Team:Lethbridge HS/Poster
Poster: Lethbridge_HS
tPectinACE: Targeting Pectin to Accelerate Compost Enzymatically
Student Members
Shada Aborawi, Rebecca Avileli, Jasmine Belisle, Thomas Byrne, Wenyu Chen, Kimoya Edwards, Olive Graham, Linda He, Livia Kadezabek, Charlin Li, Dominic Piper, Declan Sander, Lana VanGenderen, Elisha Wong, Marissa Wong, Michelle Wu
Student Advisors
Luke Saville, Dia Koupantsis, Damian La Rosa Montes, Kristi Turton
Faculty
Dr. Laura Keffer-Wilkes (Primary PI, U of L), Jalyce Heller (Primary PI, U of L)
Schools
Winston Churchill High School, Lethbridge Collegiate Institute, Chinook High School, Catholic Central High School
Abstract
As the population of the earth continues to grow, more waste is ending up in land- fills, with a large portion of the items occupying the landfill space being compostable food waste. When food waste enters landfills, there are a variety of environmental consequences such as the copious amounts of greenhouse gases emitted during the production of our food as well as the anaerobic decompositions that occur in landfills. Additionally, food shipping often utilizes cardboard and plastic, which poses harm to the environment. Although food waste disposal services are available, such services release methane and are not economically feasible. Furthermore, both residential and industrial composting facilities do assist in combating the issue of food waste but are oftentimes not readily available. Therefore, we propose a system that employs an engineered biological catalyst that breaks down food waste in a small-scale closed container system, suitable for keeping animals out, decreasing the need for space in landfills and ensuring that unsafe chemicals produced during the composting do not enter the environment. Our aim is to use readily available household materials to design an efficient small-scale composting unit, while increasing the speed of decomposition using an engineered pectin-degradation enzyme catalyst. This project will not only address a local problem within the Lethbridge community, where no city-wide composting system exists, but can also be expanded to other communities where these services are not available.
Introduction
We humans produce a lot of garbage, with much of what is wasted being compostable food and organic material. When food rots in landfills, it releases a large amount of methane gas--an extremely harmful greenhouse gas that plays a significant factor in global warming.
A survey we conducted of residents from the City of Lethbridge gave us some insight into how they feel about composting, as well as struggles with composting that we could address with our project. The following quote was voiced by a Lethbridge resident: “I’m not sure how to start/maintain my own compost & don’t have the resources/materials to do so”
We propose a system that utilizes engineered enzymes that break down food waste in a more time efficient manner. This will be accomplished by increasing the speed of decomposition of homogalacturonan, a common form of pectin, using the enzymes PelB PelC and Pnl in a pectin-degradation system. This project will address this problem of food waste locally, within the Lethbridge community and as well as worldwide.
Fig 1. Diagram demonstrating accelerated breakdown of an apple by utilizing pectinases from the organism P.amylolyticus to target homogalacturonan.
Inspiration
Our project, tPectinACE, is inspired by the amount of food waste being dumped every day and filling up landfill space that could be used for items that have a longer decomposition rate and a less negative impact on the environment. Food waste not only takes up unnecessary space, but it also plays a role in the increasing challenge of climate change. The average consumer's food takes many steps to get from farm to table, so when we waste food, we are also wasting the resources that were used in the production of that food. Greenhouse gases are emitted during the production of our food, and the cardboard and plastic used in shipping further harms our environment. When food is produced but not consumed it ends up in landfills, causing more emissions of greenhouse gases like methane. To our group, these issues presented a sufficient cause that needed attention because of how inaccessible composting is in many cities, specifically Lethbridge where our overall organic waste is much higher than in Alberta and Canada altogether. Food waste has an impact on everyone globally, but with Lethbridge specifically, there is a definite cry for food or organic waste control. Which is why our group’s aim is to make composting an easier and more efficient process that will attract everyone to constantly participate and help to gradually decrease how much food they are wasting that can be composted and used for other purposes. With more people composting, we hope to see a decrease in the amount of organic waste being dumped which is a start to creating a healthier environment to grow and live. 
Community Engagement
Amongst reaching out to local experts for feedback and insight on our system, our team engaged with our community before and during COVID-19. One of the first outreach events we took part in was the University of Lethbridge Playday event. This was before COVID-19 greatly affected Canada, and this year it highlighted agricultural themed activities. Our team had a table at this event, there we had a presentation educating parents on agriculture as well as misconceptions surrounding GMOs. Then for their children we had molecule sets, colouring pages, taught how to pipette, and even brought the vortex which absolutely mesmerized the young ones!
Our next effort to engage with the community was a virtual arts show. Team members performed through various musical mediums, and we took the opportunity to pitch our project and GoFundMe page. We are so incredibly grateful and thankful for our lovely community, despite the present day challenges, they were still able to show their support!
Throughout the season we also planned videos to upload to our Youtube channel. We wanted to continue educating people on synthetic biology and our system, so that is what we geared our videos towards. To this date, we have uploaded our project promotion video along with an at-home composting tutorial. In the future, we plan to upload more at- home experiments, educational videos and tutorials.
