the first two days of observation, having an average cell count of 8 on the first day and 9 on the second. On the third day of observation, the cell number rose to an average cell count of 35. The cell count kept rising steadily on the other days of counting; the fourth day of counting results in an average of 53 cells, and the counting period for the Control sample culminated in 64 cells. The morphology observed for the Control cells was that expected for living human breast cancer cells. The cells were of an elongated shape, with two antennae on each end. They began forming webs, but then separated to allow room for reproduction. A few cells died during the observation period, but that is to be expected. These cells were the minority, and they became round circles with no center. The 10 µmol/g of aflatoxin B1 sample observed a rise-and-fall trend, as shown in Figure 1. The counting began with an average of 11 cells, but went down to six on the second day of counting, then rose once again to 11 on day three. On day four, the number rose to an average of 19 cells, but lowered

to 13 on the last day. This was not enough to reject significance. Morphologically, cell death could be observed from the second day onwards. Living cells exhibited their expected shape and size, but the number was less than that observed in the Control. Cell death could also be more clearly observed. Due to the toxin treatment, the cell death can be classified as necrosis, a cell death due to a foreign toxin. However, the 50 µmol/g aflatoxin B1 sample showed significant change [Figure 1]. The average cell number continued to decrease to the last day, when the cell number rose from an average of two to five cells. On the other hand, the cell growth trend had showed a gradual reduction of cell numbers up to the fifth day, when the cell number rose once again. But compared to the Control sample, the number of cells in this concentration of aflatoxin B1 is significantly lower than that observed in the Control sample. In the 100 µm/hm of aflatoxin B1- treated cells, no cells were seen on any of the days of counting, with the exception of the second day when one cell was counted. This cell count proves that the 100 µmol/g concentration had an immediate effect on the MDA-MB-231 human breast cancer cells, killing them instantly after the carcinogen treatment. The cells died a necrotic death from the high concentration of aflatoxin B1, and the cells in the other concentrations also suffered from the effects of the toxin. Morphologically, the cells died immediately, and from the first day of counting all the cells that could be seen were small, empty circles- meaning they

Figure 1: Cell Count: All Samples


Young Scientists Journal | 2009 | Issue 7

were dead. As the days of counting passed, fewer dead cells could be seen, and dying cells were the only ones visible.

Environmental Health Sciences (NIEHS). Available from: http:// [last cited on 2008 Dec 20]. 3.

Conclusion The treatment of cells with the carcinogen has proved to be successful, eradicating the breast cancer cells especially in the highest concentrations of aflatoxin B1 solution. Further investigation and precision of the treatment process, along with a risk assessment of the subsequent carcinogenicity of aflatoxin will be necessary to investigate the potential of using aflatoxin B1 in the treatment of breast cancer.

Acknowledgment “Aflatoxins: essential data.” CBWInfo home. Available from aflatoxins.html. [last cited on 2009 Jan 12].


“Cancer - Prevalence, Types of cancer, Growth and spread of cancer, Causes of cancer.” Internet RFC/FYI/STD/BCP Archives. Available from: html. [last cited on 2009 Jan 20].


Cornell University Department of Animal Science. Available from: aflatoxin.html. [last cited on 2008 Aug 14]. [last retrieved on 2008 Jan 28].

6. - Breast Cancer Treatment Information and Pictures. (n.d.). Available from: [last rettrived on 2009 Jan 28].

The author would like to express much appreciation to Dr. Dipak K. Banerjee, from the University of Puerto Rico Medical Campus in Río Piedras, Puerto Rico, for his support and aid during this investigation.




Susan G. Komen for the Cure. (n.d.). Available from http:// [last retrived on 2009 Jan 28].


Breast Cancer Treatment - National Cancer Institute. (n.d.). Available from: treatment/breast/patient. [last retrieved on 2009 Jan 28]. Aflatoxin. (n.d.). Available from: aflatoxin.asp. [last retrived on 2009 Jan 28].



International Agency for Research on Cancer (IARC). “Aflatoxins (IARC Summary and Evaluation, Volume 56, 1993).” IPCS INCHEM. Available from: iarc/vol56/09-afl.html. [last cited on 2008 Dec 12]. NIEHS. “Aflatoxin and Liver Cancer.” National Institute of

10. GraphPad Software, Inc.. “GraphPad QuickCalcs: t test calculator.” GraphPad Software. Scientific graphing, curve fitting (nonlinear regression) and statistics. http://www.graphpad. com/quickcalcs/ttest1.cfm. [last cited on 2009 Jan 20].

About the Author Ana Victoria Colón is a high school senior from San Juan, Puerto Rico. She aspires to become either the first Puerto Rican woman to serve as an ambassador to the United Nations or work at the World Health Organization. Her interests include international relations, oncology, languages, history, writing, and film. Having been raised by two doctors, an interest in science has always been a significant part of her life. However, she does not want to limit herself to only one area of study, and hopes to incorporate all of her interests into a viable career.

Young Scientists Journal | 2009 | Issue 7


Original Article

Inhibiting Biofilm Formation of Pseudomonas Aeruginosa: A Two-Pronged Attack Katherine Cheng, Daniel Anderson, Andrei Dan Lisgar Collegiate Institute, National Research Council, Ottawa, Ontario, Canada. Email:


A bacterial biofilm is a community of adhered bacteria protected by an extracellular matrix of biomolecules. In the biofilm state, bacteria can be more than 1000 times more resistant to antibiotics than planktonic bacteria, and eradication is virtually impossible.[1] In this project, we investigate a particular type of bacteria, Pseudomonas aeruginosa, which is known for forming proliferative biofilms. The Soong et al. study highlights the importance of a specific enzyme called salivate in the biofilm formation of P. aerations.[2] However, the substrates for the enzyme are unknown. Our hypothesis is that this enzyme cleaves sialic-acid-like sugars from the surface of the cell and enables bacterial cohesion, forming biofilms. We tested our hypothesis through cloning of the gene PA2794 which codes for sialidase,[2,3] and purifying the expressed protein. Our results from purification of sialidases show a low yield, ~10% of that expected. Although we did not obtain enough protein to set up our assay, our results have lent us more insight into the structure of sialidase, which we hope will benefit future experiments with biofilms. In tandem to this approach of inhibiting biofilm formation, we also tested the effects of extracts of natural products on P. aeruginosa. Because sialidase promotes biofilm formation of P. aeruginosa by hydrolyzing glycoconjugate-linked sialic acid to increase adhesion, we chose 17 plant species containing compounds similar to sialic acids, to test whether then can inhibit sialidase. We did this by measuring the optical density of bacterial cultures after adding the inhibitors and staining with crystal violet. Our data show that bacteria P. aeruginosa (PAO) measured 0.938 at OD540. The top five inhibitors reduced the OD level of PAO to 50-64% at the same wavelength (lemon peel – 0.473, goji berries – 0.494, ginseng – 0.549, hawthorn – 0.585, white fungus – 0.596). The efficacy of these natural inhibitors is comparable with positive control (PAO + bleach – 0.546), which reduced the OD level of PAO to 58%. We hope these results help further studies that investigate the active compound in these top extracts, eventually leading to a therapy to prevent biofilm formation of P. aeruginosa.

Introduction Pseudomonas aeruginosa is gram-negative, opportunistic human pathogen and a leading cause of pneumonia, lung infection in cystic fibrosis.[3] 8

The bacterium infects the cells lining the respiratory tract by forming a biofilm, a structured community of microorganisms within a self developed matrix. A joint study by Xu et al. confirmed the enzyme sialidase as a facilitator of mucosal infection by participating Young Scientists Journal | 2009 | Issue 7

in biofilm production.[4] However, it is not certain what the enzyme substrates are. We believe that P. aeruginosa encodes a sialidase enzyme (PA2794) that cleaves sialic-acid analogues present on flagella and lipopolysaccharide (LPS) of P. aeruginosa. If it is proven that the enzyme sialidase cleaves sialic acid analogues from those surface polysaccharides, this enzyme would indicate a novel therapeutic target for preventing bacterial infection of P. aeruginosa. Natural plant products provide a diverse array of chemical structures and are known to possess a plethora of biological activities. Pseudomonas aeruginosa is occasionally a pathogen of plants. Seventeen different plant species were selected based on the underlying principle that plants possess non-specific chemicals used in passive resistance to pathogens.[5] Methanolic extracts were made and the inhibitor’s potency measured using a crystal violet reporter assay. Discovering potential inhibitors could lead to the identification of their relevant biofilm targets or potential therapeutics for P. aeruginosa infections. Objectives a) To determine whether surface polysaccharides on P. aeruginosa, lipopolysaccharide or flagella, are the true substrates for PA2794 sialidase enzyme. b) To identify any natural products that may contain an inhibitor of P. aeruginosa biofilms.

Materials and Methods A) Cloning, Expression, Purification of Sialidase Four sets of chromosomal DNA were extracted from Pseudomonas strain PAO1 and amplified by PCR,



consisting on a technique based in generation of millions of identical DNA copies of a specific stretch. Figure 1a shows the four genes on a gel at their expected number of base pairs: 1314 bp and 810 bp. One of these genes was then ligated into first vector PCR2.1 and the recombinant plasmid was inserted into E. coli, which was then grown and harvested. Plasmid DNA purification and digest were done and the second gel confirmed that our DNA insert was in the vector [Figure 1b]. The DNA was then cut out from the gel, purified and inserted into a second vector PET30 and grown in E. coli to express the DNA of protein sialidase. The bottom band on the gel seen in Figure 1c indicates that sialidase was successfully cloned. The bacterial cells were lysed and an attempt was made to purify the recombinant protein by using column chromatography, shown in Figure 2. We did not detect a large quantity of protein by measuring the absorbance of each fraction, and so we ran an SDS-PAGE gel to determine which fractions most likely contained the protein. The fractions were run on a blue gel and the protein was detected on a western blot, as shown in Figure 3, using an antibody to the histidine- tag that we had introduced. B) Biofilm Assays with Natural Products Methanolic extracts of selected plant materials were made by chopping up a weighed amount of each plant material and incubating in methanol (1:5 v/m) at room temperature overnight.[6] The supernatant was removed and dried down and then re suspended in H2O. To set up the biofilm assay, an overnight


Figure 1a-c: PCR and cloning of PA2794 neuraminidase gene from P. aeruginosa

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culture of PAO was diluted in fresh LB at 1/200 and 100µl was added to each well of a 96-well microtitre plate. Inhibitors were added in 1:10 ratio of inhibitor to PAO and the plates were incubated at 37°C for 24 hours. The plates were washed to remove planktonic bacteria, and 125µl of 0.1% crystal violet solution was added to each well to stain the adherent bacteria. The wash was repeated to remove crystal violet not specifically staining biofilm. Then 200 µl of 95% ethanol was added to each well to solubilise biofilms for 10 minutes, and contents of each well were transferred to an optically clear flat-bottom 96-well plate. The optical density (OD) of each sample was measured at a wavelength of 540 nm, to determine if there was a reduction in biofilm formation. Figure 2: Protein purification of C-terminal full-length protein sialidase

Results A) Preventing Biofilm Formation by Inhibiting Sialidase We have successfully cloned and expressed the protein sialidase PA2794, as shown in the PCR and cloning gels, but have not yet purified enough protein to do an assay. The C-terminus full-length tagged sialidase proved to be difficult to isolate because the yield in the elution pools from protein purification was much lower than expected. As seen in the protein analysis in Figure 2, the blue line is the absorbance level of our interested protein, sialidase. The red line shows the absorbance level of a counter ion when added at a constant gradient to elute the purified protein. As seen in the blue line, the elution pool from fractions 18-20 show a very minimal rise in absorbance, much lower than standard. There is another slight shoulder in fractions 14-16, which suggests that our protein might exist in those fractions. These fractions 18-20 and 14-16 were then concentrated 30 times and were run on Western blot. The X-ray image showed a clear band near the 47.5 kDa marker, which represents our C-terminus tagged full-length protein. It also displayed a lower band, a sign of protein degradation. B) Preventing Biofilm Formation with Natural Inhibitors Of the17 original species of plants we investigated, five demonstrated anti-biofilm properties: lemon (Citrus limon), golgi berries (Lycium barbarum), hawthorn (Crataegus monogyna), white fungus (Tremella fuciformis), and ginseng (Panax quinquefolius). These five inhibitors lowered the OD levels up to 50% [Figure 4], with an acceptable level of certainty measured by P-level as seen in Table 1. 10

≈ 48 kDa May well be full length cterninus

=Possible protein degradation

Figure 3: Detection of protein sialidase by SDS-PAGE and Western blot

To ensure the validity of our results, a systematic control was done as shown in Figure 5. A positive control of P. aeruginosa (PAO) in bleach as well as PAO in water confirmed that in these cases, biofilms did not form. Then we determined whether the concentration of the extract affects its abilities to inhibit biofilm formation. The five most effective inhibitors were diluted into 1/10 and 1/100. The results in Figure 6 indicate that the extracts were so potent that dilution did not affect their efficacy. Our last idea was to combine pairs of inhibitors to see whether they have cumulative effects. Figure 7 shows that the combination of white fungus and ginseng was promising, although most inhibitors were most effective when used alone.

