Team:Ruia-Mumbai/Human Practices

Human Practices




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Impact / Gravity of the problem

Paan is a traditional mouth freshener in Indian subcontinent. It is a part of the cultural heritage of India which is also reflected in existence of assorted varieties of paan which are vended by abundant paan sellers, folklores, Bollywood movies.

Health benefits of consuming paan:-

  • Aids in digestion.
  • Prevents swelling of gums
  • Inhibits tooth decay
  • Wards off mouth odour

  • Eating paan causes excess salivation, this saliva has a characteristic reddish brown colour and needs to be spat frequently. The haphazard spitting of paan coats the walls, monuments ; stains roads,dustbins and spreads respiratory diseases. These red coloured paan stains are not only an eyesore but also stubborn. Surprisingly, there are no specific and effective methods of cleaning paan stains.

    The preferred method to clean these paan stains is scrubbing with large amounts of water and multipurpose chemicals. A whopping 100 crores ( 13,571,000 USD) per month and 60,000 litres of clean water per day are used by Indian Railways and Central Railways respectively. Moreover, private housekeeping agencies also use massive amounts of water. In a country like India where water crisis is to such an extent that water has to be transported to some drought affected areas by trains, even a drop of water is of immense importance. It is unreasonable to use such tremendous amount of water just for cleaning.

    Our eco-friendly biological approach for cleaning the paan stains is using our synthetic biology machine ‘Catechewing coli’ which literally chews paan colour, thus getting rid of paan stains with minimal wastage of water.

  • Current Strategies

    Methods of preventions

    • “Prevention is always better than cure”.
      Anti-spitting drives and rallies are carried out to create awareness about the colossal issue of paan stain. The Government of India has recently launched a nationwide campaign known as Swachh Bharat Abhiyaan which focuses on cleanliness of public places.
      Paan stains serve as a huge hurdle to this campaign.
    • Wall painting is the most usual method to prevent spitting of paan. Usually walls of public places like railway stations, public bridges, highway roads are painted which includes beautiful artwork. Several NGOs and private agencies have taken up projects for beautification of public places using street art and graffiti.Unfortunately this strategy seems to be not so effective as spitting of paan on these wall still remains a problem.
    • The well-rooted faith in religion is also taken help of for forbidding spitting of paan stains. In the most stain prone areas like corners, side walls of staircases, ceramic tiles having pictures of different deities are put up so that people are retarded from spitting there.
    • Some efforts in tackling issue of paan stains have been done lately. Ezyspit is one such product developed by three Indian youths for reducing the occurrence of paan stains. Ezyspit is a container wherein the paan mixture can be spat. This spit instantly gets converted from liquid to harmless fragrant granules within 10 seconds.

    Painting of walls

    Paan stains on wall paint

    Ceramic tiles of deitiess

    Cleaning stains

    • Multipurpose chemicals are used presently for cleaning paan stains. The basic principle underlying it is that different dilutions of a single chemical are used for different type of surfaces to be cleaned thus giving it a multi-application property.
    • The chemicals used for cleaning paan stains are largely acid based. These chemicals are not only harmful but also threat to the environment. Recently, improvements have been made in this field wherein biodegradable green chemicals have been developed which are somewhat effective against stains.
    • Application of the acid based chemicals or detergents is not sufficient to get rid of these stains completely. Manual scrubbing for prolonged time is needed. However, this practice makes cleaners prone to airborne respiratory diseases. Lately mechanical scrubbers have replaced manual scrubbing only in some places because of its high installation and maintenance cost. Despite its high cost, these methods fail to clean effectively.
    • The non-painted paan stained walls are cleaned using jet sprays wherein water applied with high pressure is used for removing paan stains. This method is commonly used for cleaning paan stains. The capacity of a single jet spray machine is 500 litres/hour and 6-9 litres are approximately used for cleaning a single mark of paan stain.

      Mechanical scrubbers at CSMT station

      Janitors using jet spray


    • Our approach was to degrade paan stains by enzymes instead of using harsh chemicals that require thousands of liters of water and a large number of workers but still prove to be inefficient, time consuming and extremely costly.
    • To deal with this problem we interacted with relevant communities like paan vendors, cleaners and government officers who are directly or indirectly affected by this issue. They not only made us realize the depth of problem but also gave insights into the current strategies of cleaning and improvement of our product.
    • As our project has a strong social impact we tried to create awareness about the problem of paan stains by using some unique ways.
    • We tried to deeply understand every stakeholder’s viewpoint about this nuisance in India and their expectation from an ideal paan stain cleaning product. We aim to integrate their suggestions in our project so that our product is efficient and user-friendly. Also, we approached experts who suggested effective ways to create awareness in the society.



