3 in 10 people lack access to clean potable water, with water sources being microbially-contaminated. Existing water purification technologies suffer major drawbacks: high production barriers, being prohibitively expensive and requiring high energy inputs, impractical in the rural context.
PaperPure is designed with this challenge in mind. PaperPure is a novel, low-cost, eco-friendly, paper-based antimicrobial water filter for point-of-use applications. It is laden with bactericidal silver nanoparticles (AgNPs) produced from the reduction of silver nitrate (AgNO3) by grass extract.
We prototyped and tested PaperPure:
Our findings prove Ag+ is reduced to AgNPs by extract and impregnates onto filter paper, exerting strong (>99.8%) antimicrobial efficacy with negligible silver leaching. We plan to test its efficacy against more waterborne pathogens and seek regulatory approval for PaperPure's deployment.
According to the World Health Organisation, 2.1 billion people lack access to safe potable water. The consumption of contaminated water, in turn, causes high child mortality rates due to increased transmission of waterborne diseases including typhoid, dysentery and cholera. Also, in disaster situations, it may be possible that the only water source may be microbially contaminated.
We wish to tackle these problems through our innovation, PaperPure, a low-cost antimicrobial water filter which may be easily synthesized on site. PaperPure is a filter paper laden with antimicrobial silver nanoparticles (AgNPs) produced by the bioreduction of silver nitrate using grass.
Having conceptualised PaperPure, we had to experimentally test some aspects of it:
Having reviewed scientific literature, we expect that our grass extract would bioreduce silver nitrate into AgNPs which would impregnate unto the filter paper surface, allowing it to kill microbes in microbially contaminated water poured through it, though we do expect to see some silver leaching. Since the antimicrobial efficacy and extent of silver leaching is expected to be proportional to the silver nitrate concentration used for prototype preparation, we hope to find the optimal concentration for use such that antimicrobial efficacy is high while silver leaching is still within internationally established acceptable ranges.
PaperPure’s conceptualisation was inspired particularly by two advancements in scientific research.
Firstly, the increased research showing the antimicrobial properties of silver nanoparticles (AgNPs). Research has shown that AgNPs possesses an “oligodynamic” effect, with high toxicity against more than 650 microorganisms, including gram-positive and gram-negative bacteria, fungi and viruses at even low concentrations (Malarkodi et al., 2013). It achieves this through mechanisms including adhesion and penetration of microbial cells, generation of reactive oxygen species and free radicals, and microbial signal transduction pathways modulation. (Dakal, Tikam Chand et al., 2016). Given the multi-mechanistic action of AgNPs, microbes face difficulty gaining a resistance toward it.
Secondly, research on new methods of AgNP production has inspired our conceptualisation of PaperPure. Currently, there exist two main ways to synthesize silver nanoparticles: the top-bottom and the bottom-up approach. However, these methods are either hazardous to human health or environmentally detrimental due to toxic by-products and reactants (Ahmed, Shakeel et al., 2016).
Hence, recent research has proposed a new eco-friendly method of AgNP production called “green synthesis” where reductases in plant extract bioreduce Ag+ to AgNPs. Starch in plants additionally acts as capping and stabilising agents, preventing agglutination and maintaining structural stability of AgNPs. In producing PaperPure, we selected this method of AgNP production given its eco-friendly and more accessible nature.
Combining these two research advancements and adding in our own ideas led to the innovation of PaperPure. It is produced by using grass to bioreduce silver nitrate into AgNPs that impregnate a filter paper surface, exerting an antimicrobial effect on microbially contaminated water poured through it.
In conceptualising PaperPure, we also considered current products in the market that attempt to solve the same problem PaperPure tackles. There are two main products: halogen-based tablets and LifeStraw.