In addition, our team attended two zoom conferences catered to Albertans and Canadians, JulyGEM and cGEM, respectively. JulyGEM was our first opportunity this season to connect with other teams in our province, as well as pitch our project. Our pitch was judged by both attendees and esteemed judges. cGEM was a larger conference for our team, as it included participants in all of Canada, and it very closely resembled the giant jamboree. This gave us the opportunity to connect to our Canadian iGEM community and gain practice for the giant jamboree.
Overall this season, our team is proud of all community engagement and public outreach accomplished. It is evident that we have had to foster our creativity to find unique ways to continue engaging. However, we found that it has brought us so much closer to each other and the community!
Composting Survey
Objectives of the survey
In order to get effective feedback for human practices, we needed to be strategic with whom we interviewed. We’ve talked to stakeholders, such as restaurant owners, experts on pectinases, and composting specialists. We’ve received valuable knowledge from a variety of individuals that have helped shape and improve our project. But, despite this, we still recognized the value in finding the opinions of one of the most important stakeholders: the general population. Thus, we created a survey in order to gain opinions and perspectives that help to inform our project’s trajectory.
One of the major goals in the survey was to gather opinions and attitudes towards food waste and composting. A major aspect of our project is to spread awareness and change public opinion on composting and food waste. In order to do this, we first had to understand what the current attitudes were, to recognize if there was a need for our education aspect, and to narrow a target for the goal of this aspect. Thus, we designed questions such as “do you compost”, “if not, why not?”. Another major objective of our project was to understand current problems with composting, as well as barriers to composting that we could work on making better.
Methods of forming and distributing survey
A draft of our survey was initially sent to the University of Lethbridge Office of Research Ethics for review. After it was approved for distribution, we slightly revised a few questions, then formatted it in Qualtrics, a secure survey delivery platform that was suggested by the ethics officer as the most secure option. Delivery of our survey was accomplished using social media. It was distributed from our team’s social media platforms, sent to friends and family members of our team, as well as distributed from Environment Lethbridge’s social media channels, with the help of Kathleen Sheppard, the organization’s executive director. We note that, due to our distribution methods, where, perhaps,a higher number of participants are more active in the scientific community, and with environmental concerns then the general population, results may have been skewed slightly in that direction. Despite this, however, this survey was extremely useful in gathering attitudes about composting and food waste; particularly concerning the education and outreach aspect of our project.
Survey Section 1: Understanding current problems with composting/barriers to composting and where the problems with at-home composting lie, and to get opinions in order to design our project to improve at home composting accessibility.
In response to the question “on a scale of one to ten, how hard do you try to reduce your household waste”, the majority of survey participants answered above “6” on the scale. Those who answered above 5 on the scale elaborated on their methods of food waste reduction. Some responses are as follows:
“[Compost], not buying more than what we can use, give away food when we buy or make more than we can use, use food scraps in other ways,” -Survey Participant, answered 6 on the scale
“Delay grocery shopping trips as long as possible (shop my pantry and fridge), cook perishables and freeze before they go bad, freeze leftovers for lunches, make creative soups” -Survey Participant, answered 9 on the scale
“We buy only what we need and pay close attention to the state of food. Also, we participate in the flashfood program where we buy food that would normally been thrown away. Also, in some food we cut the part that is bruised and eat the rest. We save veggies scraps for broth.” -Survey Participant, answered 10 on the scale
Many people cited composting, buying only as much food as they needed, meal planning, and creative leftover food and overripe produce solutions. In general, results from this section of the survey would be beneficial to the education component of our project in particular, in that, we would have more suggestions to give about food waste reduction based on actual methods that the individuals responding to this survey have utilized. Out of 29 responses, 15 indicated that they do compost, 8 said that they do not, and 6 did not answer the question. When asked a question about the types of products generally composted, along with what challenges came along with the composting, some responses went as follows:
“We compost everything except bones, meat, dairy, grains. It is very difficult to compost at home because it attracts a large variety of garden pests which in turn devour our garden vegetables. Because there are not many people in the neighbourhood composting at this time our compost has become the bug Buffet. Because so many of our neighbours have Lawns full of chemicals with herbicides and pesticides it makes it very difficult to have an organic garden and compost.”
“We have worms at home for vermicompost and a backyard composter. The main challenge we find is that it takes a long time to get final compost that we can use in the garden.”
“All organic matter from the kitchen except meat and dairy, some paper products, I also experimented with a compostable plastic container someone brought to work. It did compost, but took 18-24 months”
“Nothing organic leaves my yard. I have a couple of ‘slow’ compost bins outside. I harvest from them once a year. Inside I have a vermicompost system and harvest every couple of months. It is my ‘fast’ composter. I really have no issues composting, it feels completely natural to me. During the summer when I have to be away from my worms my sister takes them.”
Many people described pests as one of the main challenges to composting, such as wasps and fruit flies, while a few mentioned composting freezing in the winter as an issue, and others described accessibility to outdoor space as a problem, and a couple cited the amount of time it took for the compost to break down as a challenge. As there was not a great majority of individuals who discussed the speed of compost as an issue, these results help to inform our project in directing our focus more towards industrial applications, where speed is a greater issue than it is in individuals’ compost. Additionally, the number of concerns about pests may help to direct our project in the future, to potential work on pest reduction as a system. For example, we could perhaps engineer a scent, in order to keep pests away from compost. We also asked a question directed towards people who do not compost, to describe why they do not. The two major things that came up in these responses were either people did not know how to compost, or they did not have access to it due to living arrangements (apartment buildings, renting,etc.). These responses would also help to greatly inform our education initiative to direct more attention towards teaching individuals how to compost, and finding creative solutions to compost in housing that does not generally support traditional composting.