Discussion A) We tried to purify the sialidase protein, but our Young Scientists Journal | 2009 | Issue 7

Figure 4: Bar graph showing the average OD of biofilm assays added with inhibitors in a 1:10 PAO: inhibitor ratio. The red line is the average of OD of PAO biofilms in 1/200 concentrations.

Figure 5: A systematic control was done with a negative control of PAO and two positive controls with water and bleach

Figure 6: Biofilm growth in dilute extracts of top inhibitors

Figure 7: Eects of combination of top inhibitors on biofilm growth

Table 1: Corresponding statistical analysis with Figure 4 PAO SDS

Mean Standard error Median Standard Deviation Range Minimum Confidence Level (95.0%)

0.939 0.053 0.928 0.200

1.145 0.131 0.929 0.474

Lily Green Dulse Yellow Red Bulbs Munga peas beans Beans 0.759 1.422 1.419 0.985 1.415 0.051 0.110 0.093 0.065 0.072 0.725 1.479 1.476 0.870 1.339 0.190 0.425 0.335 0.224 0.269

0.710 0.570 1.280 0.116

1.383 0.711 2.094 0.286

0.701 0.437 1.138 0.110

1.340 0.793 2.133 0.235

1.049 0.829 1.878 0.202

0.602 0.780 1.382 0.142

0.840 1.069 1.909 0.155

Lemon Lentils Ginseng Black Lily Dried Grape Seaweed White Aloe Ginger Goji peel sesame seeds haw seed fungus vera berries 0.473 0.014 0.453 0.052

1.282 0.119 1.213 0.460

0.549 0.027 0.541 0.103

0.994 0.038 0.968 0.142

0.791 0.039 0.791 0.146

0.585 0.911 0.018 0.062 0.566 0.937 0.069 0.225

0.894 0.041 0.930 0.152

0.596 0.041 0.604 0.153

0.712 0.041 0.709 0.158

1.131 0.139 0.969 0.482

0.494 0.028 0.451 0.106

0.197 0.418 0.615 0.031

1.470 0.716 2.186 0.255

0.339 0.422 0.761 0.057

0.394 0.817 1.211 0.082

0.552 0.261 0.744 0.533 0.504 0.510 1.085 0.765 1.254 0.084 0.038 0.136

0.523 0.589 1.112 0.088

0.516 0.338 0.904 0.088

0.436 0.487 0.923 0.088

1.283 0.540 1.823 0.306

0.348 0.387 0.735 0.061

results suggested a low yield of sialidase. The secondary band on the Western gel was a sign of protein degradation, which could explain our low yield of purified protein. Another explanation for the outcome lies in the location of the Histag on the protein. The position of the tag may affect protein behavior and its ability to bind to metal ions in the column chromatography. We speculate that the tag was embedded within the Young Scientists Journal | 2009 | Issue 7

core of the protein structure, and thus lowered the affinity between the metal and the His-tag. Therefore, when we purified the protein on the chromatography, much of the sialidase did not bind to the column. B) The top five inhibitors tested, lemon (Citrus limon), goji berries (Lycium barbarum), hawthorn (Crataegus monogyna), white fungus (Tremella fuciformis), and 11

ginseng (Panax quinquefolius), have all reduced biofilm formation up to 50%. These results may be due to the particular families of compounds found in those plants such as flavonoids in lemons and triterpenes in hawthorn, as well as polysaccharides in the fungus and goji berries.[7] Ginsenoside, for example, is a target component in ginseng that could have properties that inhibit biofilm formation. Mass spectrometry will determine the compounds that exhibit activity in the future.[8]

Conclusion A) Preventing Biofilm Formation by Inhibiting Sialidase We conclude that the C-terminus, full-length protein sialidase has been cloned and expressed, but methods have to be modified to increase yield in protein purification in order to set up an assay. Next steps: 1. Cloning of the N-terminal His-tagged full-length construct 2. Protein purification of the N-terminal construct 3. Setting up of separate assay with LPS and flagella of P. aerugino and testing its activity with sialidase after we have enough protein 4. Presence of activity confirms our hypothesis that bacterial sialidase does cleave to sialic acid analogues, which could be a novel therapeutic target in inhibiting biofilm formation. B) Preventing Biofilm Formation with Natural Inhibitors We conclude that methanolic extracts of lemon peel, goji berries, American ginseng, white fungus and haw inhibit the formation of biofilms in the P. aeruginosa to a similar degree as dilute bleach. They remain potent in dilutions up to 1/100 and demonstrate no synergy. Relevant Applications Anti-biofilm sprays and wipes can be synthesized from plant extracts which have shown inhibition. Said products have numerous applications throughout healthcare, increasing sanitation. Numerous P. aeruginosa infections occur every year from infected catheters and endoscope, which can be treated with anti-biofilm wipes that have no toxicity towards humans.[9] Knowledge of the inhibition of


sialidase can be translated to pharmaceuticals, which can treat P. aeruginosa infections, particularly in cystic fibrosis where common antibiotics seem to have no effect due to the added resistance of the biofilm in the mucosal membrane.

Acknowledgments The team would like to thank their distinguished mentors Dr. Susan Logan, Dr. Ian Schoenhofen, and Annie Aubry of the Institute of Biological Sciences at the National Research Council. We appreciate their time and effort in providing a research opportunity for us. This project was supported and funded by Sanofi-Aventis and the Ottawa Health Research Institute.

References 1.









Richards JJ, Ballard TE, Huigens RW 3rd, Melander C. Synthesis and Screening of an Oroidin Library against Pseudomonas aeruginosa Biofilms. ChemCioChem 2008;9:1267-79. Soong G, Muir A, Gomez MI, Waks J, Reddy B, Planet P, et al. Bacterial neuraminidase facilitates mucosal infection by participating in biofilm production. J Clin Invest. 2006;116:2297-305. Moreau-Marquis S, Stanton BA, O’Toole GA. Pseudomonas aeruginosa biofilm formation in the cystic fibrosis airway. Pulm Pharmacol Ther 2008;21:595-9. Xu G, Ryan C, Kiefel MJ, Wilson JC, Taylor GL. Structural Studies on the Pseudomonas aeruginosa Sialidase-Like Enzyme PA2794 Suggest Substrate and Mechanistic Variations. J Mol Biol 2009;386:828-40. Richards JJ, Ballard TE, Huigens RW 3rd, Melander C. Synthesis and Screening of an Oroidin Library against Pseudomonas aeruginosa Biofilms. ChemCioChem 2008;9:1267-79. Useh NM, Nok AJ, Ambali SF, Esievo KA. The Inhibition of Clostridium chauveoi (Jakari strain) Neuraminidase Activity by Methanolic Extracts of the Stem Barks of Tamarindus indicus and Combretum fragans. J Enzyme Inhib Med Chem 2004;19:339-43. Ryu YB, Curtis-Long MJ, Kim JH, Jeong SH, Yang MS, Lee KW, et al. Pterocarpans and flavanones from Sophora flavescens displaying potent neuraminidase inhibition. Bioorg Med Chem Lett 2008;6046-9. Junker LM, Clardy J. High-Throughput Screens for Small-Molecule Inhibitors of Pseudomonas aeruginosa Biofilm Development. Antimicrob Agents Chemother 2007;51:3582-90. Pastoriza Gallego M, Hulen C. Influence of sialic acid and bacterial sialidase on differential adhesion of Pseudomonas aeruginosa to epithelial cells. Colloids Surf B Biointerfaces 2006;52:154-6.

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Suggested Glossary (prepared by Pamela Barraza YSJ editor) PCR (Polymerase Chain Reaction): A laboratory technique used to synthesize large quantities of specific nucleotide sequences from small amounts of DNA using a heat-stable DNA polymerase

N-terminal (amino-terminal residue): The only amino acid residue polypeptide chain with a free amino group: defines the amino terminus of the polypeptide

Ligase: An enzyme that catalyzes the joining of two molecules.

Hydrolysis: A chemical reaction that involves the reaction of a molecule with water: the process by which molecules are broken into their constituents by adding water.

Recombinant plasmid: An extra chromosomal, independently replicating, small circular DNA molecule: commonly employed in genetic engineering DNA digestion: Enaymatic hydrolisys in DNA to yield their simple components. His-tag (Histidine-tag): Protein tag with a string of six His residues. LB (Luria- Bertani medium): Generic rich material for celluar growth. Column chromatography: Tool for protein purification that uses a mixture or proteins added to a column where a specific antibody binds its target protein and retains it on the column while other proteins are washed trough. Mass spectroscopy: a technique in which molecules are vaporized and then bombarded by a high-energy electron beam, causing them to fragment as cations.

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Glycoconjugate: A molecule that possesses covalently bound carbohydrate components (e.g., glycoproteins and glycolipids) Vector: A cloning vehicle into which a segment of foreign DNA can be spliced so it can be introduced and expressed in host cells. S D S - PA G E ( S D S - p o l y a c r y l a m i d e g e l electrophoresis): A method for separating proteins or determining their molecular weights that employs the negatively charged detergent sodium dodecyl sulphate C-terminal (carboxyl-terminal residue): The only amino acid residue in a polypeptide chain with a free carboxyl group. Eluate: The influent from a chromatographic column


Original Article

Feasibility of a Low-cost MFC for Electricity Generation and Wastewater Treatment Kartik Sameer Madiraju McGill University, Department of Chemical Engineering


Lack of electricity and clean water, in many developing parts of the world, has led to the development of unconventional technologies capable of addressing both issues simultaneously; a microbial fuel cell is one such technology. A microbial fuel cell (MFC) is a one or two-chamber device which uses microbes to produce a stable current. Although the concept of using bacteria to produce electricity has been identified many years ago, only recently has this technology been significantly developed for application as an alternative source of energy. In addition to electricity production, microbial fuel cells have also been shown to effectively oxidize organic matter in wastewater, thereby acting as treating agents. The range of power densities obtained from various MFC configurations is anywhere from 0.1 mW/m2 to 1.5 W/m2, however, an effective balance between costs and current output is required before MFCs can be used on a large scale. This study aims to compare the current producing ability of a low-cost MFC with a conventional MFC; this project also highlights the wastewater treating ability of a low-cost MFC.

Introduction Clean water and stable sources of electricity are two major needs that must be addressed in the developing world. Conventional resources are polluting and finite, and therefore not ideal avenues to pursue. Research into alternative methods of electricity production in the form of clean fuels or electricity generating batteries led to the development of microbial fuel cells (MFC). A microbial fuel cell is a device typically comprised of two chambers separated by a cation (or proton)selective membrane: an anode and a cathode chamber. In the anode chamber, microbes oxidize organic matter and release electrons and hydrogen ions. Electrons are transferred to the anode electrode and pass through a circuit, while the hydrogen ions diffuse through the cation-selective membrane into the cathode chamber. Electrons leaving the circuit recombine with hydrogen ions and oxygen in the 14

cathode chamber to form water. An electric current can be measured by applying a resistance to the circuit. Current and power production in an MFC can be optimized by using sophisticated electrodes (platinum-coated carbon cathodes, for example) and membrane materials (costly Nafion membranes) as well as using a very refined carbon source (i.e. glucose). However, to make this technology costeffective and accessible to the developing world, it is necessary to reduce costs of building MFCs without sacrificing quality of wastewater treatment or current density. This study uses an uncharacterized sludge consortium as the catalyst releasing electrons in an MFC constructed with low-cost material. The benefit of using a sludge consortium is its ability to mimic the diversity of microbes found in real wastewater treatment systems, as well as those found in the soils of various environments. S. cerivisiae and Young Scientists Journal | 2009 | Issue 7