    Paan vendors

    We interacted with many paan vendors from different areas across Mumbai right from road side paan vendors to well-established paan shop owners.

    Why did we approach them?
    Since, there is not much published data about composition of paan preparation, ingredients responsible for paan stain colour, we approached paan vendors to get some first hand information about paan preparation.

    What we learn?
    We confirmed that the two main ingredients responsible for intense red brown colour of paan are catechu and slaked lime. Based on the collective common answer from all, We got a rough idea of the relative proportions of these two ingredients used for preparing paan. From this interaction, we also learned that very little amount of chunna (slaked lime) is used for making paan.

    Station master

    We interviewed a Mumbai local Railway Station master Mr. Ganesh Swine at Sion station. Sion station is one of the locations where this issue of paan stains is very much prevalent.

    Why did we approach them?
    This interview was important for us to understand how Railway government approaches the issue of paan stains and the problems they are facing as they tackle these stubborn stains.

    What did we learn?
    Random spitting of paan is a major problem in Sion station as it is in proximity to a slum area where lack of awareness is huge. Many NGOs have tried to spread awareness by carrying out anti-spitting drives, wall paintings but there has been limited success. For cleaning and maintenance purpose, few small stations like Sion hire 4-5 cleaners while on the other hand some big and crowded stations give contracts to private cleaning agencies.
    Approximately 10 liters of acid is used per month to clean the stubborn paan stains in Sion station itself.

    Deputy Commissioner - Government Officials

    We interviewed Mr.Kiran Acharekar, Deputy Municipal Commissioner of Brihanmumbai Municipal Corporation (BMC) of zone 4.

    Why did we approach them?
    The main focus of interview was to perceive information about the various impacts of paan stains on government, how they tackles this problem and what different actions were taken by them to lessen paan spitting. Whether BMC would be interested to use different product which is more efficient.

    What did we learn?
    We realised that the paan spitting problem is increasing day-by-day and it’s getting unmanageable for the government to handle. Special drives of anti-spitting has been carried out, BMC hires marshals who catch hold the person spitting paan in public places and fines them. But in the city of mumbai where the population is more than a crore hiring the relative number of marshals is not affordable or rather impractical,therefore in some selective crowded places only marshals are employed. Using an efficient product for cleaning paan stains is always welcomed but most importantly the product should be cost effective.

    Cleaning Agencies - Krystal Integrated Services

    We interacted with the the Vice President Mr. Rahul Kamble, Vice President - Sales Mr. Milind Jadhav, Head of Training department Miss. Aasawari Vadkar of a well-known cleaning agency in Mumbai - Krystals Integrated Services Pvt. Ltd. The discussions with these experienced managers gave us valuable information about the issue of paan stains.

    Why did we approach them?
    To understand the deeply rooted problem of paan stains, it was necessary to not only interact with the cleaners who clean these stains but also with the cleaning agencies which hire them. We wished to know the problems encountered during the planning, management and execution of the strategies of dealing with the paan stains.

    What did we learn?
    We learned that Cleaning paan stains is a major challenge largely unaddressed in the housekeeping industry. It is one of the most difficult part of the cleaning regimen. The paan stains are harder to remove as the time passes due to the drying of the stains. Moreover, the nature of surfaces where paan stains are present play an important factor in deciding the cleaning regimen.
    The newspaper articles and the cleaners state that acid is generally used for cleaning of paan stains. However, it is not acid but acid based chemicals which are commonly employed for cleaning purposes.


    We interviewed the housekeeping staff of a private cleaning agency in Mumbai. This interaction with the cleaners enabled us to understand the reality and gravity of the problem.

    Why did we approach them?
    We were keen to know the practical problems faced by the cleaners who clean these filthy paan stains for their livelihood. Hence, we interviewed the housekeeping staff deployed at one of the business complexes in Mumbai where the paan stains are abundant. .

    What did we learn?
    We found that the paan stains are found in all possible places right from the corners, floor and corridors to the dustbins, metal surfaces. The vicious cycle of paan stain and its cleaning is very difficult to break as the paan spitting is deep rooted in the attitude of some people.
    The workers have to scrub vigorously for an average 10-15 minutes to clean a single paan stain covering 10cm*10cm area. This time increases with the duration for which the stain has been on the surface.


    Ajay Bio-tech
    We had a meeting with Dr.Subrata Sarkar, Mr.Bhavesh Mehta and Mr.Palak Agarwal of Ajay Bio-tech India ltd. Ajay bio-tech India ltd is a name associated with eco-friendly agro-input products essential for organic farming. Development of new biotechnology products through research is the main aim of the company.