“I compost when I’m living in Calgary (during summers) as it is easy with the provided bins. I’m not sure how to start/maintain my own compost & don’t have the resources/materials to do so”
“I've tried it once and it attracted gophers in my yard. I am single and don't produce much waste so it seems to take time for the compost to be workable. I would prefer that the city introduce brown bins to collect organic waste.”
Survey Section 2- Composting Accessibility
The diagram below outlines the proportions of all the answers to a question concerning accessibility of compost. 50% of replies indicated “faster more efficient breakdown of compost” as an option that would help make compost more accessible to them. This data supports the idea that individuals are looking to increase the rate of the breakdown of their compost, which our system accomplishes. This can indicate a need for our system, although this data slightly contradicts the fewer amounts of replies discussing speed of compost in earlier questions. One reason for this incongruence may be due to the fact that this question has the answer listed. So, people may not automatically view speed or efficiency as a real issue to their compost, but when given the option to, would recognize the value in it.
Survey part 3: Feedback on our system
This part of the survey was dedicated to getting feedback and opinions on how the biological portion of our project is perceived. We wanted to understand if people would be willing to use our system and wanted to get feedback from these important stakeholders--the public-- on how to make our system better.
At the start of this section, we included a brief description of our system’s objectives. It goes as follows:
‘Our team is working on a project to increase the rate of composting by engineering enzymes to break down pectin, a component found in the cell walls of plants that is present in the majority of fruits and plants that make up a composting pile. We are planning on having the system be administered in a lysate form, therefore there wouldn't be any live organisms.’
In this section, we included a question that asks the survey participants to describe any concerns they have with our proposed system, from a consumer standpoint. Several responses indicated no concerns, while multiple asked about the safety of it around pets, the compost and the ecosystem, and some had concerns about it being overly expensive or taxing to use.
Responses: “How will this [affect] the dirt to use? Will there be any long term negative effects on food grown with compost and therefore the people who eat that food.”
“I would just like more information about the process and product before making any changes to my home compost system. Pet friendly?”
“[If it would] be too expensive or too time consuming. ie. if I had to add some every day.”
“[N]one at this time - i think it's a great idea and believe that more efforts should be geared towards managing organic material that is wasted. I believe that no organic material should be sent to the landfills, so the more opportunities and research towards that belief is absolutely necessary and encouraged.”
These responses are extremely helpful, as they allow us to dive into other areas of research or our project, to ensure that our system is safe to use in various settings, and to look into cost analysis to ensure consumers are willing to pay for our system.
Conclusion
All in all, this survey allowed us to gauge concerns about composting, our biological system, and get valuable feedback on how to improve our system in order to best suit our stakeholders’ needs. Concerns from pests to cost were brought up, and it gave us insights into what real people want and need in food waste diversion. As we continue to develop tPectinACE, these responses will help shape the way in which we design our system so as to address the concerns of home-composters, as well as spread awareness about the benefits of composting.
In order to get effective feedback for human practices, we needed to be strategic with whom we interviewed. We’ve talked to stakeholders, such as restaurant owners, experts on pectinases, and composting specialists. We’ve received valuable knowledge from a variety of individuals that have helped shape and improve our project. But, despite this, we still recognized the value in finding the opinions of one of the most important stakeholders: the general population. Thus, we created a survey in order to gain opinions and perspectives that help to inform our project’s trajectory.
One of the major goals in the survey was to gather opinions and attitudes towards food waste and composting. A major aspect of our project is to spread awareness and change public opinion on composting and food waste. In order to do this, we first had to understand what the current attitudes were, to recognize if there was a need for our education aspect, and to narrow a target for the goal of this aspect. Thus, we designed questions such as “do you compost”, “if not, why not?”. Another major objective of our project was to understand current problems with composting, as well as barriers to composting that we could work on making better.
Methods of forming and distributing survey
A draft of our survey was initially sent to the University of Lethbridge Office of Research Ethics for review. After it was approved for distribution, we slightly revised a few questions, then formatted it in Qualtrics, a secure survey delivery platform that was suggested by the ethics officer as the most secure option. Delivery of our survey was accomplished using social media. It was distributed from our team’s social media platforms, sent to friends and family members of our team, as well as distributed from Environment Lethbridge’s social media channels, with the help of Kathleen Sheppard, the organization’s executive director. We note that, due to our distribution methods, where, perhaps,a higher number of participants are more active in the scientific community, and with environmental concerns then the general population, results may have been skewed slightly in that direction. Despite this, however, this survey was extremely useful in gathering attitudes about composting and food waste; particularly concerning the education and outreach aspect of our project.
Survey Section 1: Understanding current problems with composting/barriers to composting and where the problems with at-home composting lie, and to get opinions in order to design our project to improve at home composting accessibility.