E. coli were used in a conventional MFC to compare the performance of an expensive, pure-culture based MFC with the low-cost MFC. These species were chosen for their ease of manipulation (for later studies). In MFCs, microbes oxidize glucose as follows, a simple reaction which forms the basis of microbial fuel cells:[1] C6H12O6 + 6H2O → 6CO2 + 24H+ + 24ePreviously, artificial or natural mediators (neutral red, HNQ, methylene blue) were required to transfer electrons from the microbe to the anode surface, but after a breakthrough study it was shown that a mediator-less MFC could be operated as well.[2] The use of MFCs as a means of stable power generation is considered a potential source of energy in the long-term future. MFCs have been shown to produce power densities anywhere from less than 0.1 mW m-2 (unit power per unit anode surface area)[3] to more than 1.5 W m-2 using a variety of microbes.[2] MFCs have also been especially designed for secondary applications such as wastewater treatment, in which wastewater is provided as the sole carbon source and electron donor.[4]

was left for a Scotch-Brite filter (emulating a cationselective membrane), and another 8 cm ring for the cathode chamber. Sixty holes were made to serve as the cathode [Figure 1]. Both electrode areas were sealed with a conductive silver epoxy and copper wires were used for circuitry. Influent and effluent ports were also made for oxygenation and feeding. A Scotch-Brite sponge cut to the size of the bottle was inserted to separate the anode chamber from the cathode chamber. The cathode chamber was oxygenated and controlled using a rotameter. Tubing inserted into the anode chamber was used to feed a nutrient solution simulating wastewater. The volume of each chamber was 600 ml (cathode) and 200 ml (anode). The anode surface area was 0.225 m2 and the cathode surface area was 0.08 m2. Conventional MFC Construction The anode chamber was built by gluing a plastic endplate with screw holes to a plastic housing chamber fitted with a neoprene gasket. A moist, perforated cloth cut to fit the space within the neoprene gasket was placed on top of the gasket to prevent contact between the cation-selective membrane and carbon cloth electrode. A carbon

Materials and Methods Growth of S. cerivisiae and E. coli Cultures of E.coli K12 (ATCC) were grown at 30C in batch mode in growth medium made by dissolving 10 g tryptone, 5 g NaCl and 5 g yeast extract in one liter distilled water. The pH was adjusted to seven and the medium autoclaved for 15-20 min. at 121°C. The yeast slurry comprised of 5 ml glucose solution (from an 18% (w/v) stock) along with 3 g of yeast dissolved in 9 ml 0.5 M phosphate buffer. Since E. coli requires an external mediator, this slurry was added to 5 ml of methylene blue before inoculation. Sludge consortiums used were obtained from a waste treatment plant (Ste-Catherine’s, Quebec, Canada) and were kept under anoxic conditions during storage (30°C, N2 sparging) and operation (no headspace). Low-cost MFC Construction The bottom of a 1 L plastic bottle was cut off and the bottle was placed upside down; 165 holes were made ~10 cm from the spout and graphite rods (d = 0.7 mm, l = 6 mm) were inserted to serve as the anode. Above the anode holes, a 2 cm thick ring Young Scientists Journal | 2009 | Issue 7

Figure 1: Setup of low-cost MFC during operation


cloth was cut to fit inside the chamber with a small piece sticking out of the holes provided on top of the chamber. This strip was the piece for circuit completion. The same procedure was used to assemble the cathode component of the fuel cell. The fuel cell was screwed shut by placing both chambers together. Both electrodes had a surface area of 0.15 m2. Experimentation and Data Acquisition The conventional MFC was always operated in batch mode while the low-cost MFC was operated continuously. All experiments were conducted using a resistance of 1000 Ω (previously determined using a polarization curve (data not shown)) A 5 mL headspace was always maintained in each chamber in the conventional MFC. No headspace was maintained in the anode chamber of the low-cost MFC to ensure anaerobic conditions. The cathode chamber of the conventional MFC contained 10 ml potassium ferricyanide to act as an electron acceptor, while in the low-cost MFC, oxygen served as the electron acceptor. HOBO Data Logger was used in measuring data. After each experiment, which was ~24 h in duration, the conventional fuel cell was cleaned and sterilized by irradiating under a UV lamp for 15 minutes. The electrodes were sterilized by rinsing with 1 M HCl followed by 1 M NaOH. The low-cost MFC was not cleaned throughout the study to emulate field conditions. All experiments were conducted at 21C. Chemical Oxygen Demand Measurements To measure the reduction in organic matter, influent and effluent samples were taken from the low-cost MFC as well as jaggery, an unrefined complex carbon source used in the conventional MFC (to emulate the organic load of agricultural wastewaters). After diluting, each sample was boiled in sulfuric acid, silver sulfate, potassium dichromate and mercuric sulfate successively. The changing color of each sample due to this process was measured using a spectrophotometer at 620 nm. The absorption is correlated to Chemical Oxygen Demand (COD) (mg/L) using a calibration curve.

glucose ‘spike’ on day 13 increased voltage output to 0.18 mA, which lasted a few hours before decreasing to a stable value of 0.15 mA [Figure 2]. The increase in voltage when a simple sugar was added indicated that the complexity of the nutrient solution played a role in the efficiency of electron transfer in the lowcost MFC. It is also possible that periodic cleaning of the electrodes would have resulted in higher voltage outputs. The maximum current density achieved in the low-cost MFC was 0.76 mA/ m2. Conventional MFC Electricity Experiments Using glucose as a carbon source and yeast as the microbial catalyst, peak currents of 0.43 mA were observed within ~2h of inoculation. The low acclimatization period can be attributed to the use of a pure culture as well as the presence of a cation-selective membrane and catholyte. As the carbon source was depleted, current discharged to 0.1 mA within 20 h, and negligible amounts after 24 h. The peak current density achieved was 2.87 mA/ m2 [Figure 3a]. When jaggery was used as the carbon source a lower peak current of 0.25 mA was observed, which discharged to 0.15 mA within 15 h, and negligible amounts in 24 hours [Figure 3b]. Rather than a depletion of the carbon source, the lower peak current can be attributed to the microbes being unable to digest the complex carbon source completely. Using jaggery, maximum current densities of 1.67 mA/ m2 were achieved. When using E. coli as the microbial catalyst and glucose as the substrate, a peak current of 0.28 mA and peak current

Results and Discussion Low-Cost MFC Electricity Experiments A baseline voltage of 0.01 mA was observed during the ~ eight day acclimatization period. After this, voltage increased exponentially to a peak of 0.17 mA by day 12 using nutrient solution as the sole feed. A 16

Figure 2: Current output over time using a low-cost MFC. Glucose peak at day 13

Young Scientists Journal | 2009 | Issue 7

Figure 4: Current output over time using a conventional MFC with membrane-lysed yeast as the microbial catalyst and glucose as the carbon source

density of 1.87 mA/ m2 was achieved [Figure 3c]. To establish the need for live microbes to produce stable current, a pre-prepared yeast slurry was ‘broken’ (cells membranes were lysed) by passing the sample through a French Press at 20,000 psi; this mixture produced a stable peak current of ~0.2 mA using glucose as a substrate, followed by a decrease to almost nil within 12 h [Figure 4]. The peak current density was 1.3 mA/ m2 The stability of the current can be attributed to the lack of carbon source depletion; the current itself can be attributed to enzymes and cellular mediators shuttling electrons in solution, as well as bacterial residue that did not break under the French Press. Finally, the sludge consortium was also tested in the conventional MFC using glucose as a substrate. A stable current of ~0.25mA was achieved, with a peak of almost 0.3 mA [Figure 5]. This is almost twice as what was observed in the low-cost MFC, indicating the effect of a cation-selective membrane and catholyte in improving current outputs. The maximum current density achieved was 2 mA/ m2, also twice what was observed in the low-cost MFC.

Figure 3: Current output over time using a conventional MFC with yeast as the microbe and a) glucose as the carbon source; b) jaggery as the carbon source; and c) E.coli as the microbial catalyst and glucose as the carbon source

Young Scientists Journal | 2009 | Issue 7

COD Experiments The influent sample and effluent sample from the low-cost MFC yielded COD readings of 83 mg/L and 67 mg/L respectively [Figure 6]. This corresponds to a 20% reduction in organic matter over 15 days continuous operation of the low-cost MFC. It is possible that a reintroduction of effluent into the fuel 17

Figure 5: Current output over time using a conventional MFC with a sludge consortium as the microbial catalyst and glucose as the carbon source

Figure 6: Chemical oxygen demand (mg/L) for low-cost MFC influent and euent samples

cell as a feed would have further reduced the organic load of the solution, thereby treating the solution over repeated cycles. The influent COD is comparable to tertiary filtered municipal wastewater,[5] indicating that the low-cost MFC is an effective oxidizer of organic material. The COD of jaggery was 947 mg/L (data not shown), indicative of organic loads in less refined agricultural wastewaters.

which wastewater influents serve as the source of carbon and microbial catalysts.

Conclusion The low-cost MFC has an average construction cost of <10USD, while the conventional MFC cost more than 150USD (including catholyte and membrane costs). At 10% of the cost, assuming comparable electron transfer efficiencies, the low-cost MFC was able to achieve 25% of the peak current obtained using the conventional MFC. Use of salt bridge as a membrane and electrodes of higher surface area would have dramatically increased current outputs in the lowcost MFC, without affecting the cost greatly.[6] The materials used to build and operate the low-cost MFC are obviously not feasible in real-world applications, however, this study has shown that expensive materials are not necessary to achieve respectable current densities. COD studies support the use of an MFC to perform one stage of wastewater treatment. A system involving several microfiltration and chlorination steps as well as MFC treatment is conceivable. Such a system could lead to the development of MFCs capable of self-powered wastewater treatment, in 18

The benefit of using a sludge consortium as opposed to pure cultures extends beyond the economy: simple reactors can be constructed in developing regions by taking advantage of the microbial ecosystems present in lakes and agricultural soils. A low maintenance, low-cost MFC is ideal for providing cleaner water and stable electricity to developing regions. Obstacles that must be addressed before MFC technology is viable in real world applications include cleaning of electrode fouling, maintaining high current outputs under varying climates, and ensuring electrodes are colonized by the most efficient microbial catalysts.

Acknowledgments This study was conducted at the Biotechnology Research Institute (Montreal, CANADA), an affiliate of the National Research Council of Canada. The author wishes to acknowledge the financial and technical support of Dr. Boris Tartakovsky, Research Officer, Biotechnology Research Institute. Assistance with COD measurements was provided by Ms Michelle-France Manuel, Laboratory Technician, Biotechnology Research Institute.

References 1.

Chaudhuri SK, Lovley DR. Electricity generation by direct oxidation of glucose in mediatorless microbial fuel cells. Nature Biotechnology 2003;21:1229-32.

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2. 3.


Kim BH, Park DH. Mediator-less biofuel cell. 2003; US Patent 5976719. Cheng S, Liu H, Logan BE. Increased power generation in a continuous flow MFC with advective flow through the porous anode and reduced electrode spacing. Environmental Science and Technology 2003;40:2426–32. Liu H, Ramnarayanan R, Logan BE. Production of electricity

5. 6.

during wastewater treatment using a single chamber microbial fuel cell. Environ Sci Technol 2004;38:2281-5. Lopez, A, Pollice, A. Agricultural wastewater reuse in southern Italy. Desalination 2006;187:323-34. Min B, Cheng S, Logan BE. Electricity generation using membrane and salt bridge microbial fuel cells. Water Res 2005;39:1675-86.

About the Author Kartik Sameer Madiraju is an undergraduate student at McGill University, studying for his Bachelor’s of Engineering degree at the department of Chemical Engineering. He has competed for over five years in science competitions at all levels and currently organizes science fairs in his hometown of Montreal. When not conducting fuel cell research, Kartik enjoys playing badminton, cricket, and is an experienced debater.

Young Scientists Journal | 2009 | Issue 7


Original Article

Pulmonary Effects of Ozone -generating Air Purifiers Otana Jakpor Woodcrest Christian High School, 18401 Van Buren Blvd, Riverside, CA 92508 USA. Email:


Air purifiers are marketed to asthmatics and others to improve breathing. However, some air purifiers emit harmful ozone—a key component of smog. This study examines the hypothesis that ozone-generating air purifiers and other household devices that generate ozone may have a negative effect on pulmonary function. According to a recent study by the California Air Resources Board, 10% of California households own an air purifier that may produce ozone. No published studies on the direct pulmonary effects of these air purifiers have been found on Medline. The investigator used an ozone sensor to measure the amount of ozone generated from several types of air purifiers, food purifiers, and assorted ionizing household devices in a home environment. A room air purifier, personal air purifier, and food purifier, respectively, produced concentrations of ozone near the device of approximately 15 times, 9 times, and 3 times higher than a Stage 3 Smog Alert (range of error ±20%). A microspirometer was used to measure pulmonary function before and after exposure to each household device (range of error ±3%). A two-hour exposure to a room air purifier caused a statistically significant drop in an important measure of pulmonary function (FEV1/FVC) among asthmatic subjects, but not among the whole study sample (P<0.05) (n=24). There was a mean decrease’ of 11% in the FEV1/FVC ratio among the asthmatics. A three-hour exposure to a personal air purifier resulted in a statistically significant reduction in pulmonary function among the whole study sample, as well as in the asthmatic subset (P<0.05) (n=10). The mean reduction in FEV1/FVC ratio among the whole study sample was 9.6%, while it was 22.8% among the asthmatics. One asthmatic individual experienced a 29% drop accompanied by a severe asthma attack. A food purifier resulted in a reduction in the FEV1/FVC ratio of 4.2 and 9.6% among the whole study sample and the asthmatic subset, respectively (P<0.05) (n=32). Ozone-generating air purifiers and food purifiers that use ozone may impair pulmonary function.