Halogen-based tablets disadvantages vs PaperPure’s traits:
|Creates an unpleasant taste||No effect on the taste of water|
|Sediments not removed||Filters out small sediments given filter paper component of PaperPure|
|Long-term exposure poses health risks||Poses negligible health effects PaperPure has negligible silver leaching (shown through experiments) and silver has relatively low toxicity|
|Iodine tablets not suitable for people with thyroid conditions and pregnant women||PaperPure caters to a wider range of people|
LifeStraw's disadvantages vs PaperPure's traits:
|$19.95 straw filters maximum of 1000 litres, costing $0.02/litre filtered||$0.15 PaperPure filters 10 litres, costing $0.015/litre filtered|
|Bulky: 57 grams, 2.5cm diameter, 22.5cm length||PaperPure is lightweight paper <1g and small, decreasing transport costs|
Hence, we have found that our innovation PaperPure has a variety of advantages over other current solutions, and could act to complement or replace these solutions to address the real-world problem, depending on local contexts.
Note: Various steps were taken to ensure safety and sterility when using potentially-dangerous materials (e.g. Heating apparatus, AgNO3, bacteria). These are listed in the "Health and Safety" section and the detailed methodology.
Zoysia matrella grass was grown over a period of ~6 months. It was then collected and briefly rinsed to remove foreign particles, before being ground and crushed to powder. As seen from detailed methodology, 5g of the powder was boiled and blended, respectively, in 50ml of deionised water for 3min to obtain two types of grass extract. (Volumetric Flasks were used to produce all solutions due to their greater precision.)
The grass extracts were then mixed with 50ml of 0.1M AgNO3 solution and then left to stand. Eventually, a colour change was observed proving the formation of AgNPs, as explained in "Results". A UV-Vis Spectrophotometer was also used to calculate the lambda-max of the AgNO3 and AgNP solution for use in Section D.
A 30cm ruler was used to measure out 1.2x1.2cm pieces of filter paper(kept constant to ensure a fair experiment). 2 pieces were soaked in the boiled and blended grass extracts (respectively) for 3min, before being soaked in 100 ml 0.1M AgNO3 solution, for another 3min. After the filter paper pieces had dried, a Disk Diffusion Assay was conducted on Nutrient Agar plates lawned with E.coli and B.subtilis respectively (to act as model organisms for gram-negative and gram-positive bacteria, respectively). After 24h of incubation at 32oC, the zone of inhibition was measured with a 30cm ruler.
Using an electronic weighing balance and volumetric flasks, AgNO3 solutions of varying concentrations (0.102M, 0.051M, 0.026M and 0.013M) were produced, along with a negative control of deionised water. Filter paper was first soaked in 100 ml boiled grass extract, and then,100ml of the control/AgNO3 solution, for 3min each. It was then left to dry on foil, forming PaperPure.
100ml bacterial solutions of E.coli and B.subtilis were prepared at 0.5 McFarland standard turbidity, using absorbance at 600nm (measured by UV-Vis). To account for absorbance of the sterilised nutrient broth, it was used as the reference-blank. The resulting solution was serial diluted 7 times (Dilution Factor:2). 100 ml of this bacterial solution was poured through PaperPure. Micropipettes (used for their precision and sterility) were used to take appropriate aliquots of the filtrate, for spread-plating on nutrient agar. These plates were incubated for 36h at 32oC, and then the number of Colony Forming Units (CFU) was calculated. The percentage reduction in bacterial concentration (CFU/ml) and hence, relative effectiveness of PaperPure was determined. 7 replicates were conducted to improve reliability.
The lambda-max of AgNP and AgNO3 solution was 442nm and 301nm, respectively. The absorbance of all the filtered solutions was determined at these wavelengths to quantify silver leaching. The diluted suspension in Section C was the reference-blank, to account for absorbance caused by bacteria and nutrient broth.
The successful bioreduction of AgNO3 to AgNPs, by grass extract, leads to a colour change because of the phenomenon of Localised Surface Plasmon Resonance. This phenomenon was quantified by measuring the lambda-max of the AgNP solution (442nm), which helped us determine the nanoparticle size as being between 80-90nm(Agnihotri, Shekhar et al.,2014). This qualitative and quantitative observation demonstrated that Zoysia matrella could be used to produce AgNPs, allowing us to proceed with the remaining stages of the experiment.
Since the zones of inhibition for both boiled and blended samples were effectively the same for both E.coli and B.subtilis, both boiling and blending of Zoysia matrella in water are equally effective in bioreducing AgNO3 and thus, in their eventual antibacterial efficacy against both B.subtilis and E.coli.