In response to the question “on a scale of one to ten, how hard do you try to reduce your household waste”, the majority of survey participants answered above “6” on the scale. Those who answered above 5 on the scale elaborated on their methods of food waste reduction. Some responses are as follows:
“[Compost], not buying more than what we can use, give away food when we buy or make more than we can use, use food scraps in other ways,” -Survey Participant, answered 6 on the scale
“Delay grocery shopping trips as long as possible (shop my pantry and fridge), cook perishables and freeze before they go bad, freeze leftovers for lunches, make creative soups” -Survey Participant, answered 9 on the scale
“We buy only what we need and pay close attention to the state of food. Also, we participate in the flashfood program where we buy food that would normally been thrown away. Also, in some food we cut the part that is bruised and eat the rest. We save veggies scraps for broth.” -Survey Participant, answered 10 on the scale
Many people cited composting, buying only as much food as they needed, meal planning, and creative leftover food and overripe produce solutions. In general, results from this section of the survey would be beneficial to the education component of our project in particular, in that, we would have more suggestions to give about food waste reduction based on actual methods that the individuals responding to this survey have utilized. Out of 29 responses, 15 indicated that they do compost, 8 said that they do not, and 6 did not answer the question. When asked a question about the types of products generally composted, along with what challenges came along with the composting, some responses went as follows:
“We compost everything except bones, meat, dairy, grains. It is very difficult to compost at home because it attracts a large variety of garden pests which in turn devour our garden vegetables. Because there are not many people in the neighbourhood composting at this time our compost has become the bug Buffet. Because so many of our neighbours have Lawns full of chemicals with herbicides and pesticides it makes it very difficult to have an organic garden and compost.”
“We have worms at home for vermicompost and a backyard composter. The main challenge we find is that it takes a long time to get final compost that we can use in the garden.”
“All organic matter from the kitchen except meat and dairy, some paper products, I also experimented with a compostable plastic container someone brought to work. It did compost, but took 18-24 months”
“Nothing organic leaves my yard. I have a couple of ‘slow’ compost bins outside. I harvest from them once a year. Inside I have a vermicompost system and harvest every couple of months. It is my ‘fast’ composter. I really have no issues composting, it feels completely natural to me. During the summer when I have to be away from my worms my sister takes them.”
Many people described pests as one of the main challenges to composting, such as wasps and fruit flies, while a few mentioned composting freezing in the winter as an issue, and others described accessibility to outdoor space as a problem, and a couple cited the amount of time it took for the compost to break down as a challenge. As there was not a great majority of individuals who discussed the speed of compost as an issue, these results help to inform our project in directing our focus more towards industrial applications, where speed is a greater issue than it is in individuals’ compost. Additionally, the number of concerns about pests may help to direct our project in the future, to potential work on pest reduction as a system. For example, we could perhaps engineer a scent, in order to keep pests away from compost. We also asked a question directed towards people who do not compost, to describe why they do not. The two major things that came up in these responses were either people did not know how to compost, or they did not have access to it due to living arrangements (apartment buildings, renting,etc.). These responses would also help to greatly inform our education initiative to direct more attention towards teaching individuals how to compost, and finding creative solutions to compost in housing that does not generally support traditional composting.
“I compost when I’m living in Calgary (during summers) as it is easy with the provided bins. I’m not sure how to start/maintain my own compost & don’t have the resources/materials to do so”
“I've tried it once and it attracted gophers in my yard. I am single and don't produce much waste so it seems to take time for the compost to be workable. I would prefer that the city introduce brown bins to collect organic waste.”
Survey Section 2- Composting Accessibility
The diagram below outlines the proportions of all the answers to a question concerning accessibility of compost. 50% of replies indicated “faster more efficient breakdown of compost” as an option that would help make compost more accessible to them. This data supports the idea that individuals are looking to increase the rate of the breakdown of their compost, which our system accomplishes. This can indicate a need for our system, although this data slightly contradicts the fewer amounts of replies discussing speed of compost in earlier questions. One reason for this incongruence may be due to the fact that this question has the answer listed. So, people may not automatically view speed or efficiency as a real issue to their compost, but when given the option to, would recognize the value in it.
Survey part 3: Feedback on our system
This part of the survey was dedicated to getting feedback and opinions on how the biological portion of our project is perceived. We wanted to understand if people would be willing to use our system and wanted to get feedback from these important stakeholders--the public-- on how to make our system better.
At the start of this section, we included a brief description of our system’s objectives. It goes as follows:
‘Our team is working on a project to increase the rate of composting by engineering enzymes to break down pectin, a component found in the cell walls of plants that is present in the majority of fruits and plants that make up a composting pile. We are planning on having the system be administered in a lysate form, therefore there wouldn't be any live organisms.’
In this section, we included a question that asks the survey participants to describe any concerns they have with our proposed system, from a consumer standpoint. Several responses indicated no concerns, while multiple asked about the safety of it around pets, the compost and the ecosystem, and some had concerns about it being overly expensive or taxing to use.
Responses: “How will this [affect] the dirt to use? Will there be any long term negative effects on food grown with compost and therefore the people who eat that food.”
“I would just like more information about the process and product before making any changes to my home compost system. Pet friendly?”
“[If it would] be too expensive or too time consuming. ie. if I had to add some every day.”
“[N]one at this time - i think it's a great idea and believe that more efforts should be geared towards managing organic material that is wasted. I believe that no organic material should be sent to the landfills, so the more opportunities and research towards that belief is absolutely necessary and encouraged.”