Introduction Although air purifiers are advertised to improve breathing, certain types of air purifiers emit harmful ozone. Some reports state that certain air purifiers produce ozone in amounts about two times higher 20

than the California state outdoor health standard for ozone.[1] This would be equivalent to having a first Stage Smog Alert inside a home, 24 hours, seven days a week. Though there have been reports on how much ozone ionic air purifiers produce, Medline searches did not show any published studies on the Young Scientists Journal | 2009 | Issue 7

direct effect of ozone-generating air purifiers or other ozone-generating household devices on pulmonary function. The purpose of this research is to clarify the pulmonary effects of ozone-generating air purifiers and other ozone-generating household devices. Although indoor air pollution receives relatively little attention, the California Air Resources Board estimates that the overall indoor levels of air pollutants are often 25-62% greater than outdoor levels.[2] Ozone is one of the principle components of smog. It is a strong oxidant that oxidizes respiratory tissue causing inflammation similar to sunburn. Asthma is a disease of inflammation, so ozone is especially serious for the over 16 million asthmatics in the United States.[3] Many air purifiers are specifically marketed to individuals with asthma and allergies; the very people who are most sensitive to the harmful effects of ozone. In fact, Americans spend more than $350 million each year on air purifiers.[4] There are three basic methods of air purification: (1) filtration through filters, e.g. HEPA filtration, (2) ionization of the air and electrostatic precipitation of the charged particles onto metal electrodes, and (3) ozonolysis of air impurities. Many ionizing and ozonolysis type air purifiers produce ozone either intentionally or unintentionally as a byproduct of air ionization.[5] A recent study conducted by the California Air Resources Board showed that 10% of California households own an air cleaner that may produce ozone, placing approximately 828,000 Californians at risk.[6] Many other ionizing household devices may also create ozone unintentionally. United States federal government regulations do not require air purifiers to meet ozone limits. Neither the United States Food and Drug Administration, which regulates only what it considers medical devices, nor the federal Environmental Protection Agency, which regulates only outdoor air, have regulatory standards for indoor air purifiers. To remedy this regulatory gap, the California legislature passed the legislation in September, 2006, that gave the California Air Resources Board (CARB) the authority to adopt a regulation.[7] The data generated from this study were shared with CARB as they wrote a proposal for a regulation to limit ozone emissions from air purifiers to less than 0.050 parts per million (ppm). The investigator presented the data at the public hearing of the CARB on September 27, 2007. At the hearing, CARB adopted the first regulation in the nation to regulate ozone emissions from air purifiers. Young Scientists Journal | 2009 | Issue 7

Ozone irritates the respiratory system, and can cause coughing, shortness of breath, and chest pain. It also reduces lung function, causes bronchospasm, and aggravates asthma. Ozone causes inflammation and makes the respiratory epithelium more permeable, so an asthmatic might react to a lower dose exposure of an allergen. In addition, ozone aggravates chronic lung disease.[8] Recent studies have shown worrisome evidence that even small increases in outdoor ozone exposure are associated with increased risk of premature mortality, even when the levels are still within the current US Environmental Protection Agency’s standards.[9] In light of the plethora of negative effects that ozone has on the respiratory system, the investigator hypothesized that ozone-generating air purifiers would have a negative effect on pulmonary function, especially in people with asthma and allergies. Additionally, she also hypothesized that other ionizing household devices that produce ozone would have a similar effect. To test pulmonary function, the following common, quantifiable, and objective measures were tested before and after exposure: (1) forced expiratory volume in one second (FEV1)—a measure of how fast a person can blow out, (2) forced vital capacity (FVC)—a measure of the largest volume a person can blow out, and (3) the percent oxygen saturation (O2 sat.) of the blood. The FEV1/FVC ratio was selected as the primary focus of this study, because the American Thoracic Society states that it is “the most important parameter for identifying an obstructive impairment in patients.”[10]

Materials and Methods The investigator designed and executed eight experiments over a period of two years to test her hypothesis that the ozone-generating devices tested would have a negative impact on pulmonary function. (See Table 1.) In experiment A, the concentration of ozone produced by several ozone-generating air purifiers was measured at various distances. The amount of ozone produced by each device was measured with an ozone sensor made by Eco Sensors, Inc., Model A-21ZX, which detects ozone from zero to 10 ppm and has a range of error of ±20%. This particular device was calibrated by Eco Sensors, Inc. immediately prior to the beginning of the experiment. The amount of ozone produced by each ozone21

Table 1: Summaries of experiments Experiment A B C D E F G

Ozone emissions from air purifiers Ozone emissions from food purifier and miscellaneous household devices Pulmonary effects of ozone-generating air purifiers Pulmonary effects of ozone-generating personal air purifiers Pulmonary effects of a non-ozone-generating hepa filter (an additional control) Pulmonary effects of an ozone-generating food purifier Pulmonary effects of an ionic hair blow dryer


Pulmonary effects of a non-ionic hair blow dryer (an additional control)

generating air purifier was measured close to the source, six inches from the source, and one foot from the source. In addition, the Brand #1 room air purifier was tested at two, three, four, and five feet away from the source. The measurements were done consecutively each hour, starting from the closest distance. Each machine was tested with the ionizer set on the highest level for use with people present. Brand #2 was also tested at a higher level called the “away setting,” which is intended for use when people are not at home. The testing was done under typical conditions, in a carpeted living room rather than in a Plexiglas testing chamber, in order to simulate reallife conditions. In experiment B, the concentration of ozone produced by the following devices was measured: Brand #1 Ionizing Bathroom Air Purifier with night light, Brand #2 Ionizing Bathroom Air Purifier, Ionizing Car Air Purifier, Brand #1 Food Purifier, Brand #2 Food Purifier , Ionizing Blow Dryer, Ionizing Ceramic Hair Straightener, Ionizing Smokeless Ashtray, and Ionic Pet Hairbrush. Since the ASTM testing standard for measuring ozone produced by air purifiers is at a distance of two inches, these measurements were taken at two inches from the face of the device. For each test, the length of time and the testing location varied depending upon the device, in order to simulate average “real life” conditions. For instance, the hair appliances were tested in a bathroom after 10 minutes. The food purifiers were tested in a kitchen after completion of the heavy-duty cycle of approximately 23 minutes. The HEPA filter air purifier was tested in a carpeted living room after two hours. The ionizing smokeless ash tray was tested in a bathroom after two hours, and the car air purifier was tested after one hour in a car. In all experiments involving human subjects, informed consent was obtained, and the study subjects filled out a health questionnaire. The study subjects recruited were fellow students, friends, and relatives 22

Number of devices/Subjects tested 4 devices 9 devices 24 subjects 10 subjects 28 subjects 32 subjects 23 subjects 16 subjects

of the investigator. The ages of the study subjects ranged from 12 to 77 years of age. This research involving human subjects was conducted under the supervision of an experienced researcher, Karen Jakpor, MD, MPH, and followed state and federal regulatory guidance applicable to the humane and ethical conduct of such research. A school reviewed and approved the research proposal according to the procedures of the RIMS Inland Science and Engineering Fair. Experiment C tested the effect of an ozone-generating room air purifier (Brand #1) at its highest setting on the FEV1/FVC ratios and oxygen saturations of 24 test subjects. Among the 24 test subjects were three subjects with asthma and one subject with chronic obstructive pulmonary disease (COPD). For simplicity, the subset of patients with obstructive lung disease (either asthma or COPD) will be referred to as the “asthmatic subset” (although it included one subject with COPD). To simulate real life conditions, test subjects were permitted to walk around freely for approximately two hours in the carpeted living room of a house while the air purifier was running. Each subject’s FEV1/FVC ratio was measured before and after exposure to the room air purifier by using a Micro Direct “Micro” spirometer made by Micro Medical Ltd. This instrument has a range of error of ±3%. Spirometry was performed in the standard fashion. Each subject’s oxygen saturation was measured using a Nonin Onyx 9500 Pulse Oximeter, which has a range of error of ±2%. Experiment D tested the effect of a personal air purifier that hangs around the neck on the FEV1/FVC ratios and oxygen saturations of 10 test subjects, before and after wearing a personal air purifier for three hours. This study group included three asthmatics and one subject with COPD. One subject with severe asthma was tested every 15 minutes as a pilot test before any of the other subjects were tested. Young Scientists Journal | 2009 | Issue 7

Experiments E, F, G, and H tested the pulmonary effects of (1) a HEPA room air purifier that does not produce ozone, (2) the Brand #1 food purifier, (3) an ionic hair blow dryer, and (4) a regular non-ionic blow dryer. The numbers of study subjects in experiments E, F, G and H were 28, 32, 23, and 16, respectively. The numbers of these study subjects with obstructive lung disease (either asthma or COPD) in experiments E, F, G and H were 5, 11, 6, and 3, respectively. In experiments C through H, each subject served as his or her own control as pre-exposure and postexposure measurements for each subject were compared. Experiments E and H were also additional group control studies, performed to make sure that the non-ionic equivalents of the ionic devices tested had no adverse pulmonary effects, and that possible psychological effects of being tested were taken into consideration.

each individual’s pre-test and post-test data, since that is a stronger statistical test, making it more likely that the hypothesis would be rejected if it were incorrect.

Results and Discussion In experiment A, the amount of ozone produced by Brand #1 Air Purifier was measured at distances up to five feet. (See Table 2.) Next to the grill of the air purifier, the ozone concentration was highest—7770 parts per billion (ppb), which is approximately 86 times higher than the outdoor California ambient air quality standard for an exposure time of one hour (90 ppb). For perspective, Stage 1 Smog Alert is issued when the outdoor ozone level is at or above 200 ppb, stage 2 at 350 ppb, and stage 3 at 500 ppb.[11] The ozone concentration of Brand #1 near the source was approximately 15 times higher than the level of even Stage 3 Smog Alert. This unexpectedly high result was not a measurement error, and was verified a second and third time.

Statistical analysis with a Student’s Paired T-Test was performed in all the experiments measuring changes in pulmonary function. First, the change in each subject’s FEV1/FVC ratio was calculated. Then the mean change in FEV1/FVC ratio was calculated. Next, the standard deviation, standard error of the mean, two times the standard error of the mean, and 95% confidence intervals were determined. Standard deviation and standard error of the mean are both statistical terms which indicate how variable or spread out the data are from the mean. Standard error of the mean is the most appropriate measure when dealing with a study sample rather than the entire true population. The 95% confidence intervals indicate a range of plausible values, and means that there is only a five per cent chance that the true mean (as opposed to the mean of the test sample alone) falls outside of the confidence interval, and the sample results are a fluke. A statistical calculator found online at was used to determine the standard deviation and mean, but the standard error of the mean and the 95% confidence intervals were manually calculated. The mean change in the ratio and the 95% confidence intervals are included on the graphs. The statistical analysis of the oxygen saturation readings was performed in the same fashion.

Table 2: Experiment A: Ozone concentration (ppb) at various distances from Brand #1 room air purifier

The mean of the group’s FEV1/FVC ratio before exposure and the mean of the group’s FEV1/FVC ratio after exposure were also calculated, in order to determine the overall percentage drop in FEV1/FVC ratio. However, the statistical analysis to calculate standard error of the mean was performed by pairing

Distance from machine 0 in. 1 ft. 2 ft. 3 ft. 4 ft. 5 ft.