    Why did we approach them?
    We presented our project to get some valuable information about criteria that an industry needs to market an effective product.

    What did we learn?
    The meeting was fruitful as they actively tried to understand our project by voluntarily asking questions which triggered our mind and also gave several suggestions, some key suggestions include

    • Product should work on all types of surfaces like walls, fabrics, metals etc.
    • Versatility of the product.
    • Anti-rust
    • Effect of autoclaving on catechu.
      For industry scale, product should be optimised with parameters like
    • Time for degradation
    • Temperature range and optimum temperature
    • Concentration of enzyme required for effective cleaning


    Novozymes is a global biotechnology company primarily working on enzymes and microbes. We were glad to have a Skype meeting with Dr. Ashish Paradkar, Director & Head, R&T India and his team to get their valuable inputs for our project Catechewing coli.

    Why did we approach them?
    Novozymes too has been working on product that cleans paan stains for years. We approached them for getting valuable suggestions and feedback on our project and also possible future collaboration for commercialisation of our product.

    What did we learn?
    Novozymes too informed us that the paan stains are tough to remove or clean and the company itself was very interested in the idea of cleaning paan stains. They advised us that since all the present techniques do not clean paan stains efficiently, it is essential to devise a solution which degrade paan stains instead of cleaning it.



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    Social Media
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    Street Play
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    Synthetic Bio Outreach

    Social media challenge


    Social media is a powerful tool to engage primarily with current youth.We decided to use trending social media challenges as our approach to spread awareness about this paan stains menace. We announced our social media challenge #PaanSePareshaan (annoyed of paan stains) to highlight the huge impact of spitting haphazardly, wherein the one who accepts the challenge needs to click a photo pointing at disgusting paan stains and post it as a story/post with location on social media handles like Instagram, Facebook and Twitter and tag iGEM Ruia-Mumbai, add location of the place along with #PaanSePareshaan.
    The focus of the challenge was to make people realize the omnipresence of nasty paan stains.

    We got a positive response to the challenge from youngsters as well as elder citizens. The challenge was a learning experience for our team as we came to know the range of places where paan spits are found and the mentality of people spitting paan. For instance spitting in dustbins, on railway tracks, in corners, over potted plants, on walls that mentions DO NOT SPIT is considered as a normal practice.

    The challenge initiated critical discussion and debate among people resulting in an increased realization of the existence of this problem. (screenshot of the post). The #PaanSePareshaan challenge is still ongoing and we are trying to spread it as much as possible to create awareness on a larger scale.

    Street play and games

    Our another approach to engage with local public and create awareness was a street play. Street play is an effective way of engaging people and educating them about a social problem. In a fast paced city like Mumbai, people seldom take out time for a social cause. Moreover, retaining common public’s attention span is also a task. Ganesh festival is celebrated on a large scale in India, particularly Mumbai. During this period, Lord Ganesha’s idol is worshipped and people come together to celebrate this festival. We interacted with this gathering of people/devotees, conducted fun games relevant to our project and performed the skit in Ganesh pandal of Mulund. We performed a street play focusing on issue of paan stains and highlighted key points like why people should not spit randomly?, How much amount of money is invested in cleaning these stains. Our major focus area was amount of water in liters required to clean paan stains. Moreover to gain attention of crowd, humorous content was included and the skit was conducted in an entertaining way..The skit was performed in the regional Marathi language with an added slice of humour so as to reach a wider audience in an entertaining yet enlightening way.

    Synbio Outreach

    We had few enriching brainstorming sessions with our juniors and fellow batchmates. Exciting ideas and concepts of synthetic biology were discussed and debated upon. Not only the applicability of the ideas was considered but also its feasibility and implications.The ideas foddered our minds and developed our critical, analytical and creative abilities. Our juniors got themselves familiarized with the emerging and exciting field of synthetic biology.


    Integrated Human Practices


    We interacted with all possible communities directly and indirectly affected by the paan stains and who will influence our project. We tried to deeply understand every stakeholder’s viewpoint about the problem of paan stains in India and their comments, appreciations, suggestions and improvements about our project. We asked their expectation about an ideal paan stain cleaning product. We aim to integrate as many improvements as possible in our project so that the developed paan-stain degrading device is efficient and user-friendly.


    Paan vendors

    "More catechu less slaked lime"

    All the paan vendors we interviewed, told us that the red colour of paan stain is due to catechu and it gets intensified by slaked lime. This information confirmed and matched with our data obtained from literature survey. Moreover, we found out that while preparing paan very less amount of slaked lime is used, as more amount of slaked lime increases alkalinity which may cause burns in mouth.