These responses are extremely helpful, as they allow us to dive into other areas of research or our project, to ensure that our system is safe to use in various settings, and to look into cost analysis to ensure consumers are willing to pay for our system.
Conclusion
All in all, this survey allowed us to gauge concerns about composting, our biological system, and get valuable feedback on how to improve our system in order to best suit our stakeholders’ needs. Concerns from pests to cost were brought up, and it gave us insights into what real people want and need in food waste diversion. As we continue to develop tPectinACE, these responses will help shape the way in which we design our system so as to address the concerns of home-composters, as well as spread awareness about the benefits of composting.
Engineering
What is compost?
In its most basic form, compost is produced when organic and nitrogenous waste, such as food scraps, paper towels, and yard waste, is digested by decomposers (e.g. insects, fungi, and bacteria), resulting in the production of thermal energy, carbon dioxide and water.
Under ideal conditions, the result will be nutrient dense rich black earth, that has decreased dramatically in mass and size.
Compost goes through three main stages over time. The initial stage consists mainly of physical decomposition by insects and large organisms, causing rapid decomposition of amino acids.
Some thermophilic bacteria such as Actinomycetes begin to actively break down the more complex compounds (e.g. cellulose, lignin, chitin, and proteins) and as bacterial activity increases, more organic matter is broken down thus raising the temperature of the compost.
As the compost reaches the thermophilic phase, the majority of decomposition occurs, as it is the longest stage with the most active bacteria. This is the point where the cellulose, hemicellulose, and other parts of the plant cell walls are broken down.
Once the compost reaches a high enough temperature, the harmful pathogens are killed, sanitizing it. At this point, the compost is turned over either by turning the soil or the container to avoid it reaching temperatures above 70 °C, in which the thermophilic bacteria would die, slowing or even stopping the process all together.
The third and final maturation phase occurs after the compost has been turned. There is no longer enough food for the bacteria, causing them to die off. This is when there will be the most fungal activity in the compost, which will finish the process of decomposing cellulose and lignin. The result is a homogeneous soil conditioner around half the weight of the original waste, composed of mineral and bacterial matter, and high in organic content.
What is pectin?
Pectin is a complex family of polysaccharides found in the cell walls of vascular plants, as well as in the cell walls of other organisms, like algae.
Since pectin is present in the majority of the components that make up a compost pile, including fruits and plants, we hypothesize that by targeting the breakdown of pectin we will increase the rate of the breakdown of compost.
System Components
There are a variety of pectins, with varying complexity, but the pectin that is the most abundant in nature is homogalacturonan (commonly abbreviated as HG). HG forms the backbone to most pectins, and is the simplest form of pectin. In our system, we chose to target the breakdown of this pectin because of its commonality and structural simplicity.
In order to achieve our goal, which is to increase the rate of composting, we designed our system to increase the amount and activity of the enzymes that are involved in degradation of HG. We selected the enzymes to do this job from the organism Paenibacillus amylolyticus.
P. amylolyticus is an interesting Gram-positive bacterium with diverse plant cell wall polysaccharide deconstruction capabilities. It was originally isolated from an insect hindgut and has a unique pectin degradation pathway. The enzymes from this organism have a wider substrate range, as it can break down both methylated and unmethylated HG. It can break HG down without having to modify it first, so it doesn’t require a methylesterase, which is ideal for a system like ours as it skips an entire step in the degradation process.
Our project will take the genes from P. amylolyticus and engineer E. coli in order to produce an HG decomposition system from cell lysate or purified components. We will utilize the enzymes Pnl, PelB, PelC.
In the process of HG degradation, Pnl, is a pectin lyase, meaning this enzyme is responsible for the depolymerization of substrates that have a high degree of methylation. Because of this core role, we chose to use Pnl as one the enzymes we’ll be utilizing. On the other side, PelB, is responsible for breaking down demethylated HG, and has been found to break down unmethylated polygalacturonic acid as efficiently as the enzymes Pnl, Pel A, B, C and D combined. Lastly, PelC is required in our system as it which can make internal demethylated regions available to the enzyme PelB, which is required for the complete deconstruction of HG because PelB is an exotype enzyme and would not be able to access these demethylated regions without PelC.
We originally wanted to use a system with the three enzymes already mentioned, as well as with the enzymes PelA and PelD. However, we omitted these enzymes from our system as they were not as vital to HG degradation as the other three, and we wanted to simplify our system as much as possible. In a paper by Keggi and Doran-Peterson published in June 2020, found that under the conditions used in their experiment, the enzymes “PelA and PelD could be omitted from the enzyme mixture without compromising the rate of deconstruction or the final degree of completion”. There seems to be minimal difference between the reaction progress of all five enzymes versus PelB, PelC, and Pnl alone.
System Delivery
Using the Pnl, PelB and PelC genes from the P. amylolyticus genome, we will to create an HG decomposition system from cell lysate or purified components.
For the delivery of our system, we are considering two different methods. The first, would be to engineer an HG degradation pathway into a microbe, such as E. coli, then apply the cell lysate to compost. Secondly, we could purify each of our enzymes and create a lyophilized powder.