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Though the concentration of ozone near the grill of the air purifier was very high, the level quickly dropped off with distance. As seen in Table 2, a distance of five feet the amount of ozone was undetectable. This can probably be explained by the fact that ozone is a highly reactive molecule that reacts with other substances in the room, such as the carpet. This experiment was not performed in a small sealed plastic room such as those used in some industry tests for voluntary standards; sealed plastic rooms do not simulate real life conditions. To simulate typical conditions, this experiment was conducted in an ordinary carpeted living room with people talking and moving about. Perhaps air currents might have caused fluctuations in the measurements. These values should be viewed only as a general indication of the amount of ozone generated, since the test

Ozone ppb 7770 50 30 20 10 0


conditions were not ideal and the ozone sensor itself had a range of error of Âą20%. It is possible that in a smaller room without carpet and a longer testing time, the accumulation of ozone in the room might be greater. It is important to keep in mind that exposure to ozone can be extremely varied in real life situations. There are, for example, some people who make a point of keeping their ozone-generating air purifiers on their bedside Tables, less then two feet away from their faces while they sleep for eight hours; or on their computer desks next to them as they work throughout the day. Some photographs in advertisements for such air purifiers suggest this dangerous manner of placement for their products. In addition to distance, factors such as various fabrics in the room may play a part. For instance, ozone reacts with carpet and other substances to create a multitude of other potentially hazardous known and unknown chemical reaction products.[12] Although the ozone levels dropped off very quickly, the ozone reaction products were probably increasing, and these have unknown effects. The concentration of ozone dropped off similarly in the tests of other ozone-generating air purifiers [Table 3]. It should be noted that the personal air purifier also generated an extremely high level of ozone at its mouth: 4740 ppb, which is approximately 53 times higher than the outdoor California ambient air quality standard for an exposure time of one hour. This level is more than nine times higher than the level of a Stage 3 Smog Alert. Under typical conditions, this personal air purifier is actually run at about six inches from the face, so it is of particular concern that the amount of ozone generated at six inches is 70 ppb. To put that in perspective, 70 ppb is the California ambient air quality standard for an exposure time of eight hours. The concentration of ozone produced by the devices measured in experiment B was significantly lower than the concentration of ozone produced by the ozone-generating air purifiers tested in experiment A, with the exception of the open food purifier [Table 4]. Please keep in mind that the ozone sensor may not be accurate at measuring very low levels of ozone, such as levels below 20 ppb. 24

The Brand #1 food purifier contained the ozone fairly well during the washing of the potatoes on the heavy disinfection cycle. However, when the lid was opened in order to remove the potatoes at the completion of the wash, very high levels of ozone were released [Table 5]. These high levels declined over time but still stood at 30 ppb after five minutes. Experiment C tested the respiratory effects of a twohour exposure to Brand #1 Room Air Purifier on 24 subjects. The effect of the room air purifier on the FEV1/FVC ratio is shown in Figure 1. The room air purifier had no statistically significant effect on the whole study sample of 24 subjects. This is not surprising, considering that many of the subjects were at times moving beyond five feet from the room air purifier. As mentioned earlier, experiment A showed that the concentration of ozone produced by Brand #1 was undetectable at distances at or beyond five feet. Because individuals with obstructive lung disease (such as asthma and chronic obstructive pulmonary Table 3: Experiment A: ozone concentration (ppb) at various distances from ozone-generating air purifiers Distance from device 0 in. 6 in. 12 in.

Brand #1 7770 50 50

Brand #2 Brand #2 on Brand #3 Personal Air Away Setting Purifier 30 4210 40 4740 10 1770 40 70 10 1210 40 10

Table 4: Experiment B: ozone concentration (ppb) at 2 inches from ozone-generating household devices Device Brand #1 ionizing bathroom air purifier with night light Brand #2 ionizing bathroom air purifier Ionizing car air purifier Brand #1 food purifier Brand #1 food purifier 1 minute after opening it to remove food Brand #2 food purifier Ionizing blow dryer Ionizing ceramic hair straightener Ionizing smokeless ashtray

Ozone (ppb) 60 10 10 10 1380 10 10 10 30

Table 5: Experiment B: Ozone concentration (ppb) after opening Brand #1 food purifier Amount of time 1 minute 2 minutes 3 minutes 4 minutes 5 minutes

Ozone (ppb) 1380 80 50 40 30

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disease) are sensitive to ozone at lower levels than other individuals, a subset analysis was performed on the subjects who had obstructive lung disease. Even with a small sample size, there was a statistically significant drop in the FEV1/FVC ratio among the asthmatic subset (P<0.05). The mean of the changes in each subject’s FEV1/FVC ratio was reduced by –0.070 ± 0.023 among the asthmatics. Another way to look at this is to compare the mean FEV1/FVC ratio before and after exposure. As seen in Table 6, the mean FEV1/FVC ratio among the asthmatics dropped by 11%. Experiment D tested the respiratory effects of a three-hour exposure to a personal air purifier on 10 subjects. Figure 2 shows the effect on the mean of the subjects’ delta FEV1/FVC ratio in this experiment. The mean change in the FEV 1/FVC ratio in the whole study sample after exposure to a personal air purifier was –0.076 ± 0.066. This reached statistical significance (P<0.05). An FEV1/FVC ratio of less than 0.70 is a marker that physicians use to help identify patients with obstructive lung disease (asthma or chronic obstructive pulmonary disease). A fall of 0.076 was enough to drop some subjects’ FEV1/FVC ratios into this range. This is an important finding.

It is interesting to note that the drop in the FEV1/FVC ratio with the personal air purifier was significant in the whole study sample, despite the fact that it was not significant with the room air purifier. This is likely because in the case of the personal air purifier, the generator of ozone was consistently about six inches from the face for an extended period of time, so the subjects were exposed to a higher concentration of ozone. The mean change in the FEV 1/FVC ratio in the asthmatic subset in experiment D was -0.178 ± 0.084. (See Figure 2 above.) This was also statistically significant (P<0.05). The change was greater among the asthmatics than the general study sample. This was an expected finding, since asthmatics are known to be more sensitive to the respiratory effects of ozone. Asthma is a disease of inflammation, Table 6: Experiment C: Change in FEV1/FVC ratio after 2- hour. exposure to room air purifier Mean Before Mean After Change % Reduction

Whole sample 0.85 ±0.04 0.85 ±0.05 0 0.0%

Asthmatics 0.73 ± 0.12 0.65 ±0.12 -0.08 -11.0%



0 -0.001



Whole Study Sample


Asthmatic Study Sample

-0.06 -0.07 -0.08


-0.12 Study Sample

Figure 1: Change in FEV1/FVC ratio after 2-hour exposure to ozone-generating room air purifier

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Whole Study Sample


Asthmatic Study Sample

-0.178 -0.2


-0.3 Study Sample

Figure 2: Change in FEV1/FVC ratio after 3-hour exposure to ozone-generating personal air purifier

and ozone causes inflammation of the respiratory tract. This test shows the effects within just 3 hours. Inflammation, however, might take a day or two to show its full effects on pulmonary function, as has been shown in studies of how asthmatics respond to outdoor smog. Delayed testing could be a topic for future research. In Table 7, the effects of the personal air purifier are shown in an alternative way, comparing the group’s mean FEV1/FVC ratio before and after exposure. The FEV1/FVC ratio in the whole study sample fell by 9.6%. The ratio of the asthmatic subset fell by 22.8%. This is especially worrisome because the people in the asthmatic subset had lower than average FEV1/ FVC ratios to begin with. A fall of almost 23% in the FEV1/FVC ratio is definitely noteworthy. To put this fall in perspective, when physicians perform a specific inhalation challenge test to diagnose occupational asthma, they consider a fall of 15% in FEV1 to be significant.[13] Figure 3 shows, in detail, the drop in the FEV1/FVC ratio of one subject with severe asthma while wearing the personal air purifier. This subject began with an FEV1/FVC ratio of 0.89 and was tested every 15 minutes over a period of three hours. The ratio began 26

Table 7: Experiment D: Change in FEV1/FVC ratio after 3- hour. exposure to personal air purifier Mean before Mean after Change % Reduction

Whole sample 0.83 ±0.08 0.75 ±0.08 -0.08 -9.6%

Asthmatics 0.79 ± 0.16 0.61 ±0.09 -0.18 -22.8%

to drop after one hour of exposure, decreasing more rapidly as time went on. This was a pilot test and had originally been planned to go on for eight hours. However, the test had to be terminated at three hours, because a full-blown asthma attack occurred. By the next day the FEV1/FVC ratio had returned to baseline after multiple nebulized breathing treatments. At the point when the experiment was terminated, the FEV1/ FVC ratio had fallen to 0.63, which represents a 29% fall from the starting point. In light of the findings with this subject with severe asthma, the decision was made not to test any other subjects with severe asthma and to limit the exposure time to the personal air purifier to three hours. In fact, only one other subject with previously diagnosed asthma was knowingly tested, but his case was very mild. During the spirometric testing of this experiment, however, the investigator unexpectedly discovered two additional subjects, in year 1, with asthma who Young Scientists Journal | 2009 | Issue 7

0 min.

1 0.9

15 min. 0.89








0.85 0.80


30 min. 0.78

45 min.


0.7 1 hr.



0.6 1 hr. 15 min.

0.5 1 hr. 30 min

0.4 1 hr. 45 min.

0.3 2 hr.

0.2 2 hr. 15 min.


2 hr. 30 min.


2 hr. 45 min.

Time 3 hr.

Figure 3: The effect of a personal air purifier on FEV1/FVC ratio in an asthmatic subject over a period of three hours (Exp. D)

had previously been undiagnosed. Their physicians subsequently confirmed the diagnosis of asthma and placed them on medication. In year 2, two more previously undiagnosed subjects were found to have probable asthma/obstructive lung disease. Neither the investigator nor the supervising research scientist anticipated such a large effect on FEV1/FVC ratio, especially on the asthmatics of this study. The personal air purifier had been purchased through an allergy and asthma catalog. Also, one of the room air purifiers bore the “Seal of Truth” from the Asthma and Allergy Foundation of America. When the preliminary analysis showed evidence of a substantial negative effect on pulmonary function, the decision was made not to expand the number of subjects tested with exposure to ozone-generating air purifiers. Both asthmatics and non-asthmatics experienced some symptoms during exposure to the personal air purifier, such as chest tightness, coughing, and eye, throat, and nose irritation. See Figure 4 to compare the changes in the FEV1/ FVC ratios caused by each air purifier tested in both the whole study sample and the asthmatic subset. The largest drop, by far, occurred in the asthmatic subset with the personal air purifier. The asthmatic subset exposed to the ozone-generating room air Young Scientists Journal | 2009 | Issue 7

purifier experienced the next largest drop in FEV1/FVC ratio. There was a very slight drop in the asthmatic subset in the HEPA filter group. Though this change was statistically significant, it was probably too small to have clinical significance. A larger sample size of asthmatics would be required to ascertain if there is a true effect. It is conceivable that because asthmatics often have reactive airways, the process of blowing hard repeatedly into a spirometer to complete multiple pulmonary function tests might have had a small effect in of itself. Experiment F studied the pulmonary effects of the Brand #1 ozone-generating food purifier. Figure 5 shows a statistically significant reduction in FEV1/FVC ratio after a 23-minute exposure for both the whole study sample and the asthmatic subset. Table 8 shows the reduction in mean FEV1/FVC ratio at 4.17% among the whole study sample and 9.55% among the asthmatic subset. This food purifier Table 8: Exp. F: Change in FEV1/FVC ratio after 23-minute exposure to Brand #1 food purifier Whole sample


Mean before




± 0.0734

Mean after








% Reduction




0.05 0.000214

-0.001 0


Δ FEV1/FVC Ratio

-0.05 -0.07 -0.1

Ozone-generating room air purifier- whole study sample Ozone-generating room air purifier- asthmatic study sample


Ozone-generating personal air purifier- whole study sample Ozone-generating personal air purifierasthmatic study sample


HEPA filter - whole study sample (no ozone)



HEPA filter - asthmatic study sample (no ozone)


-0.3 Type of Air Purifier and Study Sample Figure 4: Change in FEV1/FVC ratio after exposure to various air purifiers (Exp. C, D, E )



Δ FEV1/FVC Ratio



Whole Study Sample


Asthmatic Study Sample




-0.12 Type of Study Sample

Figure 5: Change in FEV1/FVC ratio after 23-minute exposure to food purifier (Exp. F)


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Δ FEV1/FVC Ratio




Ionic Blow Dryer


Non-ionic Blow Dryer

-0.005 -0.00613 -0.01


-0.02 Type of Blow Dryer

Figure 6: Change in FEV1/FVC ratio after 10-minute exposure to blow dryer (Exp. G, H)



Δ Oxygen Saturation

0 0 -0.2



Ozone-generating room air purifier Ozone-generating personal air purifier

-0.304 -0.5

HEPA filter (no ozone) Ozone-generating food purifier Ionic blow dryer


Non-ionic blow dryer



-2 Type of Device

Figure 7: Change in oxygen saturation with exposure to various devices (Exp. C, D, E, F, G, H)

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generated high amounts of ozone that were released when the potatoes were removed at the end of the cleaning cycle. In experiments G and H, neither the ionic or nonionic blow driers caused any significant change in FEV1/FVC ratio among the whole study sample. (See Figure 6.) In experiments C-H there were no statistically significant changes in oxygen saturation among the whole study samples. (See Figure 7.) Presumably a significant amount of bronchospasm, reflecting a larger decrease in FEV1/FVC ratio, would be required to affect oxygen saturation.

