Team:IISc-Bangalore/Description
Description
Project CellOPHane seeks to create a functionalized bacterial cellulose filter to combat organophosphate pollution. Organophosphates are used as pesticides globally but are extremely potent neurotoxic agents, which cause adverse reactions on reaching living organisms through contaminated agricultural runoff. We are seeking to degrade the organophosphates present in water into relatively harmless products by using a broad-spectrum organophosphate hydrolase functionalized on a bacterial cellulose sheet. We're also seeking to create a one-of-a-kind modular plug-n-play platform to combat other kinds of pollution with minimal modifications. The platform that we seek to create can be used for any bioremediation strategy by just cloning the corresponding enzyme; one need not therefore spend resources on reimagining the exact design implementation for every individual contaminant. Project CellOPHane will thus help in reshaping the bioremediation landscape and make bioremediation strategies easy to develop and implement.
Background
The history of agriculture in India dates back to the Indus Valley Civilization. India ranks second worldwide in farm outputs. As per 2018 reports, agriculture employed more than 50% of the Indian work force and contributed 17-18% to the country's GDP. Indian farmers use a range of pesticides in order to improve agronomic productivity, and to prevent crop losses. The use of pesticides was introduced in India during the 1960s and they are now being used on a large scale, representing a common feature of Indian agriculture. India is now the second largest manufacturer of pesticides in Asia after China and ranks twelfth globally. The pattern of pesticide usage in India is different from that for the world in general. In India 76% of the pesticide used is insecticide, as against 44% globally. The use of herbicides and fungicides is correspondingly less heavy.
Organophosphates (OP) constitute a class of pesticides, mainly used as insecticides by Indian farmers. These are degradable organic compounds containing carbon-phosphorus (C-P) bonds, used primarily as an alternative to chlorinated hydrocarbons that persist in the environment. Organophosphates are organic derivatives of phosphorus, usually esters, amides or thiol derivatives of phosphoric, phosphonic, phosphinic, or thiophosphoric acids with two organic and additional side chains such as cyanide, thiocyanate, and phenoxy group.
The organophosphates registered in India can be classified as per WHO recommendation as follows:
| Extremely hazardous (Class Ia) | Highly hazardous (Class Ib) | Moderately hazardous (Class II) | Slightly hazardous (Class III) |
|---|---|---|---|
| Parathion-methyl, Phosphamidon, Terbufos | Fenamiphos, Monocrotophos, Oxydemeton-methyl, Propetamphos, Triazophos | Acephate, Anilophos, Chlorpyrifos, Diazinon, Dichlorvos, Dimethoate, Ethion, Fenitrothion, Fenthion, Phenthoate, Phosalone, Phorate, Pirimiphos-methyl, Profenofos, Quinalphos, Trichlorfon | Chlorpyriphos-methyl, Malathion, Temophos |
Most consumed organophosphate insecticides during 2005 to 2010 are tabulated below:
| Serial No. | Organophosphate types | Quantity (in metric tonnes) |
|---|---|---|
| 1 | Phorate | 10763 |
| 2 | Methyl parathion | 8408 |
| 3 | Monocrotophos | 8209 |
| 4 | Chlorpyrifos | 7354 |
| 5 | Malathion | 7103 |
| 6 | Chlorpyrifos | 6329 |
| 7 | Chlorpyrifos | 5803 |
Although these xenobiotics degrade under natural condition, their residues have been detected in soil, sediments, and water due to their non-regulated usage practice. Although they degrade faster than the organochlorines, they have acute toxicity and better solubility in water, hence posing risks to people who may be exposed to large amounts. Organophosphate insecticides can be absorbed by all routes, including inhalation, ingestion, and dermal absorption. The over-reliance on pesticides has not only threatened our environment but contaminations of organophosphate residues have been also detected in certain agricultural products (like tea, sugars, vegetables, and fruits) as well as in human/animal parts (like blood, urine, breast milk, semen, adipose tissue, amniotic fluid, and umbilical cord blood; cuticle) throughout India. The potential adverse impact on human health from exposure to pesticides is likely to be higher in countries like India due to easy availability of highly hazardous products, and low risk awareness, especially among children and women.