Thus, we decided to select boiling as our method of producing PaperPure. Blending is a less feasible method of production since blenders may be expensive, unavailable and face technical constraints(inconsistent electricity supply) in target regions. By contrast, fuel sources(including wood and biogas) are more likely to be available to boil the grass extract and allow for PaperPure to be produced in the target rural and/or water-scarce regions.
As [AgNO3] increased, the antibacterial efficacy of PaperPure increased for both E.coli and B.subtilis. At most concentrations of AgNO3, PaperPure was observed to be more effective for E.coli than B.subtilis, indicating it may be more effective against gram-negative bacteria.
The high R2 for the B.subtilis (0.9648) and fairly high R2 for E.coli (0.8715) indicates goodness-of-fit of the trendlines to the data points. However, the trendline for E.coli may be slightly inaccurate since it implies a greater than 100% decrease in bacterial concentration, which is impossible.
A maximum percentage reduction in CFU of 99.988% and 99.927% was observed for E. coli and B. subtilis, respectively, when using just 0.102M PaperPure. This is equivalent to a log 3.92 reduction (E.coli) and log 3.13 reduction (B.subtilis) in bacterial concentrations.
Statistical Testing (One-Way ANOVA)
Since p-value<<0.01 for both bacterial species, there is a >>99% probability that the results are significant. At the 1% significance level, a rejection of the null hypothesis (H0) is validated. Thus, as the concentration of AgNO3 increases, the bacterial concentration in the filtered solution decreases(implying percentage reduction in CFU increases).
Theoretically, leaching of AgNP and/or AgNO3 could occur from PaperPure.
AgNP:Due to the lack of sufficiently-advanced equipment, we could not quantify AgNP leaching but can only provide data on absorbance at 442nm, possibly such that the scientific community can help us interpret it. This only allows us to conclude that higher initial [AgNO3] leads to increased AgNP leaching.
AgNO3:No AgNO3 leaching occurred for most PaperPure samples, making it within the EPA limit of 100 ppb of silver per litre of water and thus, safe for drinking. However, leaching of 0.0000552M AgNO3 occurred for 0.102M AgNO3 PaperPure. This requires further testing to determine whether 0.102M PaperPure is safe,due to the imprecision and inaccuracy of the UV-Vis at such low concentrations.
PaperPure is a novel paper based water filter and its superiority over current technologies can be summarized as SCALE:
Simple to Use and Produce: PaperPure can be manufactured on-the-ground with rurally available resources in a simple one-step process that requires no external energy. The production process is much more environmentally friendly than existing treatment systems, requiring only millimolar concentrations of AgNO3 and releasing low levels of toxic leachates (Max:0.00005M AgNO3). Keeping ease-of-use in mind, we also developed prototypes that were suited for disaster-relief and rural use.
Climate Friendly: Zoysia matrella requires very little water and/or nutrients to grow and is extremely resilient in higher temperature conditions making it ideal for production in non-arable regions as well.
Affordable: A single PaperPure filter costs ~15¢ and can filter up to 10 litres of water. With industrial production and economies of scale we believe the cost could be reduced further, maybe as low as 10¢!
Lightweight: The paper-based nature of the product means it is extraordinarily lightweight, making it easy and cheap to transport to disaster hit and rural regions.
Effective: Silver nanoparticles are known to have potent antimicrobial effects including inhibiting the bacterial electron transport chain to block cell respiration and leading to the formation of ‘pitted’ structures in the bacterial cell wall that leads to cell lysis via uncontrolled osmosis. This bactericidal effect was validated by the high antimicrobial efficacy of PaperPure with >99% reduction in bacterial concentrations for all data points tested, supporting our hypothesis. However, PaperPure was observed to be marginally less effective for gram-positive bacteria. This was unexpected but could be explained by the thick peptidoglycan cell wall restricting the internalisation of AgNPs during the filtration period. Further, silver has been widely used as an antimicrobial agent for thousands of years without significant bacterial resistance developing, making it likely to be effective into the foreseeable future.
While preliminarily data suggests that PaperPure is mostly safe for use, access to a graphite furnace atomic absorption spectrometers would help to accurately quantify the total amount of silver (in AgNO3 and AgNP states) leaching into the filtrate, allowing for better characterisation of the environmental/health impacts of PaperPure.