Conclusion Although ionizing air purifiers are advertised to improve air quality and promote healthy breathing, some purifiers generate ozone in concentrations far higher than current State of California outdoor air quality standards. The ozone concentration dropped off quickly with distance--possibly because of the ozone reacting with carpet and other materials, perhaps creating byproducts of unknown safety. The ozone-generating room air purifiers tested clearly showed no beneficial effects on the respiratory parameters that were measured. For asthmatics there was a clinically important negative effect on lung function, in contrast to the control tests with a non-ionizing HEPA filter. The ozone-generating personal air purifier had an even greater negative effect, probably because there was a higher exposure to ozone in the immediate breathing zone. The personal air purifier caused an almost 10 % average reduction in the FEV1/ FVC ratio for all tested subjects and an almost 23% reduction for the asthmatics in the group. In light of this evidence of a strong negative effect on lung function caused by the personal air purifier, perhaps the California Air Resources Board should consider tightening their new regulation to set a stricter ozone limit for personal air purifiers. This study found that many ionic household devices, other than air purifiers, produced ozone. Unlike some air purifiers, most of these devices produced very low levels of ozone. A notable exception was the ozonegenerating food purifier which, on opening, released an amount of ozone rivaling that of the ozone30

generating air purifiers. The ozone-generating food purifier also had a negative effect on the pulmonary function of the test subjects, especially among the asthmatics. The new research on ozone-generating food purifiers further corroborates the pattern seen in my earlier findings with regard to ozone-generating air purifiers. This original research suggests that the California Air Resources Board should investigate a variety of other types of ozone-generating household devices and perhaps include them as well in their regulations. Federal regulation is also needed in the United States and in nations around the world to protect unknowing consumers from the pulmonary hazard of ozone-generating air purifiers.

Acknowledgments I would like to thank my mother Dr. Karen Jakpor, an asthmatic, for lending me her pulse oximeter and microspirometer, and for teaching me how to use them. I would like to thank Woodcrest Christian School teacher Mr. Scott Ritsema for lending me a room air purifier for testing. I also wish to express my gratitude to Mr. Ed Dominguez from Eco Sensors, Inc. (New Mexico) for donating an ozone monitor for my research. I would like to thank Woodcrest Christian School teacher Mr. Steve Kinney, my cousin Holly Hall, and my mother Karen Jakpor for hosting science experimentation parties to help me find study subjects. I would like to thank all my study subjects for their participation in this research project.

References 1.






Indoor Ozone. Epidemiology in North Carolina. 3 Nov. 2005. North Carolina Department of Health and Human Services. Available from: indoor.html. [last cited on 2006 Nov 29]. Indoor and Outdoor Air Pollution. Lawrence Berkeley National Laboratory. Lawrence Berkeley National Laboratory. Available from: [last cited on 2008 Jan 9]. Faststats Asthma. Centers for Disease Control and Prevention. Centers for Disease Control and Prevention. Available from: [last accessed on 2009 May 15]. [last reterived on 2006 Nov 29]. The Best Air Purifier Buying Advice. Find Product Reviews and Ratings from Consumer Reports. Consumer Reports, 2009. Web. Available from: appliances/heating-cooling-and-air/air-purifiers/air-purifierbuying-advice/index.htm. [last cited on 2009 Aug 12]. Britigan N, Alshawa A, Nizkorodov SA. Quantification of Ozone Levels in Indoor Environments Generated by Ionization and Ozonolysis Air Purifiers. J Air Waste Manag Assoc 2006;56:601-10. Piazza T. Survey of the Use of Ozone Generating Air Cleansers by the California Public. Air Resources Board. Jan. 2007. University

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of California, Berkeley. Available from: research/abstracts/05-301.htm. [last cited on 2007 Jan 29]. 7. California. Assembly Bill No. 2276. 29 Sept. 2006. 8. Ozone and Your Health. Available from: index.cfm?action=static.brochure. [last cited on 2006 Nov 29]. 9. Bell ML, Peng RD, Dominici F. The Exposure-Response Curve for Ozone and Risk of Mortality and the Adequacy of Current Ozone Regulations. Environ Health Perspect 2006;114:532-6. 10. Pellegrino R, Viegi G, Brusasco V, Crapo RO, Burgos F, Casaburi R, et al. Interpretative Strategies for Lung Function Tests. Eur Respir J 2005;26:948-65. 11. Britigan N, Alshawa A, Nizkorodov SA. Quantification of Ozone Levels in Indoor Environments Generated by Ionization and Ozonolysis Air Purifiers. J Air Waste Manag Assoc 2006;56:601-10. 12. Weschler CJ. Ozone’s Impact on Public Health: Contributions from

Indoor Exposures to Ozone and Products of Ozone-Initiated Chemistry. Environ Health Perspect 2006;114:1489-96. 13. California. California Code of Regulations. Treatment Guideline for Occupational Asthma. Title 8, Section 72. Available from: [last cited on 2007 Jan 14]. 14. Beware of Ozone-generating Indoor ‘Air Purifiers.’ California Air Resources Board. March 2006. California Air Resources Board. Available from: [last cited on 2006 Nov 29]. 15. Hazardous Ozone Generators Sold as Air Purifiers.” Welcome to California. Air Resources Board. 29 Nov. 2006. Available from: [last cited on 2006 May 5].

About the Author Fifteen-year-old Otana Jakpor is a senior at Woodcrest Christian High School in Riverside, California. In April 2008, President Bush presented Otana with the President’s Environmental Youth Award for EPA Region 9 at a White House ceremony in the Rose Garden. This award was given for her original medical research and public policy advocacy on the pulmonary effects of ozone-generating air purifiers. Otana presented her research findings at a California Air Resources Board public hearing on a proposed regulation for indoor air purifiers. The proposal passed and California became the first state in the United States to limit ozone emissions from air purifiers.

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Research Article

Fullerenes Chris Slaney

Since they were first isolated in 1985,[1] fullerenes have been the focus of a great deal of research, and speculation about their properties. For years it had been thought that carbon had only two allotropes in its pure form: graphite and diamond. However, Nobel laureate Harry Kroto and his team managed to isolate this compound after vaporizing graphite in a helium atmosphere. They found that the carbon[2] nucleates in the gas phase to form these closed spheres. This picture shows the C 60 identified originally. It consists of 12 pentagons, alongside a series of regular hexagons. All spherical fullerenes are characterized by these 12 pentagons which are required for the shell to close, a property discovered by Euler. C 60 is the smallest fullerene which is particularly stable, because it is the smallest fullerene in which each pentagon is isolated from each other.

Figure 1: Representation of a C60 “buckyball” fullerene


This is important, as adjacent pentagons experience increased reactivity. As such, C56 and C58 have been found to form derivatives much more easily.[3] They cannot easily be produced in bulk as a consequence. However, some derivatives have recently been found with a distinct ‘egg-shaped’ distortion[4]. C60 was determined to be the most stable compound formed by the process by mass spectroscopy. C60 leads to the single large peak corresponding to 60 carbons. There is also a smaller one corresponding to 70 carbons - another quite stable allotrope. C60 is also unusual in that its 13C nuclear magnetic resonance (NMR) spectrum contains only one peak, because every carbon atom is equivalent:[6] The C60 was originally thought to be superaromatic:

Figure 2: Mass spectrum of carbon atoms in bucky balls. Formation occurs under low energy laser power.5

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Figure 3: Example of a fullerene spectrum

Figure 4: Structure of corannulene (C20H10). It is of interest because it can be considered a fragment of a fullerene. This structure is known as a “buckybowl�.[12]

Far left: dissolved in toluene 2nd: in tetrahydrofuran (THF) 3rd: in water and THF 4th: suspension in water 5th: in water and toluene 6th: after addition of a mild oxidant, in toluene[12]

the three dimensional configuration of the molecule allows for greater delocalization of the p-orbitals than even benzene. However, research has suggested that the conjugative effect in C60 is less than might be expected, as the pentagons avoid containing double bonds[7]. The bonding is therefore better described as superalkene. They are therefore able to react with nucleophiles to a greater extent than might be assumed. Therefore the p-orbital delocalization about each hexagon can be approximated as: One of the principal reasons for interest in the fullerenes, especially in the field of material science, is that one can intuitively see how smaller molecules can be placed inside the shell.[8] This has been suggested as a possible method of drug insertion, or even as a medical treatment in its own right,[9] though great care would have to be taken with fragile endohedral Young Scientists Journal | 2009 | Issue 7

complexes, as the contents are susceptible to influence from the magnetic environment of both the inside and outside (including any added groups) of the fullerene cage.[10] Fullerenes have also been put forward as candidates for organic superconductors.[11] At low temperatures (18K), fullerenes doped with potassium have been shown to experience very little electrical resistance; near 5K, the resistance is negligible. They are also notable for their wide variety of colors in different solvents: Fullerenes need not necessarily take the form of spheres either. Nanotubes also exist, in which the sphere is replaced by a long cylinder, with hemispherical ends. Even more elaborate shapes 33

have been created, such as a ‘pillared graphene’, with many nanotube pores, designed for hydrogen storage[13]. This represents the new wave of fullerene research: moving on from the original spheres to increasingly intricate structures, with a more practical bent.

Bibliography 1. 2. 3. 4.

Nature 318, 162 - 163 (14 November 1985), H. W. Kroto, J. R. Heath, S. C. O’Brien, R. F. Curl & R. E. Smalley Made with ACD Labs/Chemsketch Nature 365, 426 - 429 (30 September 1993), Simon Petrie & Diethard K. Bohme J. Am. Chem. Soc., 2008, 130 (25), pp 7854–7855, Brandon Q. Mercado, Christine M. Beavers, Marilyn M. Olmstead, Manuel N. Chaur, Kenneth Walker, Brian C. Holloway, Luis Echegoyen and Alan L. Balch

5. 6. 7. 8. 9. 10. 11. 12. 13. BDGtext/BDGBucky.html raman-02.htm The Fullerenes, H. W. Kroto, David R. M. Walton, Royal Society (Great Britain), p87, 1993 Nature 366, 123 - 128 (11 November 1993), D. S. Bethune, R. D. Johnson, J. R. Salem, M. S. de Vries & C. S. Yannoni European Journal of Medicinal Chemistry, Volume 38, Issues 11-12, November-December 2003, Pages 913-923, Susanna Bosi, Tatiana Da Ros, Giampiero Spalluto and Maurizio Prato J. Am. Chem. Soc., 2005, 127 (49), pp 17148–17149, Yutaka Matsuo, Hiroyuki Isobe, Takatsugu Tanaka, Yasujiro Murata, Michihisa Murata, Koichi Komatsu, and Eiichi Nakamura Nature 350, 600 - 601 (18 April 1991), A. F. Hebard, M. J. Rosseinsky, R. C. Haddon, D. W. Murphy, S. H. Glarum, T. T. M. Palstra, A. P. Ramirez & A. R. Kortan htm Nano Lett., 2008, 8 (10), pp 3166–3170, Georgios K. Dimitrakakis†, Emmanuel Tylianakis‡ and George E. Froudakis

About the Author Chris Slaney already has A-level chemistry, biology and maths, and is doing further maths and economics. He has an unconditional place at Oriel, Oxford next year to read chemistry.


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Research Article

What are Tilings and Tessellations and how are they used in Architecture? Jaspreet Khaira King Edward VI High School for Girls, Edgbaston, Birmingham. Email:

What are Tilings and Tessellations and how are they used in Architecture? Tilings and tessellations are an important area of mathematics because they can be manipulated for use in art and architecture. One artist in particular, MC Escher, a Dutch artist, incorporated many complex tessellations into his artwork. Tilings and tessellations are used extensively in everyday items, especially in buildings and walls. They are part of an area of mathematics that often appears simple to understand. However, research and investigation show that tilings and tessellations are in fact complex.

What are Tilings and Tessellations? A tessellation is any repeating pattern of symmetrical and interlocking shapes. Therefore tessellations must have no gaps or overlapping spaces. Tessellations are sometimes referred to as “tilings”. Strictly, however, the word tilings refers to a pattern of polygons (shapes with straight sides) only. Tessellations can be formed from regular and irregular polygons, making the patterns they produce yet more interesting.

which means ‘to pave’ or ‘tessella’, which means a small, square stone. Tessellations have been found in many ancient civilizations across the world. They often have specific characteristics depending on where they are from. Tessellations have been traced all the way back to the Sumerian civilizations (around 4000 BC). Tessellations were used by the Greeks, as small quadrilaterals used in games and in making mosaics [Figure 2]. Muslim architecture shows evidence of tessellations and an example of this is the Alhambra Palace at Granada, in the south of Spain [Figure 4]. The Fatehpur Sikri [Figure 3] also shows tessellations used in architecture. Nowadays tessellations are used in the floors, walls and ceilings of buildings. They are also used in art, designs for clothing, ceramics and stained glass windows. Mauritus Cornelius Escher had no higher knowledge of mathematics, yet he contributed to the mathematics of tessellations significantly. He was the youngest son of a hydraulics engineer and was born in Leeuwarden in the Netherlands in 1898. At high school he was an indifferent student and the only part of school that

Tessellations of squares, triangles and hexagons are the simplest and are frequently seen in everyday life, for example in chessboards and beehives [Figure 1]. Tessellating other polygons, particularly irregular ones, is more difficult, as discussed later on.