In order to test our system, we plan on completing a colourimetric assay to test the degradation properties of the enzymes, by measuring the amount of HG degradation through the increase in galactose in solution. This method has previously been utilized by Keggi & Doran-Peterson (2000).
In its most basic form, compost is produced when organic and nitrogenous waste, such as food scraps, paper towels, and yard waste, is digested by decomposers (e.g. insects, fungi, and bacteria), resulting in the production of thermal energy, carbon dioxide and water.
Under ideal conditions, the result will be nutrient dense rich black earth, that has decreased dramatically in mass and size.
Compost goes through three main stages over time. The initial stage consists mainly of physical decomposition by insects and large organisms, causing rapid decomposition of amino acids.
Some thermophilic bacteria such as Actinomycetes begin to actively break down the more complex compounds (e.g. cellulose, lignin, chitin, and proteins) and as bacterial activity increases, more organic matter is broken down thus raising the temperature of the compost.
As the compost reaches the thermophilic phase, the majority of decomposition occurs, as it is the longest stage with the most active bacteria. This is the point where the cellulose, hemicellulose, and other parts of the plant cell walls are broken down.
Once the compost reaches a high enough temperature, the harmful pathogens are killed, sanitizing it. At this point, the compost is turned over either by turning the soil or the container to avoid it reaching temperatures above 70 °C, in which the thermophilic bacteria would die, slowing or even stopping the process all together.
The third and final maturation phase occurs after the compost has been turned. There is no longer enough food for the bacteria, causing them to die off. This is when there will be the most fungal activity in the compost, which will finish the process of decomposing cellulose and lignin. The result is a homogeneous soil conditioner around half the weight of the original waste, composed of mineral and bacterial matter, and high in organic content.
What is pectin?
Pectin is a complex family of polysaccharides found in the cell walls of vascular plants, as well as in the cell walls of other organisms, like algae.
Since pectin is present in the majority of the components that make up a compost pile, including fruits and plants, we hypothesize that by targeting the breakdown of pectin we will increase the rate of the breakdown of compost.
System Components
There are a variety of pectins, with varying complexity, but the pectin that is the most abundant in nature is homogalacturonan (commonly abbreviated as HG). HG forms the backbone to most pectins, and is the simplest form of pectin. In our system, we chose to target the breakdown of this pectin because of its commonality and structural simplicity.
In order to achieve our goal, which is to increase the rate of composting, we designed our system to increase the amount and activity of the enzymes that are involved in degradation of HG. We selected the enzymes to do this job from the organism Paenibacillus amylolyticus.
P. amylolyticus is an interesting Gram-positive bacterium with diverse plant cell wall polysaccharide deconstruction capabilities. It was originally isolated from an insect hindgut and has a unique pectin degradation pathway. The enzymes from this organism have a wider substrate range, as it can break down both methylated and unmethylated HG. It can break HG down without having to modify it first, so it doesn’t require a methylesterase, which is ideal for a system like ours as it skips an entire step in the degradation process.
Our project will take the genes from P. amylolyticus and engineer E. coli in order to produce an HG decomposition system from cell lysate or purified components. We will utilize the enzymes Pnl, PelB, PelC.
In the process of HG degradation, Pnl, is a pectin lyase, meaning this enzyme is responsible for the depolymerization of substrates that have a high degree of methylation. Because of this core role, we chose to use Pnl as one the enzymes we’ll be utilizing. On the other side, PelB, is responsible for breaking down demethylated HG, and has been found to break down unmethylated polygalacturonic acid as efficiently as the enzymes Pnl, Pel A, B, C and D combined. Lastly, PelC is required in our system as it which can make internal demethylated regions available to the enzyme PelB, which is required for the complete deconstruction of HG because PelB is an exotype enzyme and would not be able to access these demethylated regions without PelC.
We originally wanted to use a system with the three enzymes already mentioned, as well as with the enzymes PelA and PelD. However, we omitted these enzymes from our system as they were not as vital to HG degradation as the other three, and we wanted to simplify our system as much as possible. In a paper by Keggi and Doran-Peterson published in June 2020, found that under the conditions used in their experiment, the enzymes “PelA and PelD could be omitted from the enzyme mixture without compromising the rate of deconstruction or the final degree of completion”. There seems to be minimal difference between the reaction progress of all five enzymes versus PelB, PelC, and Pnl alone.
System Delivery
Using the Pnl, PelB and PelC genes from the P. amylolyticus genome, we will to create an HG decomposition system from cell lysate or purified components.
For the delivery of our system, we are considering two different methods. The first, would be to engineer an HG degradation pathway into a microbe, such as E. coli, then apply the cell lysate to compost. Secondly, we could purify each of our enzymes and create a lyophilized powder.
In order to test our system, we plan on completing a colourimetric assay to test the degradation properties of the enzymes, by measuring the amount of HG degradation through the increase in galactose in solution. This method has previously been utilized by Keggi & Doran-Peterson (2000).
At-home Composting
At Home Composting Study
As a way to have a more hands on experience and better understanding of the process of composting, our team decided to create our own composting experiment. As a way to share this process with our community we also documented the process of building your own at-home compost bin which is featured on our youtube channel. Team members created their own composting bins and recorded data on their composting practices over the period of two months while taking observations and maintaining the compost every week. The goal of this experiment was to investigate the favoured conditions applied to composts by testing aeration and moisture. The measurements we collected included pH, smell, bacterial composition, the amount of compost added and temperature.