Organophosphates mainly leach into waterbodies through agricultural runoff. Ultimately, they reach the bodies of various life forms including humans, where they act as potent neurotoxins by inhibiting acetylcholinesterase. The major presenting symptoms in human body owing to acute OP toxicity are cholinergic crisis, pulmonary oedema, aspiration pneumonia, vomiting, nausea, miosis, excessive salivation, blurred vision , giddiness, headache, lethargy, anxiety, depression, fatigue, and irritability. Even chronic effects cannot be neglected: Some (like monocrotophos and ethion) are suspected to be carcinogenic, some cause attention deficit hyperactivity disorder (ADHD), and some (like parathion) are even lethal. The associated neurobehavioral changes are described by the umbrella term: 'chronic organophosphate-induced neuropsychiatric disorders' (COPIND). Organophosphate toxic effects have also been reported in some other vertebrate and invertebrates in India. Malathion causes histopathological changes in earthworm Eisenia foetida (used in vermicomposting), and developmental abnormalities in zebrafish (i.e. Danio rerio).
Hence, organophosphates have faced stringent restriction in many countries, but not yet in India. As a result, many OPs banned outside India still find rampant usage in the Indian soils. For example, according to WHO's report monocrotophos is 'widely available' in the Indian market while it has already been banned in Australia, Cambodia, China, the European Union, Indonesia, Laos, Philippines, Sri Lanka, Thailand, Vietnam and the United States!
Effective, eco-friendly and economically efficient solution to this problem of contamination of waterbodies by organophosphates has not been developed yet. Mitigation of OP pollution using free water surface (FWS) constructed wetlands can be extremely cumbersome. Utilizing microorganisms to achieve biodegradation has not proved to be efficient yet.
To address the same, Team iGEM IISc 2021 presents 'CellOPHane'! We seek to develop a filter, made of bacterial cellulose and subsequently post-hoc functionalized with organophosphate degrading enzyme molecules, which can be used as a modular plug-n-play bioremediation platform.
Approach
Based on past approaches to mitigate OP pollution in soil, heavy metal pollution in water, and pesticide molecules in air, CellOPHane seeks to offer a remedy to water pollution owing to OP, by cleaving the organophosphate molecules that come in contact with the functionalized bacterial cellulose (BC) filter. Gathering recent advances in synthetic biology, microfluidics and materials engineering, we attempt to offer a cost-efficient, reliable, sustainable solution to organophosphate pollution in waterbodies. This year, we begin from solving a local problem to finally making a global impact. For that, our proposal is divided into four different (but often overlapping) stages:
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Enzymes: Production of enzymes (preferably, one with a broad substrate range) that would chop off OP molecules into relatively benign chemicals.
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Sheet: Production of BC sheet of suitable dimensions and microstructure.
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Filter: Functionalization of the BC sheet with the enzymes in such a way that modularity is maintained.
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Experimentation: Conduct experiments and assays to quantify the efficiency of this novel filter.
Inspiration
When we newly formed this iGEM Team of Indian Institute of Science, we had no idea of what kind of project we wanted to work on. We were not really familiar with the type of real-life problems that iGEM teams generally work to address. So. we went through the wikis created by the teams that had participated in previous editions of iGEM, where we were inspired by so many ingenious ideas. Our mentors further encouraged us to let our ideas flow freely.
Soon, we sat down together to brainstorm for ideas. We kept in mind that with synthetic biology, everything is possible and no idea is too daring. Among the ideas for issues to work on were acid mine drainage, inefficient traditional process of jute retting, preterm birth, hydrochlorofluorocarbon pollution, fibromyalgia, drying out of soil, and pesticide pollution. With these loose ideas we started to get more concrete.
