Something's fishy
Wake up! It's food o' clock. Cooking is a lot more than just food, it brings people closer together. Throughout history, fish has been utilised by humans as a food source. Fishes are integrated into many traditions throughout the world being the traditional food for Rosh Hashanah, the Jewish New Year, or through fish festivals etc. It plays a huge role in ceremonial traditions, creating important ties between families and individuals and embodying their symbolic ties to the environment in communities residing near coastlines. Many countries have fishes as a part of their primary diet.
However, methylmercury and its prevalence in seafood is a major deterrent. Hence, in an attempt to create awareness as well as celebrate different recipes from around the world we have curated Something's fishy.
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Taxonomy of Hanoi
In order to spread the beauty of biology during the COVID-19 pandemic, we decided to develop an Android game. The game developed follows the concept of Tower of Hanoi, one of the most popular puzzles. The aim of the game is to transfer the tower of n disks from the first rack to the third (last) one. This has to be done by ensuring that while stacking the disks, a smaller disk is always placed over a larger disk. The reverse is forbidden. Our version of the game tries to put out a fun twist to learning taxonomic ranks. The disks represent the rank. Young children can learn the taxonomic classifications for widely known creatures like humans, butterflies and lions.
Newspaper Article
In order to spread information about methylmercury poisoning: its source, exposure, long term effects, treatment and its prevalence in fishes, we have written a newspaper article in the local language of Kannada which is to be published in a newspaper.
To download this document, click here.
Data
Fish Consumption
Having a long coastline, fish is a staple food for many people in India. Some of the major fishing communities are the Kolis in Maharashtra, Mogaveeras in Karnataka, Jalaris and Rajbhansis in Orissa, Mukkuvars and Paravas in Tamil Nadu etc. The Tangkhul people of Manipur celebrate Khairi Kashao in the dry months of April or May. This festival is a means to invoke the rain gods, it is also parallelly an occasion for mass fishing. The traditional fishing festival of the Jaunpuri community of Tehri Garhwal, Maund Matsya Mela is what the festival in Uttarakhand is called. This festival was started as a means to reduce exploitative fishing all year in the river. The Chang people of Nagaland celebrate Khiliinyie, which is a week-long harvesting festival. This shows the high relevance of our project in our native country.
Figure 1: High Consumption
Figure 2: Moderate Consumption
Figure 3: Low Consumption
Complex Nitrogen sources
Nitrogen is one of the primary nutrients critical for the survival of all living organisms. It is a necessary component of many biomolecules, including proteins, DNA, and chlorophyll. There are a variety of complex nitrogen sources available in India.
Corn steep liquor is a by-product of the corn wet milling industry. It is rich in nutrients and is available at a much cheaper price point than most traditional alternatives such as yeast extract. Soybean Liquor, Groundnut meal and Cottonseed Flour are all high protein concentrates that are produced as by products of their respective oil productions. Yeast extract is one of the most popular nitrogen sources that is used in the industry. It is a by-product of the beer brewing industry.
Figure 4: Map of Complex Nitrogen Sources
Population Enumeration Data - Census 2011.
Methylmercury and Fishes
Rivers are a high risk source for methylmercury containing fishes due to the presence of industries, mines, etc near them. We have depicted some of the popular fishes in this ecosystem and their methylmercury content.
Figure 5: Affected Ecosystems
Figures from Pal M. et al, 2012.
Estimation of production
In order to design the bioreactor we conducted a literature study as well as a survey to estimate the production requirements.
We estimated the number of people who would consume the pills based on their location (rural or urban) and possible willingness to take the medication. We used the information we got through the survey about the number of people who would be willing to take the probiotic regularly and modified the values considering that the people who filled our survey were educated.
Figure 6: Map of expected consumption
| Consumption | Dosage | Total pills per month | Total amount of active component (Child Variant) in g | Total amount of active component (Adult Variant) in g |
|---|---|---|---|---|
| High consumption | 6 pills per month | 464999748 | 3347998.186 | 7672495.842 |
| Moderate consumption | 2 pills per month | 42077976 | 302961.4272 | 694286.604 |
| Low consumption | 1 pill per month | 90802634 | 653778.9648 | 1498243.461 |
| Note: Active component for in child variant 16mg and 30mg in adult variant | ||||
Survey 1
A survey was conducted to understand the level of awareness about methylmercury poisoning amongst the general public. We had 138 responses and most of the people who filled the survey did not have a background in biological sciences.