Access to a Scanning Electron Microscope would help in imaging the bacteria passing through the filtrate and better determine the mechanisms by which PaperPure exerts antimicrobial effects.
PaperPure’s efficacy can be tested across a wider range of water-borne microorganisms like other bacterial species (e.g.Vibrio cholerae), protozoans, cysts (e.g.Toxoplasma gondii). However, this would require access to a laboratory with a higher biosafety level than our school laboratory.
Testing of other grass species for PaperPure production, especially grass varieties common in parts of the world where Zoysia matrella is not. We could test the effect of varying the mass of grass used since we theorise that this would help to limit silver leaching (by bioreducing more AgNO3 to AgNPs and by helping to stabilise the AgNPs on the cellulose matrix because of the presence of complex plant fibers), especially at the 0.102M data point.
Hi! We are Pulkit and Gideon from Singapore. For both of us, science was our biggest passion while growing up. We met at the Accelerated Class for Science, a specialised school program, that exposed us to advanced scientific concepts. It was there that our interests and aptitude in STEM were developed, leading us to start this project almost 2 years ago.
Having frequently gone overseas to South/Southeast Asia, we realised how scarce clean water was for so many. Moreover, bottled water often littered countrysides with plastic waste. Most people wish to make a positive impact in the world, and we believe that providing clean water is a key way of making this happen.
Initially drawn to STEM by a child-like curiosity about how the universe functions, Pulkit and Gideon's passion is now driven by the realisation of the huge impact they can make through innovation. Moving forward, Gideon aspires to be a clinician-scientist, constantly researching and innovating to produce new medical solutions while also caring for patients directly. Meanwhile, Pulkit wants to pursue research in the fields that interest him: biochemistry and cellular biology, researching on topics that can directly impact and improve the lives of ordinary people.
Winning at GSF would provide the highest level of validation for all the effort we put into this project over the last 2 years. It would also incentivise us (and other kids!) to keep researching and innovating to improve the lives of those around us.
All experiments for this project were conducted in our school laboratory independently. The lab is located in our school at the address: 121 Dover Road, Singapore 139650. The teacher supervisor's email is firstname.lastname@example.org.
As a lab with Biosafety Level 1 (BSL 1) clearance, non-pathogenic Escherichia coli, and Bacillus subtilis were available and used for our experiments. A list of microorganisms approved for use in our school lab is shown below. In conducting these experiments, health and safety guidelines of the lab, shown in the image below, and local laws were followed.
Despite conducting experiments in our school lab, all investigations and experiments were done independently. The following additional safety precautions were taken:
Hence, extensive health and safety procedures were taken in addition to the basic guidelines laid out by the lab.
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All testing was carried out using facilities provided in the IB Biology Laboratory of our school, Anglo-Chinese School (Independent). No special equipment was used during the process of experimentation. However, a summary of the key equipment we used is provided below:
We would like to thank our school laboratory technician, Madam Reena Quah, who helped to provide, on the part of the lab, the chemicals/materials required for our experiments, namely, solid Silver nitrate (AgNO3) salt, 95% Ethanol solution, Nutrient Broth Powder (Oxoid CM0001) and Nutrient Agar Powder (Oxoid CM0003). We then used this to prepare the solutions at the requisite concentrations and volumes set out in our methodology. Mdm Quah also provided us with the E.coli and B.subtilis solutions that were maintained by the lab. We subsequently used these solutions to produce our own stock of E.coli and B.subtilis bacteria (grown in Nutrient Broth), which we then used in order to test the antimicrobial efficacy of PaperPure.
We would like to thank our teacher-supervisor, Madam Yong Lee Har, for giving us access to carry out our experimentation in the school laboratories. Mdm Yong helped to act as a crucial sounding board for us when we were attempting to finalise the details of our methodology as well as encouraging us to focus on the real-world applications of our project such that PaperPure could eventually be used to provide clean drinking water, at low-cost, for all.
Last but not least, we would like to thank our parents and friends, especially John and AJ, for their support throughout this project, as well as providing us with occasional insights into how we could improve on PaperPure and its real-world applications.