    In order to check the degradation of paan stains by our catechewing coli it was essential for us to know the actual components and their approximate ratio in the paan that gives maximum red-brown colour.
    We conducted several experiments for preparation of catechu extract using different ratios of catechu and slaked lime. We observed maximum red colour formation in 10:1 ratio of catechu to slaked lime confirming the information provided.

    For more information refer media formulation protocol


    Dr. Phadke

    “Why to concentrate on multiple component?”
    Focus on degrading the naturally occuring substrate

    Dr. Ravindra Phadke is an emeritus faculty of Department of Microbiology, Ramnarain Ruia Autonomous College,Mumbai. He has done his PhD from Institute of Chemical Technology (ICT),Mumbai in 1995. He loves teaching biochemistry and has unique methods of instilling inquisitiveness amongst students.

    When we were struggling with quantification of paan stain colour due to lack of trusted information about multiple components of catechu, Dr. Phadke came to our rescue. Catechu and slaked lime stock is a mixture of numerous components. When we determined the wavelength of maximum absorption of this stock, it was found to be 354 nm. This indicated that the major component is a colourless compound absorbing in UV range which was later found to be catechol. So, to determine the component which gives colour to the stain and determine its wavelength of maximum absorption was a challenge. Lack of scientific literature on paan stains further provided resistance to find solution to this challenge. we were reluctant to use LC-MS or HPLC due to lack of funds.
    When we consulted Dr.Phadke about this issue, he suggested to focus on catechu extract as a substrate for colour quantification instead of finding its coloured component.


    Cleaning agencies - Krystals

    “We desire a liquid formulation, green certified chemical product will surely add cherry on the cake”
    Krystal Cleaning agency is a leading cleaning agency in India. We had a fruitful meeting with the managing committee of Krystal agency Head of Training department Miss. Aasawari Vadkar who gave us valuable feedback about our project . When asked about the desirable nature of product, Sales Manager of the Company advised us to make the paan stain cleaning agent in liquid form as it becomes easy to volumetrically dilute and distribute it in small quantities among the janitors. This advice helped us in eliminating the thought of making our product in solid or spray form.
    Moreover, we also found that housekeeping Industry is majorly a manpower based industry wherein the chemicals and cleaning agents constitute a meagre 2-3% of the budget which suggested that it is of immense importance to price our cleaning agent appropriately.
    Cleaning agencies are pushed by the Government to use green certified chemicals. Green certified chemicals are non-toxic, biodegradable and do not contain any volatile components and meets the standard of environmental responsibility. Currently used chemicals produce toxic smell while cleaning which becomes not only an irritant but also are harmful for the cleaners. This inspired us to have our product.
    Thus, we learned about the present methods and recent developments in cleaning paan stains from this interaction with Krystal Integrated Services. The managers of Krystal Integrated Services encouraged us to take plunge into solving the problem of paan stains.


    Ajay Bio-tech

    “Is your product versatile? Does autoclaving affects catechu”
    Ajay Bio-tech suggested that instead of a product that degrades only paan stains, industry would want something that is more versatile. They suggested that we incorporate multiple enzymes in our formulation to enhance the versatility. To check the versatility of product first we tried to test it on common laboratory dye safranin. We got a positive degradation result for safranin. We got a positive degradation result for congo red dye,safranin. For more details refer to our result section.
    They also had an interesting question which was whether we autoclave the catechu extract before incorporating it into the media. In practical autoclaving affects the stability of some compounds that may affects the reaction with enzymes and the colour intensity of compound. We thought on this question and checked the stability of catechu before and after autoclave. For more details see protocols - Effects of autoclaving on catechu.



    "Focus on enzyme and
    Why don’t try GRAS?"

    Skype meeting with novozymes bestowed us some key suggestions which are totally worth incorporating in our project for it to be a successful product.
    Our chassis for expression of paan stain degrading genes is Escherichia coli. E.coli is a LPS (lipopolysaccharide producer) and an intestinal flora. Hence, industries like novozymes do not suggest using it as a chassis for enzyme production. Instead, GRAS (generally recognized as safe) organisms like yeast, Aspergillus niger, Bacillus sp, Trichoderma sp. are used as chassis for industrial production of enzymes.
    This is an incredibly valuable suggestion for the future of the project. We were especially delighted because A. niger was one of the isolates we found to be degrading paan stains. However, for the purpose of iGEM we continued to use E. coli as a chassis.
    Novozymes also directed us to focus on the right enzyme and ensure that the breakdown products of paan stain do not attract more dirt.
    They strongly advised us to purify paan stain degrading enzymes and use it for industrial applications instead of the Genetically modified organism (GMO).
    They also suggested that they prefer enzymes in their natural forms i.e without any tags. For the purpose of iGEM we have used enzymes with tags such as histag but in the future we desire to incorporate this suggestion by Novozymes.
    They were of the opinion that our product would have a great market in the housekeeping and detergent industry.