Awareness about methylmercury poisoning:
Figure 7:
Figure 8:
Figure 9:
Concern about GMOs:
Figure 10:
Figure 11:
Willingness to consume probiotics:
Figure 12:
Survey 2
In order to have a better perception of the views of doctors and pharmacists about probiotics, we conducted a survey where we went to clinics and drug stores and requested them to do the same. Majority of the stakeholders were in favour of probiotics as shown
Figure 13:
Using the survey we found out about the most common probiotics in the market - Darolac, Vivagut, Totalis, Bifilac, Enterogermina, Floristore, Vibact etc. Most of these probiotics are sold in capsule form enabling us to gain insight that it is the most preferred method of delivery. As you can see in the following figures, the preferences of our stakeholders are highly in the favour of pills.
Figure 14:
Figure 15:
Our probiotic pill is proposed as a preventative measure which would be recommended to be taken regularly. As shown, most of our stakeholders would consider doing so. As opposed to our first survey, Concerns about GMOs were few, which could also be due to the lack of awareness amongst their customers about what GMOs are. The most common concern was about the long term effects of GMOs. Our stakeholders themselves have a mixed perception of GMOs.
Figure 16:
Figure 17:
Figure 18:
In order to expand on the survey we also had an interview with Dr. Sudha Somayaji, a gynaecologist and obstetrician practicing in Mangalore.
What is your general opinion about probiotics?
I highly recommend probiotics over other medication for the same problems as I believe it is extremely safe, effective and reliable. I also believe that it is safe for pregnant women.
Do you or any of your patients express concerns about the contents of probiotics like GMOs?
My patients are not of the background who would know what the contents are. In India, most patients trust their doctors on the medications. My opinion of Genetically modified organisms is in favour of it for therapeutic purposes.
Which is the most preferred method of consumption of probiotics for you?
Capsules would be preferred for adults. However, I am concerned about capsules for children as it can get stuck in the throat but if appropriate sizes are used, then I would prescribe it.
How often do you prescribe probiotics? Are the reactions positive?
I prescribe probiotics very often - Monthly about 1 box (100 capsules). The reactions are overwhelmingly positive, with some complaints about the capsule getting stuck in the throats for children.
Conversations
In order to keep in check the information and design relevance of our project as well as make improvements, we consulted many experts. They helped in innumerable ways to shape our project from a problem statement to a solution.
Dr. Keyur Raval:
Figure 19: Dr. Keyur Raval
Design of a bioreactor can make or break the effective implementation of a project. Hence, we sought advice from Dr. Keyur Raval, an associate professor in the Department of Chemical Engineering, National Institute of Technology, Karnataka. His knowledge and expertise in scale-up of cell cultures, bioreactor design, bioprocess development and optimization enabled us to understand the considerations involved in these areas.
At first we had decided to remove the antibiotic-resistance genes from the final product considering the safety aspects. However, he said that the presence of antibiotic-resistance genes would create a plasmid conservation pressure during production which would ensure that plasmid loss would not occur as well as prevent contamination of media. Hence, we modified the final design of each plasmid to have a different antibiotic resistance gene in the final strain developed for production.
However, inclusion of such genes in the final strain design poses the threat of imparting unnecessary antibiotic resistance to the natural gut microbiome. When we voiced our concerns about this, he suggested the usage of CRISPR Cas 9 technology to integrate our design to the genome of the bacteria. However, we decided against it as the long term effects of this recent technology are not known. In addition, Escherichia coli Nissle 1917 cannot conjugate and thus cannot participate in Horizontal Gene Transfer which made us more confident about the traditional method.