History of Tessellations The Latin root of the word tessellations is tessellare, Young Scientists Journal | 2009 | Issue 7

Figure 1: Tessellations of hexagons and squares


Figure 3: Fatehpur Sikri (palace), India, Tessellations can be seen on the balconies Figure 2: Mosaic using tessellations, dining room floor, Chedworth Roman villa

interested him was art. After leaving school, Escher briefly studied under an architect before leaving to study ‘decorative arts’. He then began using graphic techniques in his own sketches. Although Escher had no deep knowledge of mathematics his meticulous research into tilings of the plane was extensive and showed thorough mathematical research. In fact he once said, “Although I am absolutely innocent of training or knowledge in the exact sciences, I often seem to have more in common with mathematicians than with my fellow artists.” (The Graphic Work of MC Escher, New York, 1967, p.9). Mathematicians have been fascinated by Escher’s work [Figure 5]. The Alhambra Palace in Granada, Spain with its ceilings Figure 4: Alhambra palace, Granada

Figure 5: Examples of Escher’s work


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and walls covered with beautiful ornamentation, sparked Escher’s interest in tessellating patterns. During his lifetime Escher created and designed over 100 tessellated patterns, many of which are still admired today.

Mathematics Behind Tessellations Which Shapes can Tessellate and Why The simplest type of tessellation is formed from regular polygons. Regular tessellations are tessellations that are made up of only one kind of regular polygon. After some experimentation it can be deduced that equilateral triangles, squares and regular hexagons form regular tessellations. However, pentagons and heptagons cannot do this without leaving gaps or producing overlaps. In order for a shape to tessellate, the interior angles must fill all of the space around a vertex (i.e., their angles must add up to 360°). It is therefore important for us to work out the interior angles of a shape. In order to do this we must know that the exterior angles of any polygon will add up to 360°. From this we can deduce the interior angles of the polygon and check to see if they are factors of 360. If they are, we know that they will completely fill the space around a point, called a vertex. A vertex is the point at which adjacent sides of polygons meet. If this space is filled, the shape will tessellate. An algebraic formula summarizes this. The formula

to work out the interior angle of a regular polygon is: a = 180 - 360/n, where a is the interior angle of the polygon and n is the number of sides of the polygon. This is because 360 divided by the number of sides of the polygon gives the exterior angle, and when the exterior angle is subtracted from 180, we get the interior angle of the polygon. To determine how many polygons are needed to fill the space around a vertex and allow the polygon to tessellate, another formula is used: k(n)= 360/a = 2n/ (n-2), where k(n) is the number of polygons needed. Therefore, it can be deduced that regular polygons that can fill the space around a vertex can tessellate. In more mathematical terms, regular polygons with interior angles that are a factor of 360, can tessellate. Because of this, only regular polygons with 3, 4 or 6 sides – equilateral triangles, squares and regular hexagons – can perfectly fill 360° and tessellate by themselves. Symmetry and Transformations Symmetry is the process of taking a shape and through certain movements, matching it exactly to another shape. A tessellation is created through this, by repeating the same motion a number of times. For example, the straight section of a railway line forms a tessellation as it uses the simplest type of symmetry. The same shape is repeated, moving a same fixed distance in the same fixed direction each time a movement is made.

Triangle – The three exterior angles must add to make 360°. Therefore each one must be (360/3) 120°. The interior angles are (180-120) 60°. 60 is a factor of 360 and so the equilateral triangle will tessellate.

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Square – The four exterior angles must add to make 360°. Therefore each one must be (360/4) 90°. The interior angles are (180-90) 90°. 90 is a factor of 360 and so a square will tessellate.

Pentagon – The five exterior angles must add to make 360°. Therefore each one must be (360/5) 72°. The interior angles are (180-72) 108°. 108 is not a factor of 360 and so a pentagon will not tessellate.

Hexagon – The six exterior angles must add to make 360°. Therefore each one must be (360/6) 60°. The interior angles are (180-60) 120°. 120 is a factor of 360 and so a hexagon will tessellate.

Heptagon – The seven exterior angles must add to make 360°. Therefore each one must be (360/7) 51.4°. The interior angles angles are (180-51.4) 128.6°. 128.6 is not a factor of 360 and so a heptagon will not tessellate.


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The diagram shows the original point is moved in a certain direction. In this case, it is the angle shown. It is also moved a certain magnitude. This shows how far it has been moved from the original point.

This show the translation of whole shapes, not just one point. This is one of the main principals of how tessellations are formed, once the basic pattern is known

The techniques of forming symmetry are called transformations. These include translations, rotations, reflections and glide reflections. One of the simplest types of symmetry is translational symmetry. A translation is simply a vertical, horizontal or diagonal slide. The shape must be moved a certain magnitude in a certain direction. If a shape is marked on a square grid, vectors can be used to state a translation. Vectors appear as two numbers inside brackets e.g. . These numbers refer to the movement of the shape along the x and y axis. Therefore the vector tells us to move the shape five units along the x-axis and four units up the y-axis. Vectors can also have negative values, to show the shape is being moved in the opposite direction. 5 4

5 4

Another type of symmetry is rotational symmetry. This is where a shape is moved a certain number of degrees around a central point, called the centre of rotation. The amount that the shape is turned is called the angle of rotation. Rotations are used in tessellations to make shapes fit together. For example, the trapeziums above, Young Scientists Journal | 2009 | Issue 7

once rotated and translated, fit together to produce rectangles. These rectangles have interior angles of 90° and so four of them (4 Ă— 90) will completely fill the space around a vertex, producing a tessellation. If a tessellation has rotational symmetry it means that the tessellation can be rotated a certain number of degrees (other than 360°) to produce the same tessellation. The most familiar type of symmetry is reflective symmetry. Reflections occur across a line called an axis. The distance of a point from this axis must be the same in the reflection. Therefore corresponding 39

tessellations when the basic, repeating unit pattern is known. They are also used to make shapes fit into one another in a tessellation of non-regular polygons.

Polygon Arrangement Around A Vertex In order to understand the polygon arrangement around a vertex, we need to be able to specify exactly which polygons are found around the particular vertex. To do this, each tessellation is given a code which shows the number of sides of each polygon around the vertex. A triangle, for example, is a 3-sided shape and would therefore have the number 3. The tessellation on the right has reflective symmetry.

points should be the same distance away from the axis of reflection. The last type of symmetry is glide reflection. A glide reflection is a reflection and a translation combined together. It does not matter which of the transformations happens first. The shape that emerges as a result of a reflection and translation is simply called the glide reflection of the original Figure. In order for a glide reflection to take place an axis is needed to perform the reflection, and magnitude and direction are needed to perform the translation. In this example of a glide reflection, the reflection is performed first and the translation is performed second. All of the above transformations are used to produce 40

When writing the polygon configuration around a vertex, we begin with the shape with the least sides and proceed clockwise around the vertex. Example: Pick a vertex. Find the polygon with the smallest number of sides. Write down the number of sides that it has. Above are the regular tessellations of equilateral triangles, squares and regular hexagons. They contain only one type of shape and are often referred to as periodic tessellations or tilings.

Semi-regular Tessellations and Tilings Semi-regular tessellations are non-periodic i.e., they Young Scientists Journal | 2009 | Issue 7

contain more than one type of polygon. In a semiregular tessellation the arrangement of polygons around a vertex must be the same at all vertices. However, only certain polygon arrangements will form semi-regular tessellations. This is because the interior angles of the polygons must fill 360°. If two convex polygons met at a vertex, then the sum of their interior angles must be 360°. This would assume that one of the angles was 180° or more. This is impossible, however, leading to the conclusion that there must be at least 3 different types of polygon around a vertex. In conclusion, semi-regular tessellations are more complex tessellations, with the same configuration of at least 3 polygons at each vertex. Examples of semi-regular tessellations: Other types of tessellation are demi-regular tessellations. These are similar to semi-regular tessellations, except there can be more than one different polygon arrangement at a vertex. Tilings and Tessellations of Non-Regular Polygons Non-regular polygons are those in which the interior angles are not the same, nor are the sides of the polygon of equal length. Non-regular polygons are slightly more difficult to tessellate, as they first Young Scientists Journal | 2009 | Issue 7

To prove that this is a semi-regular tessellation, we must look at the polygon arrangement at more than one vertex.



The configuration at vertex 1 is, and the configuration at vertex 2 is also This proves that it is a semi-regular tessellation.

require various transformations before they fit into a shape that will tessellate automatically. Non-regular tessellations are those in which there is no restriction on the order of the polygons around vertices. All triangles and quadrilaterals will tessellate, but not all pentagons and hexagons will. When tessellating, we must remember that we are trying to find a polygon arrangement that will fill the 360° around a vertex. Triangles All interior angles of all triangles, whether equilateral, isosceles or scalene, will add up to 180°. Therefore we can fill the space around a vertex, if we use two of each of the angles of the triangle. 41

These three angles sum to 360 degrees (540 - 180 = 360).

There are many different ways to arrange the angles of a triangle around a point to fill 360 degrees. Note that around each vertex, there are two copies of angle 1, two copies of angle 2, and two copies of angle 3. The above examples will not tessellate because the sides are not lined up. If we line the sides up, however, we can tessellate any type of triangle. In the diagram below, two copies of each angle are still used, to fill the space around the vertex. There is another method that can be used to tessellate any type of triangle. This method is fairly similar to the one described above, except that after three triangles are arranged to form a 180° angle, the pattern is reflected, producing a slightly different tessellation. At this point, however, the triangles are reflected (flipped)

Quadrilaterals The method used to tessellate quadrilaterals is very similar to the method of tessellating triangles. We must first take into account that the interior angles of a quadrilateral add up to 360°. Therefore, we only need one of each angle to fill the space around a vertex. Again there are many possibilities that will not tessellate. We need therefore need to line up the sides to create a pattern that will tessellate. Pentagons Pentagons, as seen before, become much more difficult to tessellate because the sum of the interior angles of a pentagon is 540°, not 360°. Therefore, Note the arrangement of angles around the central vertex

Rotatiing the quadrilateral around the midpoints of its sides produces an arrangement that will tessellate.


the angles will not fill the space around a vertex. However it is possible to tessellate pentagons, under certain conditions. If we think of the interior angles of a pentagon as 180° + 360°, rather than 540°, we can tessellate some pentagons. There are three techniques of tessellating a pentagon. The first method requires the pentagon to contain 2 adjacent angles that add up to 180°. This method uses translations, rotations and reflections to extend the pentagon into a tessellation. These two angles sum to 180 degrees.

As the two adjacent angles add up to make 180°, we can translate the two angles to form a straight line, along the tops of the pentagons.

As the remaining three angles sum to 360°, they can be rotated and translated to fill the space around a vertex.

Now, to extend the pattern, we can either translate (move) the pattern:

In order to form a tessellation, the pattern can then be translated or reflected. The second method requires the pentagon to contain two non-adjacent right angles, as well as two pairs of sides of equal length (as shown in the diagram). This method ensures that all of the right angles meet at a vertex, in order to fill the 360°. This method uses multiple reflections, and takes advantage of the sides of equal length, to tessellate the pentagon. Young Scientists Journal | 2009 | Issue 7

The final technique of tessellating a pentagon begins with a triangle. One side of this triangle is broken up and two new sides are created from this, as shown in the diagram.

This new pentagon is then rotated around the midpoint, producing a parallelogram. This can be expanded as a quadrilateral (see earlier section) to form a tessellation. Hexagons and other non-regular polygons can also form tessellations after various reflections, rotations and translations, which produce new shapes that can easily form tessellations. The mathematics behind tessellations requires the space around a vertex to be filled completely without gaps or overlaps and in more complex shapes (pentagons etc.), this can be achieved by manipulating the shape.

Tessellations in Nature Tessellations can be found everywhere we look. Nature contains many different tessellations, some being arrangements of polygons. Figure 6 shows some examples of tessellations found in nature.

Tessellations in Architecture Tessellations are used extensively in architecture, both two-dimensional and three-dimensional. Tessellations are easy to use in architecture, especially in two-dimensional, because even the simplest repeating pattern can look astonishing when it covers a large area. Tessellations are used in Islamic architecture, for example the Taj Mahal [Figure 7], Agra and Fatehpur Sikri (a palace in India) [Figure 7]. Tessellations were also used by the Greeks and Romans in mosaics. The top pictures in Figure 8 are of Hagia Sophia in Istanbul. They are examples of Islamic tessellations and tilings. The dome uses a three-dimensional tessellation of rectangles to cover it. There are further Young Scientists Journal | 2009 | Issue 7

Figure 6: Turtle shell, honeycomb, pineapple, spider web, girae markings and fish scales-examples of tessellations in nature


Figure 7: (Left to right) Mosaic at Chedworth Rooman villa, decorative panel at Fatehpur Sikri, Patterns on the Taj Mahal

Figure 8: (Above) Hagia Sophia, Istanbul and (below) the Blue Mosque, Istanbul

Figure 9: Federation Square, Melbourne – this building is made up of a range of right-angled triangles. As can be seen, it is a very complex structure and is made out of many different materials.