Meanwhile, Dr. Bitasta Das, our humanities course (UH201) instructor, shared a documentary with the class about "cancer express". It is the infamous nickname of Passenger Express 339. The cancer patients from Malwa region of Punjab travel almost 326 km on it daily to reach Acharya Tulsi Regional Cancer Treatment and Research Centre in Bikaner (in Rajasthan), the closest government cancer hospital that is affordable. The Green Revolution that started in the mid-1960s has turned Punjab into the breadbasket of India - contributing more than 95% of the food grains that feed deficit areas in other states - but it has also turned the water table into a poisonous aquifer. Malwa consumes 75% of the pesticides used in Punjab. The abundance of cancer cases has been shown to bear some strong epidemiological correlation with heavy usage of organophosphates in Malwa region and its leaching into the waterbodies.
All this information made a powerful impression on our team during our ideation process, where we were first introduced to the effects of organophosphate pollution on human health and the ecosystem in general. When our team got to know one another more, we learned that our team had a personal stake and this inspired us even more to investigate the possibilities of utilizing synthetic biology to address organophosphate pollution. Throughout our brainstorming sessions, we realized that bioremediation of organophosphate pollution has not been paid enough attention by the SynBio community in the past. After further research and discussions, meetings with a number of experts, and exertion of democracy, we came up with CellOPHane!
As for our goals, we cannot make the huge amount of organophosphate pesticide present in various waterbodies disappear at once. As this is not our job as scientists, we set the goal of providing a proof of principle for our novel bioremediation platform. We intended to prove that we could build a functionalized plug-n-play bacterial cellulose filter, degrade organophosphate molecules present in some water sample by using the filter, and quantify the bioremediation efficiency. These goals appeared to us as ambitious enough for half a year of time.
We firmly believe that to drive synthetic biology forward, we have to make it attractive in the eyes of policy makers and the public. This can be done by presenting possible solutions to problems that so far seem unmanageable. That is partly why we chose organophosphate bioremediation as our project, as the issue of the ongoing plastic pollution is highly topical, however there is no solution in sight.
References
- "India economic survey 2018: Farmers gain as agriculture mechanisation speeds up, but more R&D needed". The Financial Express. 29 January 2018. Retrieved 8 January 2019.
- Aktar MW, Sengupta D, Chowdhury A. Impact of pesticides use in agriculture: their benefits and hazards. Interdiscip. Toxicol. 2009; 2(1): 1-12. DOI:10.2478/v10102-009-0001-7.
- Kumar S, Kaushik G, Villarreal-Chiu JF. Scenario of organophosphate pollution and toxicity in India: A review. Environ. Sci. Pollut. Res. 2016; 23: 9480-9491. DOI:10.1007/s11356-016-6294-0.
- Roberts JR, Reigart JR (2013). Recognition and Management of Pesticide Poisonings (6th ed.). Environmental Protection Agency. p. 43. Archived (PDF) from the original on 13 May 2017. Retrieved 10 March 2018.
- Krause K, Thriel CV, De Sousa PA, Leist M, Hengstler JG. Monocrotophos in Gandaman village: India school lunch deaths and need for improved toxicity testing. Arch. Toxicol. 2013; 87: 1877-1881. DOI: 10.1007/s00204-013-1113-6.
- Sahin C, Karpuzcu ME. Mitigation of organophosphate pesticide pollution in agricultural watersheds. Sci. Total. Environ. 2020; 25(710): 136261. DOI: 10.1016/j.scitotenv.2019.136261.
- Dar MA, Kaushik G, Villareal Chiu JF. Chapter 2: Pollution status and biodegradation of organophosphate pesticides in the environment. Abatement of Environmental Pollutants 2020; 25-66. DOI:10.1016/b978-0-12-818095-2.00002-3.
- "Cancer Express". Hindustan Times (by Praveen Donthi). 17 January 2010.