Initially our media formulation used glucose and tryptone as the carbon source. However, he directed us towards the fact these components can be a source of food for humans and animals and tryptone would be too expensive on a large scale - thus increasing final product cost. Hence, we modified our media formulation as well as cryo and lyoprotectant composition to contain glycerol instead. Our nitrogen source was fixed to be only yeast extract at first, however, he asked us to consider the costs of doing so. He said that since the main cost driver of bioprocessing is incurred due to raw materials, different regions might prefer different nitrogen sources depending on cost and availability. Hence, our media formulation was changed to be variable according to availability. He also suggested the use of SCaBa impellers as they are the current industry standard.
We also presented the design of SAUL to him. He noticed that our retention time in the device was too low as compared to the retention time of food in the gut. This enabled us to improve the experimentation aspects further.
Dr. Abdul Ajees:
Figure 20: Dr. Abdul Ajees
Due to the pandemic, the modelling aspects of our project became even more important. To visualize the structure of our protein and understand its aspects, we consulted Dr. Abdul Ajees, associate professor, Department of Atomic and Molecular Physics, Manipal Academy of Higher Education. He guided us about the various visualization techniques such as molecular dynamics. We wanted to see how methylmercury interacts with alkylmercury lyase and what are the conformational changes in the structure due to the interactions. He suggested the use of the Morph command on the PyMol software which enabled us to do the same and guided us on what the changes suggested about the proteins.
Dr. Sudha Somayaji:
Figure 21: Dr. Sudha Somayaji
As a therapeutic project, one of our most important stakeholders are doctors who would be prescribing the medicines, we interviewed Dr. Sudha Somayaji, Gynaecologist and Obstetrician practicing in Mangalore, Karnataka, India. Her concerns about large capsules being a difficult mode of delivery for children helped us to survey more and keep two variants of our product - for adults and for children.
Dr. Vytla Ramachandra Murty:
Figure 22: Dr. Vytla Ramachandra Murty
At the beginning stages of designing a bioreactor, we were considering several options for the type of reactor to be chosen. We explained our project and needs to Dr. Vytla Ramachandra Murty, a professor in the Department of Biotechnology, Manipal Institute of Technology who recommended stirred batch fermenters as an appropriate choice.
Ms. Archana Mahadev Rao:
Figure 23: Ms. Archana Mahadev Rao
In order to develop a product, innumerable aspects have to be considered. To ensure we have done the same, Ms. Archana Mahadev Rao, professor in department of biotechnology, Manipal Institute of technology, gave us a good manufacturing practices checklist. She said that given a product and a desired annual production rate bioprocess design endeavours to answer several questions, them being
- What are the required amounts of raw materials and utilities?
- What is the required size of process equipment and supporting utilities?
- What is the manufacturing cost?
- What is the optimum batch size?
- How long does a single batch take?
- How much product can be generated per year?
- What is the demand for resources in the course of a batch?
- What is the amount of resources consumed?
- Are there any bottlenecks?
Following this, we were able to design several preliminary experiments (refer to iGEM MIT_MAHE Product Development Experiments Handbook) which we had not considered initially.
Dr. Saran Kumar:
Figure 24: Dr. Saran Kumar
In order to ensure that we have considered all possibilities during our design phase, we approached Dr. Saran Kumar, Postdoctoral fellow, Faculty of Medicine, Hebrew University of Jerusalem, Israel. He asked us innumerable questions of the various possibilities and ensured that we have chosen the best possible design. He expressed his concerns about the plasmid length which is also one of our concerns. Hence we designed an experiment which would measure the rate of plasmid loss due to length.
Dr. Subbalaxmi Selvaraj:
Figure 25: Dr. Subbalaxmi Selvaraj
Optimal media formulation is one of the most essential steps in bioprocess design. In order to improve our design, we consulted Dr. Subbalaxmi Selvaraj, professor in the Department of Biotechnology, Manipal Institute of Technology. She suggested we calculate the respiratory and yield coefficient as well as the theoretical oxygen demand to better understand our requirements. She also asked us to formulate experiments to determine the nitrogen content in the nitrogen sources which would be used so that optimal amounts can be added.
Downloads
Raw Data about estimated demand, region-wise distrbution of complex nitrogen sources, fish consumption, and methylmercury content in fishes can be downloaded below.
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