    Social media

    "Smartly decide the time to start social media challenge"
    To make common people realize and spread awareness about the depth of this social problem we decided to start with a social media challenge. Our main concern here was how to maximize the reach of challenge and reach out to the policy makers. We seeked guidance from Miss Nidhi Kamdar, Officer on Special Duty at Chief Minister of Maharashtra office. We got several tips on extending the reach of our challenge.

    Some key points include:-

    • Announcing our challenge at specific active time 8:00 am - 11:00 am and 6:00 pm -9:00pm when people are usually travelling and scrolling through social media.
    • Start with at least 50-100 tweets or posts for #PaanSePareshaan before tagging policy makers to get the challenge to their notice.
    • Tag some policy makers and celebrities on all social media handles.
    • Nudge theory.

    Miss.Nidhi Kamdar is a young and talented lady officer who manages all social media handles of Chief Minister of state of Maharashtra Hon. Mr.Devendra Fadnavis


    Street Play

    “A street play should be crowd pulling”
    Street play is a key tool in generating awareness effectively in a shorter span of time. It was a wholesome experience for our team to step out of the lab and engage with the society through the street play. For a street play to become successful, it is essential to give a power-packed crowd-engaging performance. Under the guidance and direction of veteran actress Mrs. Sushma Rege, we tried to put our best foot forward in exploring this art arena. Rege Ma’am helped us to effectively deliver a regional dialect and how to modulate our voice throughout the play. She also helped us to improve the overall impact of street play making it a success.

    Sushma Rege is a veteran stage TV actress and a teacher who has won recognition and awards at various state and national forums. She has also written and directed numerous plays that spread awareness about various social issues.





    Many laboratories across the world make use of disposable requirements such as plastic petri plates, plastic pipettes, etc. generating a lot of non-biodegradable plastic waste.
    Team Ruia-Mumbai aims at dealing with this issue of increasing plastic waste in a sustainable way. For that, we tried using three approaches which are as follows;

    • 1. Reusing glassware by washing and autoclaving wherever possible.
    • 2. Using reusable glassware instead of using disposable plasticware.
    • 3. Reusing plasticware and checking for its cleanliness.



    Microbiology department of Ramnarain Ruia college comprises of in all 4 UG batches which have on an average 40 students per batch performing practicals on 6 week days.
    If on an average every student requires 5 glasswares of each type listed below, then the total requirement of each type of glassware is as follows:
    40 students *4 batches = 160 students
    160 students*5 glasswares of each type* 6 days = 4800 glasswares
    Hence, we need a total of 4800 glasswares of each type.
    Let us go through the number of each type of glassware we have for UG students in our department

    From the above data it is evident that there is a huge difference in the amount of glassware required as compared to what is actually available in our lab. .

    Then how do we fulfil this requirement ?
    Solution :

    If we replace all these glasswares with disposable plasticware, then the amount of plastic waste that will be generated is;

    4800*10 types of plasticware= 48000 plasticware per week
    Hence, we save on a huge amount of plastic waste generated otherwise.


    Our First Approach :


    Fig.1: From left: Negative control vial (not incubated), vial placed near the lid of the autoclave & vial placed at the base of the autoclave. Comparing the test vials with the negative control vial, it clearly suggest that our autoclave is capable of efficiently sterilizing media.

    Our second approach :


    Our third approach:
    We decided to check whether we can reuse the plasticware by cleaning them and confirming their cleanliness by performing some chemical tests.
    Chemical test:

    • Clean the plasticware with water in tub instead of using running tap water
    • Add 1 ml of phenolphthalein to it.
    • Again wash the plasticware with water.
    • Add 1 ml of NaOH to the same washed apparatus
    • If the solution turns pink that means phenolphthalein is not effectively washed off. Hence, the plasticware cannot be reused.

    All of our plasticware failed the above test, even after giving 3-4 washes, making them non-reusable. However, all the glasswares showed negative results, i.e. the solution did not turn pink, making them considerably reusable.


    Our co-ordinates:

    L .Nappo Road, Matunga
    Dadar East,Mumbai,
    Maharashtra 400019,