Key Takeaways from Study
However, although observations and measurements were taken there is an insufficient amount of quantitative data for our team to draw to a conclusion about our results. Instead this experiment allowed our team to experience the process of composting for ourselves to determine any unsatisfactory outcomes from composting and what processes worked better for us as well as how we could resolve these problems to provide tips and advice on creating a positive result from composting. Some important observations taken by our team about the composting process pertained to the importance of aeration and maintaining airflow done by stirring the contents of the compost. Another relevant piece of advice is to attempt to achieve the 3:1 ratio of brown (soil) to green (kitchen scraps) material in order to sustain a well incorporated mixture and a satisfactory end product. Our team also learned it was wise to cut food scraps into smaller pieces in order to increase degradation rate.
As a way to have a more hands on experience and better understanding of the process of composting, our team decided to create our own composting experiment. As a way to share this process with our community we also documented the process of building your own at-home compost bin which is featured on our youtube channel. Team members created their own composting bins and recorded data on their composting practices over the period of two months while taking observations and maintaining the compost every week. The goal of this experiment was to investigate the favoured conditions applied to composts by testing aeration and moisture. The measurements we collected included pH, smell, bacterial composition, the amount of compost added and temperature.
Key Takeaways from Study
- Proper composting needs to have the right amount of moisture, oxygen, and carbon: nitrogen material ratios in order to decompose efficiently.
- If these specific needs are not met, the compost will not decompose at an efficient rate.
- Aeration is a critical aspect of maintaining a successful compost bin
- Organic waste breakdown can be sped up due to pectinases contributing to the decomposition process. As we are building a thermostable pectinase variant, the climate or specifically the heat of the compost will not cause a loss in enzymatic activity.
However, although observations and measurements were taken there is an insufficient amount of quantitative data for our team to draw to a conclusion about our results. Instead this experiment allowed our team to experience the process of composting for ourselves to determine any unsatisfactory outcomes from composting and what processes worked better for us as well as how we could resolve these problems to provide tips and advice on creating a positive result from composting. Some important observations taken by our team about the composting process pertained to the importance of aeration and maintaining airflow done by stirring the contents of the compost. Another relevant piece of advice is to attempt to achieve the 3:1 ratio of brown (soil) to green (kitchen scraps) material in order to sustain a well incorporated mixture and a satisfactory end product. Our team also learned it was wise to cut food scraps into smaller pieces in order to increase degradation rate.
Homology Modelling
The enzymes PelB, PelC and Pnl from P. amylolyticus do not have crystal structures available. To gain a better understanding of how our proteins function, we decided to generate a homology model using related, known structures of pectin degradation enzymes. The structures will also allow us to complete molecular dynamics simulations as well. Pnl, PelB and PelC were modeled using SWISS-MODEL and iTASSER. For Pnl, the sequences identified showed only 23% identity and also very low similarity.
The structure of Pnl shows the polypeptide strand forming into the canonical 𝛃-helix fold, common among pectate lyase enzyme families. Due to the low sequence similarity the N-terminus was not able to be modeled through these methods.
The sequence used to model PelB had 93% identity and 96% similarity, meaning that this structure could be used much more reliably for function predictions.
Both SWISS-MODEL and iTASSER modeling of PelB resulted in an overall 𝛃-helix structure, typical of PL1 enzymes. Several alpha-helices decorate the exterior of the inner 𝛃-sheets. Similar to Pnl, PelB has additional amino acids at the N-terminus that were unable to be modelled using this software. However, predictions using QUARK, has shown that this region in both proteins fold into an extended alpha-helix. Unfortunately, PelC also had low sequence identity and similarity, 34 and 47%, respectively with the amino acid sequence that was used to predict the structure. As with Pnl, the low sequence similarity between these enzymes amino acid sequences means that we have to be careful when making any interpretations from the structures.
Improving the Thermostability of Pectin Degradation Enzymes
Thermostability is extremely important for our project as composting piles can reach temperatures above 45 degrees Celsius. These high temperatures may appear to benefit us - the optimal temperatures of all the proteins are between 55 and 70 degrees Celsius - but it is important to ensure that our system can remain stable over long periods of time. Bearing this in mind we wanted to improve the thermostability of our system.
We completed multiple sequence alignments of Pnl enzymes from many different species to create a general consensus sequence. We then analyzed this sequence with Popmusic to determine the sites at which point mutations would be most effective at improving thermostability. Team members then implemented these changes to the Pnl sequence. Two different sets of point mutations were implemented, one that resulted in increases in predicted thermostability greater than fifteen degrees, and one with increases limited to between five and eight degrees.
With these new sequences we have hopefully created a more thermostable version of our project that is better suited to the high temperatures compost piles can reach.