Figure 10: London City Hall – This building projects a 2-D tessellation onto a ring. Each ring of the building is then slightly displaced


Figure 11: Louvre, Paris – Again the Louvre projects a 2-D tessellation onto a 3-D structure. Each separate face of the pyramid contains a section of a 2-D tessellation. A rhombus is used as the main shape

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Figure 12: London Swiss Re Building. This building looks like a giant gherkin. It was made using computer modelling and without the use of complex computer models, buildings like this would not be possible. The gherkin uses rhombus shapes, which are manipulated to curve around the shape of the building

Figure 13: British Museum. Ceiling of Great Hall. The grand court of the British Museum, London contains this beautiful, glass, tessellated roof. It uses triangles to fill the curved roof. This three-dimensional tessellation, like the one above, is very complex as it is not simply a projection of a two-dimensional tessellation onto a three-dimensional object. The question that arises from studying such a complex structure is: How can we manipulate a triangular tessellation to form a curve?

examples of Islamic tiling inside the building.


The beautiful tilings shown in Figure 8 are more examples of Islamic tilings at the Blue Mosque, also in Istanbul. The tilings are extremely intricate and delicate, covering the walls and ceiling.

The following diagrams were taken from the ‘Totally Tessellated’ website and were not the author’s own: The source of the figures are as in the biography section.

The above examples are all of two-dimensional tessellations in architecture. Three-dimensional tessellations have also been used in various buildings and some examples are given below. Instead of using rectangular building blocks, the basic shape of the building blocks has been manipulated to produce interesting shapes. This is a difficult principle to master, but there have been some attempts. Buildings that use three-dimensional tessellations, once completed, generally are elegant and eyecatching. However, extensive planning and the use of complex computer models are required.


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Websites regular.2.html tessellate/?version=1.6.0-oem&browser=MSIE&vendor=Sun_ Microsystems_Inc. Pictures (in order of appearance in article) Chessboard [Figure 1]:


information/glossary/c_summ.htm Honeycomb [Figure 1]: Chedworth Roman Villa [Figure 2] and Fatehpur Sikri [Figure 3]: J. Khaira Alhambra Palace, Granada [Figure 4]: architecture/1/0/K/i/ALHAMBRA_Int_lassic.H.jpg Escher’s work [Figure 5]: 1. MathE302/escher.jpg 2. Colored diagrams on right (pages 3, 4 and 5): Totally tessellated website - polygons/regular2.html#Anchor-Analysis-16576 (diagrams on left by J. Khaira) Diagrams of transformations: background/symmetry.1.html (trapeziums by J. Khaira) Images of tessellations: Diagrams explaining triangle, quadrilateral and hexagon tessellations: polygons/regular2.html#Anchor-Analysis-16576 Fish scales [Figure 6]: Pineapple [Figure 6]: image/26435878 Honeycomb [Figure 6]: Turtle Shell [Figure 6]: Giraffe [Figure 6]: Spider Web [Figure 6]:


Decorative panel at Fatehpur Sikri [Figure 7]: enback/swastika7/jpg Taj Mahal [Figure 7]: a7f26616df.jpg?v=0 Chedworth Roman Villa [Figure 7]: J. Khaira Blue Mosque [Figure 8]: Hagia Sophia [Figure 8]: hagia-sophia-photos/ Federation Square [Figure 9]: images/gallery/im19.htm London City Hall [Figure 10]: london-city-hall-green.jpg Louvre, Paris [Figure 11]: wp-content/uploads/2008/04/louvre-glass-pyramid-paris-prlouve3. jpg Gherkin [Figure 12]: British Museum Ceiling [Figure 13]: algorithms-for-architecture/ Books “Fearful Symmetry” by Ian Stewart and Martin Golubitsky “Penrose Tiles to Trapdoor Ciphers” by Martin Gardner “Geometric Symmetry” by E.H. Lockwood and R.H. Macmillan “The Golden Ratio” by Mario Livio “The Changing Shape of Geometry” edited by Chris Pritchard “Indra’s Pearls – The Vision of Felix Klein” by David Mumford, Caroline Series and David Wright.

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Book Review

João Magueijo’s “Faster than the Speed of Light”: A review Christopher Barry The King’s School, Canterbury, United Kingdom. Email:

Magueijo’s book is the story of his journey in physics with the revolutionary, if not heretical, speculation that the speed of light may not actually be constant with respect to time. The book takes the reader from his original spark of inspiration in St. John’s College, Cambridge, through the years that followed in Imperial College and other locations around the world pursuing the idea with his research partners.

somewhat paradoxically, you will see them at their best – I have always felt that the most brilliant textbook ideas are best explained by their negatives. Forcing them to undergo cynical challenge, a counterpart to courtroom crossexamination, brings them to life. For these reasons, I believe that you should still read this book even if in the end VSL does not deliver the goods. However, it is obvious that this story will be far more interesting should the goods indeed be abundantly delivered.”

The varying speed of light (VSL) theory is still very much alive in physics four years after the book was first published and anyone with an interest in cosmology should read this book. In order to give the reader the background, Magueijo first explains the other important ideas in cosmology – relativity, the beginnings of the universe, inflation, and cosmological problems – before telling the story of his own. As he says:

As well as explaining the science, the author recounts his experiences of a career in science, giving revealing insights into both the excitement of being on the forefront of research and the flaws in the system and some people which scientists must battle against.

“Narrating the VSL story will force me to explain in detail the very ideas that theory contradicts or bypasses: relativity and inflation. Therefore,

Magueijo’s literary style is grabbing, if a little provocative. You will not be bored by the literature, the story, or the science.

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Darwin’s Theory of Evolution Nelson Bridgford United Kingdom. E-mail:

Darwin’s Theory of Evolution is the widely held notion that all life is related and has descended from a common ancestor: the birds and the bananas, the fishes and the flowers – are all related. Darwin’s general theory presumes the development of life from non-life and stresses a purely naturalistic (undirected) “descent with modification”. That is, complex creatures evolve from more simplistic ancestors naturally over time. In a nutshell, as random genetic mutations occur within an organism’s genetic code, the beneficial mutations are preserved because they aid survival - a process known as “natural selection.” These beneficial mutations are passed on to the next generation. Over time, beneficial mutations accumulate and the result is an entirely different organism (not just a variation of the original, but an entirely different creature). In this case study I intend to question whether Darwin’s theory is as relevant today as it was in 1859, and how it matches up to evidence from the past 150 years since the publication of The Origin of the Species. I intend to look at recent evidence for and against this groundbreaking theory, which is controversial even today. Evolution’s biggest rival is creationism. Creationism is one of the oldest theories in the world, and one of the most believed. Creationism is a religious belief that a higher being created the world and all that resides within it. This theory completely opposes Darwin’s theory, which cites no divine intervention. Darwin found many roadblocks while writing and researching his theory, and faced much opposition from the church and many religious leaders as this theory directly contradicted their teachings. 48

Creationists would argue that all life seems designed and scientists can’t explain why, so they use the explanation of divine intervention; and they would also argue that people’s brains and minds are separate from one another which could lead them to dwell on, yet again, divine intervention. Darwin first became interested in species while on a navy ship heading to the Galapagos Islands. The Galapagos Islands have species found in no other part of the world, though similar ones exist on the west coast of South America. Darwin was struck by the fact that the birds were slightly different from one island to another. He realized the reason this difference existed was connected with the fact that the various species live in different kinds of environments. This was the first part of his road to discovery. Darwin identified 13 species of finches in the Galapagos Islands. This was puzzling since he knew of only one species of this bird on the mainland of South America, nearly 600 miles to the east, where they had all presumably originated. He observed that the Galapagos species differed from each other in beak size and shape. He also noted that the beak varieties were associated with diets based on different foods. He concluded that when the original South American finches reached the islands, they dispersed to different environments where they had to adapt to different conditions. Over many generations, they changed anatomically in ways that allowed them to get enough food and survive to reproduce [Figure 1]. Nineteenth century critics of Darwin thought that he had misinterpreted the Galapagos finch data. They Young Scientists Journal | 2009 | Issue 7

said that God had created the 13 different species as they are and that no evolution in beak shape had ever occurred. It was difficult to conclusively refute such counter arguments at that time. However, 20th century field research has proven Darwin correct. Today we use the term adaptive radiation to refer to this sort of branching evolution in which different populations of a species become reproductively isolated from each other by adapting to different ecological niches and eventually become separate species [Figure 2]. Darwin came to understand that any population consists of individuals that are all slightly different from one another. Those individuals with a variation that gives them an advantage in staying alive long enough to successfully reproduce are the ones that pass on their traits more frequently to the next generation. Subsequently, their traits become more common and the population evolves. Darwin called this “descent with modification”. But as always to every argument there is a counter

argument, as creationists believe they can disprove spontaneous generation of life -“Louis Pasteur disproved spontaneous generation of life. Sir Fred Hoyle and Charles Wickramasinghe stated in their book, Evolution from Space, that “they estimated the probability of forming a single enzyme or protein at random, in a rich ocean of amino acids, was no more than one in 10 to the 20th power.” Next, they calculated the likelihood of forming all of the 2000+ enzymes used in the life forms of earth. This probability was calculated at one in 10 to the 40,000th power. They popularized the following cliché: “belief in the chemical evolution of the first cell from lifeless chemicals is equivalent to believing that a tornado could sweep through a junkyard and form a Boeing 747.” (Quote: The latest theory regarding the evolution of man (sapiens sapiens) suggests that modern humans and apes originate from an apelike ancestor that resided on earth. The theory states that man, through a mixture of environmental and genetic factors, emerged as a species to produce the diversity of ethnicities seen today, while modern apes evolved on a separate evolutionary pathway. Mankind’s origin has usually been explained from an evolutionary perspective. In addition, the theory of man’s evolution has been and continues to be modified as new findings are discovered, revisions to the theory are adopted, and past concepts proven incorrect are discarded [Figure 3]. “One of the foremost evidences for the evolution of man is homology, that is, the resemblance of either anatomical or genetic features between species. For instance, the similarity in the skeleton structure of apes and humans has been correlated to the homologous genetic sequences inside each species as strong confirmation for common ancestry.

Figure 1: Shows adaptive radiation in Galapagos finches

Figure 2: Shows a diagram depicting natural selection

Young Scientists Journal | 2009 | Issue 7

Fig 3: Ascension of man, showing how modern humans might have evolved.


This argument contains the major assumption that similarity equals relatedness. In other words, the more comparable two species appear, the more closely they are related to one another. This is known to be a poor assumption. Two species can have homologous anatomy even though they are not related in any way. This is called “convergence” in evolutionary terms. It is now known that homologous features can be generated from entirely different gene segments within different unrelated species. The reality of convergence implies that anatomical features arise because of the need for specific functionality, which is a serious blow to the concept of homology and ancestry.” (Quote modified: In conclusion, I think evolution is the most logical explanation for the creation of man but creationism


still has many valid points and it comes down to choosing what you believe in. Even today the word “evolution” is not used in the state of Kansas’s science curriculum. To fully understand your own view you need to understand a different view and I feel that some creationists are being very close-minded when it comes to learning and understanding a viewpoint different to their own.

Resources - - - htm - (finch diagram) -http:// (ascension of man diagram) Evolution 101 podcast

Young Scientists Journal | 2009 | Issue 7

In Brief

Formulae of Squares and Square Roots Rittik Gautam, Age 13 103 Ram Vihar, Ballupur, Dehradun, India. Email:

Here is a new and short way to find the square of a given number if the square of the predecessor and successor is given. To find the square of next number we add a particular odd number. If we know the square of x and we have to find the square of next number then we add the odd number which is in the place of the next number of x. E.g. if we know the square of 10 then we can find the square of the next number which is 11. We will add the 11th odd number to square of 10. Example First step- (10)2+11th odd number = (11)2 Second step-100+21= (11)2 Third step-121= (11)2 Formula in terms of x (x+1)2 =x2 +2x+1 To find the square of previous number we subtract a particular odd number. If we know the square of x and we have to find the square of previous number then we subtract the odd number which is in the place of the previous number of x. E.g. if we know the square of 11 the we can find the square of the previous number which is 10. We will subtract the 11th odd number to square of 11.

Young Scientists Journal | 2009 | Issue 7

Formula First step- (11)2-11th odd number = (10)2 Second step-121-21= (10)2 Third step-100= (10)2 Where x is any positive integer (x-1)2= x2 +2(x-1) +1 Short cut for odd numbers- If we have to find a big odd number then we double the predecessor number because 1st odd number is 1 and the 1st even number is 0 so odd numbers are 1 point bigger than the even numbers occurring at same place. E.g. 20th odd number is 39 and 20th even number is 38 so the odd number occurring at the same place is bigger than the even number in that same place. Here place means the position where they lie. E.g. they both lie in the 20th place. (as per example above). E.g. of Short cut for odd numbers -124th even number is 246.We doubled the predecessor 124 to get the 124th even number. First step-124th even number= 2(124-1) Second step-124th even number=2(123) Third step-124th even number=246


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