Wet Lab Experiments
Cloning P. amylolyticus Genes
Future Directions
Contributions to iGEM Community
Parts
Basic and Composite parts consisting of:
Parts Review (Bronze Requirement)
Basic and Composite parts consisting of:
- Pnl (BBa_K3349000)
- PelB (BBa_K3349001)
- PelC (BBa_K3349002)
- T7 promoter (BBa_I712074)
- medium strength RBS (BBa_J61100)
- double terminator (BBa_B0014)
Parts Review (Bronze Requirement)
- Rose Odor Generator Device (Geranoil) (BBa_K727007), AUC Turkey 2012
- Pectinase (BBa_K2377003), Lanzhou 2017
- Lumazine Synthase (BBa_K249002 ), Lethbridge 2009
References and Acknowledgements
References
- Aggie Horticulture. (n.d.). Chapter 1, The Decomposition Process: Earth-Kind® Landscaping. Retrieved November 09, 2020, from https://aggie-horticulture.tamu.edu/earthkind/landscape/dont-bag-it/chapter-1-the-decomposition-process/
- Altschul, S., Gish, W., Miller, W., Myers, E., and Lipman, D. (1990). Basic local alignment search tool. Journal of Molecular Biology. 215:403-410.
- Bell, J. (2015). An organic waste inventory for Alberta’s agrifood sector. Retrieved November 09, 2020, from https://provisioncoalition.com/Assets/ProvisionCoalition/Documents/Library%20Content/Food%20Waste%20Management/An%20Organic%20Waste%20Inventory%20for%20Alberta.pdf
- Boland, W., Henriksen, E., and Doran-Peterson, J. (2010). Characterization of two paenibacillus amylolyticus strain 27C64 pectate lyases with activity on highly methylated Pectin. Applied and Environmental Microbiology. 76: 6006-6009
- CIEI-China iGEM team (2017). Terminator of Food Waste. Retrieved November 09, 2020, from https://2017.igem.org/Team:CIEI-China/Project/Background.
- Environment Lethbridge (n.d.). Wasteless: An Environment Lethbridge Project. Retrieved November 09, 2020, from http://wasteless.ca/waste-in-yql
- Food and Agriculture Organization of the United Nations. (n.d.). Food wastage: Key facts and figures. . Retrieved November 09, 2020, from http://www.fao.org/news/story/en/item/196402/icode/
- Frischmann, C. (2019, April 01). Opinion | The climate impact of the food in the back of your fridge. Retrieved November 09, 2020, from https://www.washingtonpost.com/news/theworldpost/wp/2018/07/31/food-waste/
- Gasteiger E., Gattiker A., Hoogland C., Ivanyi I., Appel R.D.. and Bairoch A. (2003). ExPASy: the proteomics server for in-depth protein knowledge and analysis Nucleic Acids Research. 31:3784-3788
- Madeira, F., Park, Y., Lee, J., Buso, N., Gur, T., Madhusoodanan, N., Basutkar, P., Tivey, A., Potter, S., Finn, R., and Lopez, R. (2019). The EMBL-EBI search and sequence analysis tools APIs in 2019. Nucleic Acids Research. 47: W636-W641.
- Government of Canada. (2019, November 01). Food Loss and Waste. Retrieved November 09, 2020, from https://www.canada.ca/en/environment-climate-change/services/managing-reducing-waste/food-loss-waste.html
- Heiss-Blanquet, S., Foyelle-Guichard, F., Lombard, V., Hébert, A., Coutinho, P., Groppi, A., Barre, A., and Henrissat, B. (2016). Composting-like conditions are more efficient for enrichment and diversity of organisms containing cellulase-encoding genes than submerged cultures. PLos One 11: e0167216
- Janus, A. (2019, January 17). More than half of all food produced in Canada is lost or wasted, report says | CBC News. Retrieved November 09, 2020, from https://www.cbc.ca/news/canada/toronto/food-waste-report-second-harvest-1.4981728
- Keggo, C., and Doran-Peterson, J. (2020). The homogalacturonan deconstruction system of Paenibacillus amylolyticus 27C64 requires no extracellular pectin methyltransferase and has a significant industrial potential. Applied and environmental microbiology. 86:e02275-19
- Nideh, D., Rogowski, A., Cartmell, A., Luis, A., Baslé, Gray, J., Venditto, I., Briggs, J., Zhang, X., Labourel, A., Terrapon, N., Buffetto, F., Nepogodiev, S., Xiao, Y., Field, R., Zhu, Y., O’Niell, M., Urbanowicz, B., York, W., Davies, G., Abbott, W., Ralet, M., Martens, E., Henrissat, B., and Gilbert, H. (2017). Complex pectin metabolism by gut bacteria reveals novel catalytic functions. Nature. 544: 65-70
- Silva, I., Larsen, D., Jets, C., Derkx, P., Meyer, A., and Mikkelsen, J. (2013). Enhancing lyase thermostability by targeted single point mutations. Applied Microbiology Biotechnology. 97: 9727-9735
- Zeng, G., Zhang, L., Dong, H., Chen, Y., Zhang, J., Zhu, Y., Xie, Y., and Fang, W. (2017). Pathway and mechanism of nitrogen transformation during composting: Functional enzymes and genes under different concentrations of PVP-AgNPs. Bioresource Technology. 253:112-120
Acknowledgements
Thank you to:
Nicole Robinson, City of Lethbridge
Bill MacMillan, City of Lethbridge
Kathleen Sheppard, Environment Lethbridge
Anni Huang, Fusion Sushi Owner
Dr. Wade Abbot, Scientist, Agriculture and AgriFood Canada
Christina Seidel,
Dr. Michael Stingl, University of Lethbridge Philosophy Professor